VL_Grundlagen Stdtebau

MagMAX ™ FFPE DNA/RNA Ultra KitAutomated or manual isolation of total nucleic acid (TNA) from FFPE samples using AutoLys tubesCatalog Number A31881Pub. No. MAN0017538 Rev.A.0WARNING! Read the Safety Data Sheets (SDSs) and follow thehandling instructions. Wear appropriate protective eyewear,clothing, and gloves. Safety Data Sheets (SDSs) are available from /support .Product descriptionThe Applied Biosystems ™ MagMAX ™ FFPE DNA/RNA Ultra Kit is designed to isolate both DNA and RNA from the same section offormaldehyde- or paraformaldehyde-fixed, paraffin-embedded (FFPE)tissues. The kit also allows for flexibility to isolate DNA only, RNA only or total nucleic acid (TNA). The kit uses MagMAX ™ magnetic-bead technology, ensuring reproducible recovery of high-quality nucleic acid through manual or automated processing. The isolated nucleic acid is appropriate for use with a broad range of downstream assays, such as quantitative real-time RT-PCR and next-generation sequencing.In addition to the use of traditional solvents, the kit is compatible with Autolys M tubes that enable a faster and more convenient means of deparaffinizing FFPE samples by eliminating the need for organic solvents such as xylene or CitriSolv and ethanol. Samples are put into the tubes for protease digestion, tubes are lifted with the Auto-pliers or Auto-Lifter and then samples are spun down. The wax and debris are contained in the upper chamber while the lysate is passed through.Afterwards, the clarified lysate can be directly purified with the MagMAX ™ FFPE DNA/RNA Ultra Kit.For guides without using AutoLys M tubes for sequential DNA and RNA isolation, or DNA isolation, or RNA isolation only, seeMagMAX ™ FFPE DNA/RNA Ultra Kit User Guide (sequential DNA/RNA isolation) (Pub. No. MAN0015877), or MagMAX ™ FFPE DNA/RNA Ultra Kit User Guide (DNA isolation only) (Pub. No. MAN0015905), or MagMAX ™ FFPE DNA/RNA Ultra Kit User Guide (RNA isolation only)(Pub. No. MAN0015906), respectively.This guide describes isolation of TNA from FFPE tissue blocks or FFPE slides using AutoLys M tubes. Three optimized methods for sections or curls both up to 40 µm using AutoLys M tubes are included:•Manual sample processing.•KingFisher ™ Flex Magnetic Particle Processor with 96 Deep-Well Head (DW96; 96-well deep well setting).•KingFisher ™ Duo Prime Magnetic Particle Processor (12-well deep well setting).For sequential DNA and RNA isolation, or DNA isolation, or RNA isolation only, see MagMAX ™ FFPE DNA/RNA Ultra Kit User Guide (sequential DNA/RNA isolation) (Pub. No. MAN0017541), MagMAX ™FFPE DNA/RNA Ultra Kit User Guide (DNA isolation only)(Pub. No. MAN0017539), or MagMAX ™ FFPE DNA/RNA Ultra Kit User Guide (RNA isolation only) (Pub. No. MAN0017540), respectively.Contents and storageReagents provided in the kit are sufficient for 48 TNA isolations from sections up to 40 µm with the AutoLys workflow.Table 1 MagMAX ™ FFPE DNA/RNA Ultra Kit (Cat. No. A31881)Additional reagents are available separately; Protease Digestion Buffer, Binding Solution, and DNA Wash Buffer are also available as Cat. No. A32796. [2]Shipped at room temperature. [3]Not used in TNA workflow[4]Final volume; see “Isolate TNA“ on page 4.Required materials not suppliedUnless otherwise indicated, all materials are available through . MLS: Fisher Scientific ( ) or other major laboratory supplier.Table 2 Materials required for nucleic acid isolation (all methods)Table 3 Additional materials required for manual isolationTable 4 Additional materials required for automated isolationIf needed, download the KingFisher ™ Duo Prime or Flex programThe programs required for this protocol are not pre-installed on theKingFisher ™Duo Prime Magnetic Particle Processor or on theKingFisher ™Flex Magnetic Particle Processor 96DW.1.On the MagMAX ™ FFPE DNA/RNA Ultra Kit product web page,scroll down to the Product Literature section.2.Right-click on the appropriate program file(s) for your samplesize to download the program to your computer:3.4.Refer to the manufacturer's documentation for instructions forinstalling the program on the instrument.Procedural guidelines•Perform all steps at room temperature (20–25°C) unless otherwise noted.•When mixing samples by pipetting up and down, avoid creating bubbles.•When working with RNA:–Wear clean gloves and a clean lab coat.–Change gloves whenever you suspect that they are contaminated.–Open and close all sample tubes carefully. Avoid splashing or spraying samples.–Use a positive-displacement pipettor and RNase-free pipette tips.–Clean lab benches and equipment periodically with an RNase decontamination solution, such as RNase Zap ™ Solution (Cat.No. AM9780).•Incubation at 60°C can be extended overnight to increase DNA yields, followed by incubation at 90°C for 1 hour.•Volumes for reagent mixes are given per sample. We recommend that you prepare master mixes for larger sample numbers. To calculate volumes for master mixes, refer to the per-well volume and add 5–10% overage.Before you beginBefore first use of the kit•Prepare the Wash Solutions from the concentrates:–Add 46 mL of isopropanol to RNA Wash Buffer Concentrate ,mix, and store at room temperature.–Add 168 mL of ethanol to Wash Solution 2 Concentrate , mix,and store at room temperature.Before each use of the kit•Equilibrate the Nucleic Acid Binding Beads to room temperature.•Pre-heat the incubators or ovens to 60°C and 90°C.•Prepare the following solutions according to the following tables.Table 5 Protease solutionTable 6 TNA Binding BufferPrepare the FFPE samples•For curls from FFPE tissue blocks: proceed to “Prepare the curls from FFPE tissue blocks“ on page 3.•For FFPE slide-mounted sections: proceed to “Prepare samples from FFPE slides“ on page 3.Prepare the curls from FFPE tissue blocksa.Cut sections from FFPE tissue blocks using a microtome.Note: For miRNA extraction, we recommend using sections of 10 µm or thicker.b.Collect each section in an AutoLys M tube.1Section FFPE tissue blocks a.Add 235 µL of the Protease Solution (see Table 5).Note: If working with curls, they might stick straight up so make sure to submerge samples in theProtease Solution with a tip or a 1 mL syringe plunger or do a quick spin down at 3000 rpm for 1 minute prior to the addition of buffer to collapse the curl. Time may be extended.b.Incubate at 60°C for 1 hour or longer.Note: Use the AutoLys racks and place in an incubator or oven.c.Incubate at 90°C for 1 hour.Note: For automated isolation, set up the processing plates during the incubation.·For isolation using KingFisher ™ Duo Prime Magnetic Particle Processor, proceed to “Set up the processing plate“ on page 4.·For isolation using KingFisher ™ Flex Magnetic Particle Processor 96DW, proceed to “Set up the TNA processing plates“ on page 5.2Digest with Protease a.Allow samples to cool down for 3–5 minutes before proceeding to lift the tubes.e the Auto-plier for individual tube lifting or the Auto-lifter for multiple tube lifting of up to 24 tubes.c.Lock the tubes in position by hand or use the locking lid.d.Centrifuge at 2000 × g for 10 minutes in a benchtop centrifuge with plate adapters.e.Unlock the tubes by hand or remove the locking lid.e the Auto-plier or Auto-lifer to lift the inner tube for sample access.g.Proceed to purification. See “Isolate TNA“ on page 43Lift the tubesPrepare samples from FFPE slidesa.Pipet 2–4 µL of Protease Digestion Buffer depending on the tissue size evenly across the FFPE tissuesection on the slide to pre-wet the section.Note: You can adjust the volume of Protease Digestion Buffer if the tissue is smaller or larger.b.Scrape the tissue sections in a single direction with a clean razor blade or scalpel, then collect the tissueon the slide into a cohesive mass.c.Transfer the tissue mass into an AutoLys M tube with the scalpel or a pipette tip.d.Add 235 µL of the Protease Solution (see Table 5).Note: Be sure to submerge samples in the Protease Solution with a tip or a 1 mL syringe plunger e.Incubate at 60°C for 1 hour or longer.Note: Use the AutoLys racks and place in an incubator or oven.f.Incubate at 90°C for 1 hour.Note: For automated isolation, set up the processing plates during the incubation.·For isolation using KingFisher ™ Duo Prime Magnetic Particle Processor, proceed to “Set up the processing plate“ on page 4.·For isolation using KingFisher ™ Flex Magnetic Particle Processor 96DW, proceed to “Set up the TNA processing plates“ on page 5.4Scrape the samples and digest with Proteasea.Allow samples to cool down for 3–5 minutes before proceeding to lift the tubes.e the Auto-plier for individual tube lifting or the Auto-lifter for multiple tube lifting of up to 24 tubes.c.Lock the tubes in position by hand or use the locking lid.d.Centrifuge at 2000 × g for 10 minutes in a benchtop centrifuge with plate adapters.e.Unlock the tubes by hand or remove the locking lid.e the Auto-plier or Auto-lifer to lift the inner tube for sample access.g.Proceed to purification. See “Isolate TNA“ on page 45Lift the tubesIsolate TNA•To isolate TNA manually, proceed to “Isolate TNA manually“ on page 4.•To isolate TNA using the KingFisher ™Duo Prime Magnetic Particle Processor, proceed to “Isolate TNA using KingFisher ™ Duo Prime Magnetic Particle Processor“ on page 4.•To isolate TNA using the KingFisher ™Flex Magnetic Particle Processor 96DW, proceed to “Isolate TNA using KingFisher ™ Flex Magnetic Particle Processor 96DW“ on page 5.Isolate TNA manuallyUse microcentrifuge tubes to perform manual TNA isolations.a.After the Protease digestion is complete, add 20 µL of Nucleic Acid Binding Beads to the samples.b.Add 900 µL of TNA Binding Buffer (see Table 6) to the sample.c.Shake for 5 minutes at speed 10 or 1150 rpm.d.Place the sample on the magnetic stand for 2 minutes or until the solution clears and the beads are pelleted against the magnet.e.Carefully discard the supernatant with a pipette.1Bind the TNA to beadsa.Wash the beads with 500 µL of RNA Wash Buffer.b.Shake for 1 minute at speed 10 or 1150 rpm until the mixture is thoroughly chocolate brown in color.c.Place the sample on the magnetic stand for 2 minutes or until the solution clears and the beads arepelleted against the magnet.d.Carefully discard the supernatant with a pipette.e.Repeat steps a-d.f.Wash the beads with 500 µL of Wash Solution 2.g.Shake for 1 minute at speed 10 or 1150 rpm until the mixture is thoroughly chocolate brown in color.h.Place the sample on the magnetic stand for 2 minutes or until the solution clears and the beads arepelleted against the magnet.i.Carefully discard the supernatant with a pipette.j.Repeat steps f-i.k.Shake for 1–2 minutes at speed 10 or 1150 rpm to dry the beads.Do not over-dry the beads. Over-dried beads results in low TNA recovery yields.2Wash TNA on the beadsa.Add 50 µL of Elution Solution to the beads.b.Shake for 5 minutes at speed 10 or 1150 rpm and at 55°C until the mixture is thoroughly chocolatebrown in color.c.Place the sample on the magnetic stand for 2 minutes or until the solution clears and the beads arepelleted against the magnet.The supernatant contains the purified TNAThe purified TNA is ready for immediate use. Store at –20°C or –80°C for long-term storage.3Elute the TNAIsolate TNA using KingFisher ™ Duo Prime Magnetic Particle ProcessorDuring the protease incubation, add processing reagents to the wells of a MagMAX ™ Express-96 Deep WellPlate as indicated in the following table.Table 7 TNA plate setupRow on the MagMAX ™ Express-96 Deep Well Plate.4Set up the processing plate a.Ensure that the instrument is set up for processing with the deep well 96–well plates and select theappropriate program A31881_DUO_large_vol_TNA on the instrument.b.At the end of the protease incubation, add 200 µL of sample to each well in Row G of the TNA plate.c.Add 20 µL of Nucleic Acid Binding Beads to each sample well in Row G.d.Start the run and load the prepared processing plates when prompted by the instrument.5Bind, wash, rebind, and elute the TNA5Bind, wash, rebind,and elute the TNA(continued)e.At the end of the run, remove the Elution Plate from the instrument and transfer the eluted TNA(Row A of TNA plate) to a new plate and seal immediately with a new MicroAmp ™ Clear Adhesive Film.IMPORTANT! Do not allow the purified samples to sit uncovered at room temperature for more than 10minutes, to prevent evaporation and contamination.The purified TNA is ready for immediate use. Store at –20°C or –80°C for long-term storage.Isolate TNA using KingFisher ™ Flex Magnetic Particle Processor 96DWDuring the protease incubation, add processing reagents to the wells of MagMAX ™ Express-96 Plates asindicated in the following table.Table 8 TNA plates setupPosition on the instrument6Set up the TNA processing platesa.Ensure that the instrument is set up for processing with the deep well magnetic head and select theA31881_FLEX_large_vol_TNA program on the instrument.b.At the end of the protease incubation, add 200 µL of sample to each well in Plate 1.c.Add 20 µL of Nucleic Acid Binding Beads to each sample well in Plate 1.d.Start the run and load the prepared processing plates in their positions when prompted by theinstrument (see “Set up the TNA processing plates“ on page 5).e.At the end of the run, remove the Elution Plate from the instrument and seal immediately with a newMicroAmp ™ Clear Adhesive Film.IMPORTANT! Do not allow the purified samples to sit uncovered at room temperature for more than 10minutes, to prevent evaporation and contamination.The purified TNA is ready for immediate use. Store at –20°C or –80°C for long-term storage. .7Bind, wash, rebind, and elute the TNALimited product warrantyLife Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life Technologies' website at /us/en/home/global/terms-and-conditions.html . If you have any questions,please contact Life Technologies at /support .Manufacturer: Life Technologies Corporation | 2130 Woodward Street | Austin, TX 78744The information in this guide is subject to change without notice.DISCLAIMER : TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE,MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.Important Licensing Information : This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited Use Label Licenses.©2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified./support | /askaquestion 。

Eliminate Secondary Labeling Steps. Matrix 2D Barcoded ScrewTop Storage tubes are available pre-printed with 1D and human-readable codes that match the tube’s 2D code. Laser etched 2D, linear and human readable barcodes are also included on the sides of the standard latch racks. The additional barcoding on tube and rack eliminates the need for the secondary application of labels, streamlining storage procedures.Enhanced Secure Tracking. A permanently bonded, unique 2D barcode is laser etched onto the base of every tube to securely identify and track samples. The linear and human readable codes match this unique 2D barcode, allowing 1D scanning or visual identification of samples across satellite or collaborative sites without2D readers. Complementary Thermo Scientific 2D and 1D barcode readers scan and instantly decode each tube’s barcode into any application or database. Laser etched 2D, linear, and human readable barcodes are included on three sides of the standard latch rack, providing traceability at the rack level and providing readability to laboratories with or without barcode decoding equipment. The 2D barcode included on the bottom of the rack serves as both an orientation and identification feature for automated and benchtop storage equipment.Superior Storage Format. Matrix 2D Barcoded ScrewTop storage tubes are available in specially designed, barcoded, stackable, microplate-footprint Latch Racks to save precious space in storage and on the bench top while maintaining traceability. Lid canbe positioned to rest on the rack, preventing contamination during manual pipetting. Or remove lid completely for robotic handling. Flexibility. Side-printed Matrix ScrewTop tubes and barcoded latch racks maintain compatibility with existing ScrewTop tube storage systems and accessories. ScrewTop Removal Tool caps/ decaps tubes individually to enable one-handed pipetting. The Thermo Scientific ScrewTop Cap Tray can be used to cap/decap several tubes at once with automated capping systems or to fill tubes on an automated liquid handling platform, then cap an entire rack at once. Seven color cap options allow easy identification of samples at a glance.Stringent Quality Control. Every 2D barcoded storage tube is scanned to ensure readability. All barcodes are checked against our database of previously assigned codes to prevent duplicates.S A M P L E S T O R A G EThermo Scientific™ Matrix™2D Barcoded ScrewTop Tubes in Barcoded RacksMatrix 2D Barcoded ScrewTop Storage tubes are now available in a standard barcoded latch rack. The barcoded latch rack provides permanent 2D, linear,and human readable codes on 3 sidesof the rack. A 2D barcode on the rack bottom enables orientation detection and identification when using automated storage equipment. These additions to the Matrix ScrewTop Storage tube portfolio allow visual sample identification ina range of laboratory procedures and eliminate the need for secondary labeling. Identify and track your samplesfor optimal sample storageProduct SpecificationsThermo Scientific MatrixBarcoded Tubes and RacksDescription 500 µl ScrewTop Storage Tubes 1.0 ml ScrewTop Storage Tubes Item Number3743, 3744, 37453744-WP; 3744-WP1D; 3745-WP; 3745-WP1D 3740, 3741, 37423741-WP; 3741-WP1D; 3742-WP; 3742-WP1D Figure 1A: Tube Side ViewWidth (1A)7.40±0.10 [0.290±0.002]7.40±0.10 [0.290±0.002]Height (1A)30.60±0.20 [1.204±0.007]44.00±0.10 [1.732±0.003]Figure 1B: Tube Side View w/ ScrewTop Width (1B)8.70±0.10 [0.344±0.003]8.70±0.10 [0.344±0.003]Height (1B)39.20 [1.543]52.60 [2.071]Figure 2: Rack Side ViewHeight (2A)29.50±0.10 [1.160±0.005]38.10±0.10 [1.500±0.005]Height (2B)29.10±0.10 [1.145±0.005]42.50±0.20 [1.673±0.008]Height (2C)32.60±0.30 [1.282±0.012]46.00±0.20 [1.809±0.008]Height (2D)41.30±0.40 [1.626±0.017]54.60±0.30 [2.149±0.110]Figure 3: Rack Top View Length 127.75±0.13 [5.029±0.005]127.75±0.13 [5.029±0.005]Width85.46±0.13 [3.364±0.005]85.46±0.13 [3.364±0.005]Figure 4: Stacked Rack ViewSingle Height (4A)44.32±0.24 [1.745±0.010]58.42±0.25 [2.300±0.010]Incremental Height (4B)43.31±0.25 [1.705±0.010]57.30±0.25 [2.220±0.010]Stacked Height (4C)87.63±0.51 [3.450±0.020]114.81±0.51 [4.520±0.020]Description Storage Tubes Barcoded RackItem Number3743-BR, 3744-BR, 3745-BR, 3744-WP-BR, 3744-WP1D-BR, 3745-WP-BR, 3745-WP1D-BR, 3740-BR, 3741-BR, 3742-BR, 3741-WP-BR, 3741-WP1D-BR, 3742-WP-BR, 3742-WP1D-BR, 3748-BR Figure 5: Rack Bottom ViewDistance to barcode from short side (5A)120.48 ± 0.254 [4.74 ± 0.010]Distance to barcode from short side (5B) 1.443 ± 0.203 [0.057 ± 0.008]Barcode length5.83 ± 0.127 [0.230 ± 0.005]Distance to barcode from long side (5C)71.98 ± 0.254 [2.834 ± 0.010]Distance to barcode from long side (5D)8.18 ± 0.203 [0.322 ± 0.008]Barcode width5.30 ± 0.127 [0.209 ± 0.005]Description ScrewTop Cap Tray Item Number 4906, 4477Figure 6: Tray Top View Length 127.75±0.25 [5.03±0.010]Width85.58±0.25 [3.37±0.010]Figure 7: Stacked Tray ViewSingle Height (2A)19.81±0.13 [0.78±0.005]Incremental Height (2B)18.29±0.25 [0.72±0.010]Stacked Height (2C)38.10±0.25 [1.50±0.010]Tube Material Virgin Class VI Medical Grade Polypropylene Rack Material Polycarbonate with acetal latchesCap MaterialVirgin Class VI Medical Grade Polypropylene with Santoprene™ gasket ScrewTop Cap Tray Material Acrylonitrile Butadiene Styrene (ABS)Contaminant-free All tubes and tray are supplied free from DNA, RNAse, DNAse and endotoxinsAutoclavable Racked and unracked tubes are autoclavable with the caps loosened; ScrewTop trays are not autoclavable 2D CodeNon-proprietary, 12x12 Data-matrix with ECC200 Built-in Error Correction Tube/Cap Temp Range -196°C to 121°C; Autoclaving and boiling to vapor phase liquid nitrogen storage Latch Rack Coding 2D Datamatrix; Linear EAN Code 128; Human Readable Matrix Barcoded ScrewTop TubesProduct SpecificationsBarcode WidthDistance to barcodefrom short side (5B)Barcode LengthDistance to barcode from short side (5A)Distance to barcode from long side (5C)s Figure1: Tube Side View Width (1B)s Figure 2: Rack Side ViewSSSST2DTUBES 0615© 2015 Thermo Fisher Scientific Inc. All rights reserved. Santoprene is a registered trademark of Exxon Mobil Corporation. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries./samplestorageCat. No.Non-Barcoded Rack Cat. No.Barcoded Rack DescriptionMax Working Volume, µL Cap Color Qty37443744-BR Tube, ScrewTop, 500 µL 2D, V-Bottom 590Colorless 5 racks of 96/case Sterile3744RED 3744RED-BR Tube, ScrewTop, 500 µL 2D, V-Bottom 590Red 5 racks of 96/case Sterile 3744YEL 3744YEL-BR Tube, ScrewTop, 500 µL 2D, V-Bottom 590Yellow 5 racks of 96/case Sterile 3744BLU 3744BLU-BR Tube, ScrewTop, 500 µL 2D, V-Bottom 590Blue 5 racks of 96/case Sterile 3744GRE 3744GRE-BR Tube, ScrewTop, 500 µL 2D, V-Bottom 590Green 5 racks of 96/case Sterile 3744WHI 3744WHI-BR Tube, ScrewTop, 500 µL 2D, V-Bottom 590White 5 racks of 96/case Sterile 3744PUR 3744PUR-BR Tube, ScrewTop, 500 µL 2D, V-Bottom 590Purple 5 racks of 96/case Sterile 3744AMB 3744AMB-BR Amber Tube, ScrewTop, 500µL 2D, V-bottom 590 5 racks of 96/case Sterile 3743AMB 3743AMB-BR Amber Tube, ScrewTop, 500µL 2D, V-bottom 590Red5 racks of 96/case Sterile 37453745-BR Tube, ScrewTop, 500 µL 2D, V-Bottom, No Caps 590 5 racks of 96/caseSterile 3744-WP 3744-WP-BR Tube, ScrewTop, 500 µL 2D, WP, V-Bottom 590Colorless 5 racks of 96/case Sterile 3744-WP1D 3744-WP1D-BR Tube, ScrewTop, 500 µL 2D, WP/1D, V-Bottom 590Colorless5 racks of 96/case Sterile 3745-WP 3745-WP-BR Tube, ScrewTop, 500 µL 2D, WP, V-Bottom, No caps 590 5 racks of 96/case Sterile 3745-WP1D 3745-WP1D-BRTube, ScrewTop, 500 µL 2D, WP/1D, V-Bottom, No caps 590 5 racks of 96/caseSterile 3743Tube, ScrewTop, 500 µL 2D, V-Bottom 590Colorless Bulk, 480/case Sterile 3743RED Tube, ScrewTop, 500 µL 2D, V-Bottom 590Red Bulk, 480/case Sterile 3743YEL Tube, ScrewTop, 500 µL 2D, V-Bottom 590Yellow Bulk, 480/case Sterile 3743BLU Tube, ScrewTop, 500 µL 2D, V-Bottom 590Blue Bulk, 480/case Sterile 3743GRE Tube, ScrewTop, 500 µL 2D, V-Bottom 590Green Bulk, 480/case Sterile 3743WHI Tube, ScrewTop, 500 µL 2D, V-Bottom 590White Bulk, 480/case Sterile 3743PUR Tube, ScrewTop, 500 µL 2D, V-Bottom590Purple Bulk, 480/case Sterile 37413741-BR Tube, ScrewTop, 1.0 mL 2D, V-Bottom 940Colorless 5 racks of 96/case Sterile 3741RED 3741RED-BR Tube, ScrewTop, 1.0 mL 2D, V-Bottom 940Red 5 racks of 96/case Sterile 3741YEL 3741YEL-BR Tube, ScrewTop, 1.0 mL 2D, V-Bottom 940Yellow 5 racks of 96/case Sterile 3741BLU 3741BLU-BR Tube, ScrewTop, 1.0 mL 2D, V-Bottom 940Blue 5 racks of 96/case Sterile 3741GRE 3741GRE-BR Tube, ScrewTop, 1.0 mL 2D, V-Bottom 940Green 5 racks of 96/case Sterile 3741WHI 3741WHI-BR Tube, ScrewTop, 1.0 mL 2D, V-Bottom 940White 5 racks of 96/case Sterile 3741PUR 3741PUR-BR Tube, ScrewTop, 1.0 mL 2D, V-Bottom 940Purple 5 racks of 96/case Sterile 3741AMB 3741AMB-BR Amber Tube, ScrewTop, 1.0mL 2D, V-Bottom 940Red5 racks of 96/case Sterile 3742AMB 3742AMB-BR Amber Tube, ScrewTop, 1.0mL 2D, V-Bottom 940 5 racks of 96/case 37423742-BR Tube, ScrewTop, 1.0 mL 2D, V-Bottom, No Caps 940 5 racks of 96/caseSterile 3741-WP 3741-WP-BR Tube, ScrewTop, 1.0 mL 2D, WP, V-Bottom 940Colorless 5 racks of 96/case Sterile 3741-WP1D 3741-WP1D-BR Tube, ScrewTop, 1.0 mL 2D, WP/1D, V-Bottom 940Colorless5 racks of 96/case Sterile 3742-WP 3742-WP-BR Tube, ScrewTop, 1.0 mL 2D, WP, V-Bottom, No caps 940 5 racks of 96/case Sterile 3742-WP1D 3742-WP1D-BRTube, ScrewTop, 1.0 mL 2D, WP/1D, V-Bottom, No caps 940 5 racks of 96/caseSterile 3740Tube, ScrewTop, 1.0 mL 2D, V-Bottom 940Colorless Bulk, 480/case Sterile 3740RED Tube, ScrewTop, 1.0 mL 2D, V-Bottom 940Red Bulk, 480/case Sterile 3740YEL Tube, ScrewTop, 1.0 mL 2D, V-Bottom 940Yellow Bulk, 480/case Sterile 3740BLU Tube, ScrewTop, 1.0 mL 2D, V-Bottom 940Blue Bulk, 480/case Sterile 3740GRE Tube, ScrewTop, 1.0 mL 2D, V-Bottom 940Green Bulk, 480/case Sterile 3740WHI Tube, ScrewTop, 1.0 mL 2D, V-Bottom 940White Bulk, 480/case Sterile 3740PURTube, ScrewTop, 1.0 mL 2D, V-Bottom940PurpleBulk, 480/caseSterileORDERING GUIDEAsia: Australia: 1300-735-292; New Zealand: 0800-933-966; China +86-21-6865-4588 or +86-10-8419-3588;China Toll-free: 800-810-5118 or 400-650-5118; Singapore +65-6872-9718; Japan: +81-3-5826-1616; Korea +82-2-2023-0640; Taiwan + 886-2-87516655; India: +91-22-6680-3000 Europe: Austria: +43-1-801-40-0; Belgium: +32-2-482-30-30; Denmark: +45-4631-2000; France: +33-2-2803-2180; Germany: +49-6184-90-6000; Germany Toll-free: 0800-1-536-376;Italy: +39-02-95059-554; Netherlands: +31-76-571-4440; Nordic/Baltic/CIS countries: +358-10-329-2200; Russia: +7-(812)-703-42-15; Spain/Portugal: +34-93-223-09-18; Switzerland: +41-44-454-12-12; UK/Ireland: +44-870-609-9203 North America: USA/Canada +1-585-586-8800; USA Toll-free: 800-625-4327South America: USA sales support: +1-585-586-8800 Countries not listed: +49-6184-90-6000 or +33-2-2803-2000Sterile ScrewTop Tube Caps Cat. No.DescriptionCap Color Qty/Case Cat. No.Description Cap Color Qty/Case 4906Tray, ScrewTop Cap, Empty 5 trays4470Caps, ScrewTop Tube Colorless Bulk, 5004477Tray, ScrewTop Cap Coloreless 5 trays of 96 caps 4470RED Caps, ScrewTop Tube Red Bulk, 5004477RED Tray, ScrewTop Cap Red 5 trays of 96 caps 4470YEL Caps, ScrewTop Tube Yellow Bulk, 5004477YEL Tray, ScrewTop Cap Yellow 5 trays of 96 caps 4470BLU Caps, ScrewTop Tube Blue Bulk, 5004477BLU Tray, ScrewTop Cap Blue 5 trays of 96 caps 4470GRE Caps, ScrewTop Tube Green Bulk, 5004477GRE Tray, ScrewTop Cap Green 5 trays of 96 caps 4470WHI Caps, ScrewTop Tube White Bulk, 5004477WHI Tray, ScrewTop Cap White 5 trays of 96 caps 4470PURCaps, ScrewTop TubePurpleBulk, 5004477PURTray, ScrewTop CapPurple5 trays of 96 caps。

Therapeutic Products DirectorateMedical Devices BureauRoom 1605, Main Statistics CanadaBuildingTunney’s Pasture, P.L. 0301H1Ottawa, OntarioK1A 0L2June 19, 1998To: Medical Devices StakeholdersSubject: Guidance for the Labelling of In Vitro Diagnostic Devices (Draft)The proposed Medical Devices Regulations set out the requirements governing the sale, importation and advertisement of medical devices. The goal of the Regulations is to ensure that medical devices distributed in Canada are safe, effective, and meet quality standards. It is the intention of the Therapeutic Products Programme to have these proposed Regulations published in Canada Gazette II in May 1998 and begin implementation on July 1, 1998.This draft document, titled Guidance for the Labelling of In Vitro Diagnostic Devices, sets out the Programme’s guidance for Industry on the subject. It is being provided now in a draft format so that interested stakeholders can comment and participate in its development.This guidance document is intended to assist manufacturers in understanding and complying with the regulatory requirements for labelling in vitro diagnostic devices.For more information on in vitro diagnostic devices please contact:Maria CarballoHead, In Vitro Diagnostic Devices Section, Device Evaluation Divisionphone: (613)-954-9391To comment on this document or to get more information on how to label an IVDD please contact by July 15, 1998:Maria CarballoDevice Evaluation Division, Medical Devices Bureau1605 Main Statistics Canada Building,Postal Locator: 0301H1Tunney’s Pasture, Ottawa, Ontario K1A 0L2phone: (613) 954-9391, fax: (613) 946-8798e-mail: Maria_Carballo@hc-sc.gc.caThank you for providing yourcomments.Beth PietersonA/DirectorMedical Devices Bureau AttachmentsTherapeutic Products Programme Programme des produits thérapeutiquesOUR MISSION: To ensure that the drugs, medical devices,NOTRE MISSION: Faire en sorte que les médicaments, les matériels and other therapeutic products available in Canada are safe,médicaux et les autres produits thérapeutiques disponibles au Canada effective and of high quality.soient sûrs, efficaces et de haute qualité.DRAFTTherapeutic Products ProgrammeGUIDANCE DOCUMENTGuidance for the Labelling of In Vitro Diagnostic DevicesGuidance Document for the Labelling of In Vitro Diagnostic Devices GD012/RevDR-MDBDraft file: labl_ivd_e.wpd June 24, 1998 Printed: December 11, 2002Page 1 of 15 Table of Contents1Introduction (3)1.1Purpose (3)1.2Scope (3)1.3Definitions (3)2Labelling requirements for IVDDs (4)3Labelling information for IVDDs as required by Section 21 Subsection (1) Paragraphs (a) to (j) (4)3.1Label (4)3.2Labelling requirements for a package insert (5)3.2.1Name of the IVDD (5)3.2.2Name and address of the manufacturer (5)3.2.3Intended use (5)3.2.4Summary and explanation (6)3.2.5DIRECTIONS FOR USE (6)3.2.5.1Components (6)3.2.5.2Warnings and precautionary statements (7)3.2.5.3Specimen collection and handling (8)3.2.5.4Test procedure (9)3.2.5.5Results (9)3.2.5.6Interpretation of results (9)3.2.5.7Limitations (10)3.2.5.8Expected values (10)3.2.5.9Disposal (10)3.2.6Performance characteristics (10)3.2.7Storage instructions (10)3.2.8Identifier (11)3.2.9Date of issue (11)3.2.10Bibliography (11)3.3Immediate container LABEL requirements (11)3.3.1Name of the IVDD (11)3.3.2Intended use (11)3.3.3Contents of kit (11)3.3.4Warnings and precautions (11)3.3.5Storage instructions (11)3.3.6Expiration date (12)3.3.7Name and address of the manufacturer (12)Guidance Document for the Labelling of In Vitro Diagnostic Devices GD012/RevDR-MDBDraft file: labl_ivd_e.wpd June 24, 1998 Printed: December 11, 2002Page 2 of 153.3.8CONTROL NUMBER (12)3.3.9Identifier (12)3.3.10Specific operating instructions (12)3.4Reagent LABEL requirements (12)3.4.1Name of the IVDD and reagent (12)3.4.2Contents (12)3.4.3Warnings and precautions (12)3.4.4Storage instructions (13)3.4.5Expiration date (13)3.4.6Name and address of the manufacturer (13)3.4.7CONTROL NUMBER (13)3.4.8Identifier (13)4Labelling information for IVDDs as required by Section 21Subsection (2) (13)5Labelling information for IVDDs as required by Section 22 (13)6Labelling information for IVDDs as required by Section 23 (14)7Labelling for IVDDs containing explosive materials or components (15)8Bibliography (15)Guidance Document for the Labelling of In Vitro Diagnostic Devices GD012/RevDR-MDBDraft file: labl_ivd_e.wpd June 24, 1998 Printed: December 11, 2002Page 3 of 151Introduction1.1PurposeThis guideline is intended to assist manufacturers in the labelling of in vitro diagnostic devices (IVDDs) to meet current Canadian regulatory requirements.1.2ScopeThis guideline addresses the labelling requirements of Part 1, Sections 21, 22, and 23 of the Medical Devices Regulations, for all products deemed to be IVDDs under these Regulations. This may apply to IVDDs intended for research use if they are also labelled or otherwise represented by manufacturers for a specific diagnostic, investigational or therapeutic application. The guidance document “Guidance for the Classification Rules For In Vitro Diagnostic Devices GD007/RevDR-MDB” provides additional information on these topics.Additional information regarding the labelling requirements for all medical devices can be found in the document “Guideline for the Labelling of Medical Devices Sections 21 to 23 of the Medical Devices Regulations GD011/RevDR-MDB.”Although this guideline does not specifically address the labelling for IVDDs intended for near-patient use, the information for these products required by Sections 21 to 23 of the Medical Devices Regulations should be expressed and presented with the intended user of the device in mind. Directions for use should be clearly written in a step by step format and include illustrations and drawings where appropriate. The user should be clear as to what action is to be taken in the case of a particular result and on the possibility of a false positive or false negative result.The Medical Devices Bureau reserves the right to ask for more labelling information than is indicated in this guideline if is felt that such labelling will impact on the safe and effective use of this device.1.3Definitions1.3.1LABEL: as defined in the Food and Drugs Act "..."label" includes any legend, word ormark attached to, included in, belonging to or accompanying any food, drug, cosmetic,device or package... ."1.3.2In vitro diagnostic device: A medical device or a product subject to section 3.1 of theMedical Devices Regulations that is to be used in vitro for the examination of specimens derived from the human body.1.3.3DIRECTIONS FOR USE: defined in the Medical Devices Regulations as " ...full informationas to the procedures recommended for achieving the optimum performance of the device and includes cautions, warnings, contraindications and possible side effects."Guidance Document for the Labelling of In Vitro Diagnostic Devices GD012/RevDR-MDBDraft file: labl_ivd_e.wpd June 24, 1998 Printed: December 11, 2002Page 4 of 151.3.4CONTROL NUMBER: defined in the Medical Devices Regulations as "...a uniquecombination of letters or symbols that is assigned to a medical device by the manufacturer and from which a complete history of the manufacture, control, packaging anddistribution of a production run or lot of the device can be determined.”1.3.5TEST KIT: an IVDD that contains reagents or articles or both, manufactured, sold orrepresented for use in combination to conduct a specific test.2Labelling requirements for IVDDsThe labelling of all medical devices is governed by Part 1, Sections 21, 22, and 23 of the Medical Devices Regulations. The following sections of this guideline (Sections 3,4,5 and 6) indicate the regulation in italics followed by the requirements specific to IVDDs.3Labelling information for IVDDs as required by Section 21 Subsection (1) Paragraphs (a) to (j) of the Medical Devices RegulationsSection 21 Subsection (1) Paragraphs (a) to (j): No person shall import or sell a medical device unless the device has a label that sets out the following information:(a) the name of the device;(b)the name and address of the manufacturer;(c)the identifier of the device, including the identifier of any medical device that ispart of a system, test kit, medical device group, medical device family or medicaldevice group family;(d)in the case of a Class III or IV device, the control number;(e) if the contents are not readily apparent, an indication of what the packagecontains, expressed in terms appropriate to the device, which may include thesize, net weight, length, volume or number of units of the device;(f) the words “Sterile” and “Stérile”, if the manufacturer intends the device to besold in a sterile condition;(g) the expiry date of the device, if the device has one, to be determined by themanufacturer on the basis of the component that has the shortest projected usefullife;(h) unless self-evident to the intended user, the medical conditions, purposes and usesfor which the device is manufactured, sold or represented, including theperformance specifications of the device if those specifications are necessary forproper use;(i) the directions for use, unless directions are not required for the device to be usedsafely and effectively; and(j) any special storage conditions applicable to the device.3.1LabelAll IVDDs must have a LABEL which provides the information specified in Section 21Guidance Document for the Labelling of In Vitro Diagnostic Devices GD012/RevDR-MDBDraft file: labl_ivd_e.wpd June 24, 1998 Printed: December 11, 2002Page 5 of 15Subsection (1) Paragraphs (a) to (j) of the Medical Devices Regulations. The LABEL as defined in the Food and Drugs Act includes any legend, word or mark attached to, included in, belonging to or accompanying any food, drug, cosmetic, device or package. Labelling for IVDDs includes, but is not limited to, the immediate device container label, the reagent/component label and package insert.3.2Labelling requirements for a package insertPackage inserts are essential for most IVDDs. The requirements for a package insert indicated in this section of the guideline apply to the majority of TEST KIT s for all classes of IVDDs. It is recognized that the extent of the information required in the package insert may depend upon the complexity and safety considerations of the test.The information required for a package insert may be presented in a different format than that indicated in 3.2.1 to 3.2.10 of this guideline.3.2.1Name of the IVDD [Section 21 Subsection (1) Paragraph (a)]The name of the IVDD on the label should enable the user to identify the device and distinguish it from other similar devices.3.2.2Name and address of the manufacturer [Section 21 Subsection (1) Paragraph (b)] The name and mailing address of the manufacturer is required.3.2.3Intended use [Section 21 Subsection (1) Paragraph (h)]The package insert should clearly indicate intended use(s)and indications for use of the IVDD. The following information should be included:C Nature of the intended use (e.g. screening, monitoring, diagnostic, etc.). Class IV IVDDsnot intended for donor screening must indicate “Not for donor screening” on the device container label and package insert.C Technology of the IVDD (e.g. ELISA, chromatographic, etc.).C Type of test: qualitative or quantitative.C The specific disorder, condition, or risk factor of interest for which the test is intended,i.e. the analyte to be measured.C Description of the patient population the IVDD is to be used in.C Indicate if the device is for use in clinical laboratories, alternative care sites, or home use.Note: The Limitations section of the package insert should include any specific training required for test performance or use.C Type of specimen(s) required (e.g. serum, plasma, etc.).C Indicate if the IVDD must be used in combination with or installed with or connected toother medical devices or equipment.C Specific contraindications for use, e.g. “Use of this device is contraindicated in recentinfluenza vaccine recipients...” when considerable cross-reactivity can be expected inrecent influenza vaccine recipients, etc.Guidance Document for the Labelling of In Vitro Diagnostic Devices GD012/RevDR-MDBDraft file: labl_ivd_e.wpd June 24, 1998 Printed: December 11, 2002Page 6 of 15An example of an intended use statement is the following:[Manufacturer’s Name]’s [Assay Name] Enzyme Immunoassay (EIA) is used for thequalitative (or quantitative) detection of Antibody to Human Immunodeficiency VirusTypes 1 and/or 2 (HIV-1 and HIV-2) in human serum or plasma, and is indicated as ascreening test for serum or plasma (or “ Not for donor screening”) and as an aid in thediagnosis of infection with HIV-1 and/or HIV-2.3.2.4Summary and explanation [Section 21 Subsection (1)0 Paragraph (h)]Indicate a brief summary and explanation of the test and how it works, including the clinicalbenefits and limitations of the test with respect to intended use. Describe the technique(s) andreactions (biological, chemical, microbiological, immunochemical, etc.) used, citing literaturereferences where appropriate. The summary should include descriptions of the types ofantibodies and antigens used in the test, (e.g. synthetic peptide, monoclonal, recombinant, etc.),and purification methods.3.2.5DIRECTIONS FOR USE [Section 21 Subsection (1) Paragraph (i)]Section 21 Subsection (1) Paragraph (i) requests DIRECTIONS FOR USE unless directions are notrequired for the device to be used safely and effectively. Most IVDDs will require DIRECTIONS FOR USE. Directions for use are defined in the Medical Devices Regulations as the procedures recommended for achieving the optimum performance of the device, including warnings andprecautions, contraindications, and possible side effects.The required information may be presented in a package insert in a format different from thatindicated in Section 3.2.5. For example, warnings and precautions may be indicated under aseparate heading. Components of a TEST KIT may be indicated in a table format along withinstructions for preparation and use, storage conditions, stability information, warnings andprecautions, etc.3.2.5.1Components (reagents, supplies, etc.)a)The description of a component should include the following:C Name of the component.C Contents in terms of quantity (e.g. number of vials, if applicable), massand/or volume or concentration. For reagents, indicate the following:(a)Quantity, proportion, concentration or activity of each reactiveingredient. For biologicals, indicate the source and measure ofactivity.(b) A statement indicating the presence of catalytic or non-reactiveingredients, such as buffers, preservatives or stabilizers, where thisinformation is needed for the safe and effective use of the test.C Specify the maximum number of tests that can be performed with statedcontents.Guidance Document for the Labelling of In Vitro Diagnostic Devices GD012/RevDR-MDBDraft file: labl_ivd_e.wpd June 24, 1998 Printed: December 11, 2002Page 7 of 15C Complete directions for preparation (reconstitution, mixing or dilution).C Storage instructions for both opened and unopened reagents. Note:Thisinformation can also be provided in a separate section of the packageinsert.C Information regarding possible deterioration of the reagent, i.e. indicatorsof reagent, calibrator or quality control material deterioration, whereapplicable.C Appropriate warnings and precautions. This information can also beprovided in a separate section of the package insert.b)Indicate any essential components and/or special equipment or instruments notprovided. Include details such as sizes, numbers, types, quality, etc. Examples are:incubators, precision pipettes, calibrated thermometers, appropriate disinfectantsand disinfection procedures, appropriate reaction vessels (specify glass,polystyrene, polypropylene), etc. For instruments such as microplate readers,indicate required specifications such as wavelength, band width, absorbance,precision, filters, etc.c)Indicate any dedicated instruments/equipment/software. Include the following:C Name of the instrument.C Model number(s)/version number(s).C Brief description of use or function, performancecharacteristics/specifications, warnings and precautions, limitations, etc.3.2.5.2Warnings and precautionary statementsIndicate appropriate warnings and precautionary statements for the safe and effective use of the IVDD. Warnings alert the user to potential serious adverse reactions and safety hazards that can occur in the proper use, or misuse, of an IVDD. Precautions alert the user to the special care or procedures necessary for the safe and effective use of the IVDD. The use of international symbols and signal words such as “warning” and “caution” are effective in alerting the user to a hazard.For all classes of IVDDs, indicate the statement:For in vitro diagnostic use.Biological Hazards:IVDDs containing material of human or animal origin are required to have a statement to the effect:CAUTION: the device contains material of human or animal origin and should be handled as a potential carrier and transmitter of disease.Guidance Document for the Labelling of In Vitro Diagnostic Devices GD012/RevDR-MDBDraft file: labl_ivd_e.wpd June 24, 1998 Printed: December 11, 2002Page 8 of 15For IVDDs containing potentially infectious agents, indicate whether any antigens and/or control sera have been inactivated and provide a complete description of what tests have been performed on positive and negative controls, and results obtained, for HCV, HBV, HTLV and HIV. If the testing revealed the presence of an infectious agent, a hazard statement should be included to the effect:HAZARD: The device may transmit [infectious agent] and should be handled with extreme caution. No known test method can offer complete assurance that products derived from human blood will not transmit infectious agents.Section 21 Subsection (1) Paragraph (f) the Medical Devices Regulations, requires the word Sterile, if the manufacturer intends the device or components to be sold in a sterile condition. Examples of appropriate warnings and precautions:C Do not pipette by mouth.C Do not smoke, drink, or eat in areas where specimens or kit reagents are being handled.C Wear protective clothing and disposable gloves while handling the kit reagents.C Wash hands thoroughly after performing the test.C Use in ventilated area.C Avoid contact with eyes; use safety glasses; in case of contact, flush with waterimmediately and contact a doctor.C Avoid contact with skin; use gloves; in case of contact with skin, flush immediately andthoroughly with water.C Handle DMSO containing reagents with care, since DMSO is readily absorbed throughthe skin.C For acids, include appropriate warnings for spills such as “wipe up spills immediately andflush with water” and “should the reagent contact eyes or skin, flush with copiousamounts of water and consult a physician”.C For biological spills, indicate appropriate disinfectants and disinfection procedure.C Dispose of all specimens and components of the kit as potentially infectious agents.C Do not use the kit or any kit component past the indicated expiry date.C Do not use any other reagents from different lots in this test, unless the reagent isdesignated to be used with other lots of the same kit.C Do not use any reagent in other TEST KIT s, unless the reagent is designated to be used withother kits.C Avoid microbial contamination of reagents.C Bring all reagents or components to room temperature before use.C For manual pipetting of samples and controls, use individual pipette tips to eliminatecarryover.3.2.5.3Specimen collection and handlingIndicate the following:Guidance Document for the Labelling of In Vitro Diagnostic Devices GD012/RevDR-MDBDraft file: labl_ivd_e.wpd June 24, 1998 Printed: December 11, 2002Page 9 of 15C Description of the specimen.C Criteria for acceptance or rejection of samples.C Patient preparation, precautions and procedure for specimen collection (e.g. removal ofparticulate matter by centrifugation, etc.).C Additives and preservatives to be added to the specimen, to preserve the integrity of thespecimen.C Storage and handling requirements.C Any known interferences.3.2.5.4Test procedurea)For the test method:Instructions for use must provide complete information relevant to the safe andeffective use of the IVDD. The following information should be included:C Description of the required amounts of reagents, samples, and controls;incubation schedules, temperature, wavelengths used for measurement,and other relevant environmental conditions under which the device is tobe used.C Sample selection and handling.C Performance/ turnaround time.C Calibration information: controls, reference samples, blanks, preparationof standard curve, indication of the maximum and minimum levels ofdetection, etc.C Stability of the final reaction product.C Quality control procedures and materials required. Indicate whetherpositive and negative controls are required and what are considered to besatisfactory limits of performance.b)For the individual reagents:C Complete instructions for preparing use-dilutions or mixing of individualreagents, unless provided in an alternate section of the package insert.C Test volumes and DIRECTIONS FOR USE, unless provided in an alternatesection of the package insert.3.2.5.5ResultsIndicate the step by step procedure for calculating the value of the test sample, including appropriate formulae and a sample calculation.3.2.5.6Interpretation of resultsIndicate the criteria for acceptance or rejection and whether further testing is required if a particular result is obtained. For example, requirements for duplicate tests if the initial test isGuidance Document for the Labelling of In Vitro Diagnostic Devices GD012/RevDR-MDBDraft file: labl_ivd_e.wpd June 24, 1998 Printed: December 11, 2002Page 10 of 15reactive.Indicate the significance of the test results obtained, including information as to what degree a negative test does or does not exclude the possibility of exposure to, or infection with, the organism, etc. A positive or negative result must be clearly defined with cutoff levels where appropriate.If the test is designed to provide qualitative results, provide an explanation of expected results. If the test requires the interpretation of “visual” results, e.g. colorimetric reactions, include a high quality photograph or reproduction of results.3.2.5.7LimitationsIndicate test limitations and all known contraindications, if not stated in a previous section of the package insert, with references if appropriate. This section may include qualifications of personnel performing the test and/or interpreting test results; an indication that results should only be used in conjuction with other clinical and laboratory data; various patient and clinical factors that may affect marker levels; and factors that should be considered when interpreting test results.3.2.5.8Expected valuesIndicate the range of expected values based on studies of test results from various populations. Indicate how the range was established and clearly identify the population(s) which were used for the testing. Include literature references where appropriate.3.2.5.9DisposalIndicate appropriate decontamination and disposal procedures of used or expired kits and/or reagents. Disposal of all specimens and kit components must comply with all applicable waste disposal requirements.Note: Decontamination and disposal information may also be provided in the “Warnings and precautions” section of the package insert.3.2.6Performance characteristics [Section 21 Subsection (1) Paragraph (h)]The Performance characteristics section must include a summary of data from clincial trials upon which the performance of the test is based.Performance characteristics such as sensitivity, specificity, predictive values, reproducibility, repeatability, stability, limits of detection and measurement range, earliest clinical detection in comparison with tests of reference, etc., are required. Indicate 95% confidence intervals where appropriate.3.2.7Storage instructions [Section 21 Subsection (1) Paragraph (j)]a)Indicate the storage conditions necessary to ensure the stability of the product inthe unopened state for both device and individual reagents. Recommended storagetemperature intervals and other conditions for storage such as light, humidity, etc.should be stated. Examples of appropriate statements are: 2 o C to 8 o C, 2...8 o C, -Guidance Document for the Labelling of In Vitro Diagnostic Devices GD012/RevDR-MDBDraft file: labl_ivd_e.wpd June 24, 1998 Printed: December 11, 2002Page 11 of 1520 o C or below, < -20 o C, protect from freezing, do not freeze, store in the dark,store dessicated, etc.b)Indicate storage conditions as outlined above for opened or reconstituted/mixedreagents.3.2.8Identifier [Section 21 Subsection (1) Paragraph (c)]The identifier or catalogue number should be indicated on the package insert.3.2.9Date of issueThe date of issue of DIRECTIONS FOR USE or of any revision should be indicated.3.2.10BibliographyThe Bibliography should include pertinent up-to date references for information cited in the text and any other references related to the subject matter.3.3Immediate container LABEL requirementsThe Manufacturer should refer to Section 3.2 of this guideline for a complete description of the abbreviated requirements indicated below.3.3.1Name of the IVDD [Section 21 Subsection (1) Paragraph (a)]3.3.2Intended use [Section 21 Subsection (1) Paragraph (h)]An example of an appropriate statement for the immediate container LABEL is the following: [Assay name] for the detection of antibodies to Human Immunodeficiency VirusTypes I and II (HIV-1/HIV-2) in human serum or plasma. Not for donor screening.Note: Class IV IVDDs not intended for donor screening must indicate “Not for donorscreening” on the device immediate container LABEL and package insert.3.3.3Contents of kit [Section 21 Subsection (1) Paragraph (e)]List of kit contents, including quantities, descriptions, volumes, number of tests, etc. If more than a single determination may be performed using the product, any statement of the number of tests must be consistent with instructions for use and amount of material provided.3.3.4Warnings and precautions [Section 21 Subsection (1) Paragraph (i)]Warnings or precautions for users appropriate to the IVDD, including the statement “For In Vitro Diagnostic Use” for all IVDDs, and “Sterile”, if the manufacturer intends the kit to be sold in a sterile condition.For IVDDs containing potentially infectious agents, whether inactivated or not, indicate a statement to the effect:Handle all the reagents as though capable of transmitting infection.。

Atg8,a Ubiquitin-like Protein Required for Autophagosome Formation,Mediates Membrane Tethering and HemifusionHitoshi Nakatogawa,1,2Yoshinobu Ichimura,1,3and Yoshinori Ohsumi1,*1Department of Cell Biology,National Institute for Basic Biology,Okazaki444-8585,Japan2PRESTO,Japan Science and Technology Agency,Saitama332-0012,Japan3Present address:Department of Biochemistry,Juntendo University School of Medicine,Bunkyo-ku,Tokyo113-8421,Japan. *Correspondence:yohsumi@nibb.ac.jpDOI10.1016/j.cell.2007.05.021SUMMARYAutophagy involves de novo formation of double membrane-bound structures called autophagosomes,which engulf material to be degraded in lytic compartments.Atg8is a ubiq-uitin-like protein required for this process in Saccharomyces cerevisiae that can be conju-gated to the lipid phosphatidylethanolamine by a ubiquitin-like system.Here,we show using an in vitro system that Atg8mediates the teth-ering and hemifusion of membranes,which are evoked by the lipidation of the protein and reversibly modulated by the deconjugation enzyme Atg4.Mutational analyses suggest that membrane tethering and hemifusion ob-served in vitro represent an authentic function of Atg8in autophagosome formation in vivo.In addition,electron microscopic analyses indicate that these functions of Atg8are in-volved in the expansion of autophagosomal membranes.Our results provide further insights into the mechanisms underlying the unique membrane dynamics of autophagy and also in-dicate the functional versatility of ubiquitin-like proteins.INTRODUCTIONAutophagy is an evolutionally conserved protein degrada-tion pathway in eukaryotes that is essential for cell survival under nutrient-limiting conditions(Levine and Klionsky, 2004).In addition,recent studies have revealed a wide variety of physiological roles for autophagy(Mizushima, 2005)as well as its relevance to diseases(Cuervo,2004). During autophagy,cup-shaped,single membrane-bound structures called isolation membranes appear and expand,which results in the sequestration of a portion of the cytosol and often organelles.Eventually,spherical, double membrane-bound structures called autophago-somes are formed(Baba et al.,1994),and then delivered to and fused with lysosomes or vacuoles to allow their contents to be degraded.Studies in S.cerevisiae have identified18ATG genes required for autophagosome formation,most of which are also found in higher eukary-otes(Levine and Klionsky,2004).Recent studies have shown that Atg proteins constitutefive functional groups: (i)the Atg1protein kinase complex,(ii)the Atg14-contain-ing phosphatidylinositol-3kinase complex,(iii)the Atg12-Atg5protein conjugation system,(iv)the Atg8lipid con-jugation system,and(v)the Atg9membrane protein recycling system(Yorimitsu and Klionsky,2005).The mechanisms by which these units act collaboratively with lipid molecules to form the autophagosomes,how-ever,are still poorly understood.Atg8is one of two ubiquitin-like proteins required for autophagosome formation(Mizushima et al.,1998;Ichi-mura et al.,2000).Because it has been shown that Atg8 and its homologs(LC3in mammals)localize on the isola-tion membranes and the autophagosomes,these proteins have been used in various studies as reliable markers for the induction and progression of autophagy(Kirisako et al.,1999;Kabeya et al.,2000;Yoshimoto et al.,2004). In S.cerevisiae,Atg8is synthesized with an arginine resi-due at the C terminus,which is immediately removed by the cysteine protease Atg4(Kirisako et al.,2000).The resulting Atg8G116protein has a glycine residue at the new C terminus and can serve as substrate in a ubiqui-tin-like conjugation reaction catalyzed by Atg7and Atg3, which correspond to the E1and E2enzymes of the ubiq-uitination system,respectively(Ichimura et al.,2000). Remarkably,unlike other ubiquitin-like conjugation sys-tems,Atg8is conjugated to the lipid phosphatidylethanol-amine(PE),thereby Atg8is anchored to membranes (Ichimura et al.,2000;Kirisako et al.,2000).Immunoelec-tron microscopy revealed that Atg8,probably as a PE-conjugated form(Atg8-PE),is predominantly localized on the isolation membranes rather than on the complete autophagosomes(Kirisako et al.,1999),suggesting that Atg8-PE plays a pivotal role in the process of autophago-some formation.The precise function of Atg8-PE,how-ever,has remained unknown.The conjugation of Atg8to PE is reversible;Atg4also functions as a deconjugation enzyme,resulting in the Cell130,165–178,July13,2007ª2007Elsevier Inc.165release of Atg8from the membrane(Kirisako et al.,2000). This reaction is thought to be important for the regulation of the function of Atg8and/or the recycling of Atg8after it has fulfilled its role in autophagosome formation.We reconstituted the Atg8-PE conjugation reaction in vitro with purified components(Ichimura et al.,2004). Here,we show using this system that Atg8mediates the tethering and hemifusion of liposomes in response to the conjugation with PE.These phenomena observed in vitro are suggested to reflect a bonafide in vivo function of Atg8 in the expansion of the isolation membrane.Based on mutational analyses and structural information,the mech-anisms of Atg8-mediated membrane tethering and hemi-fusion as well as its regulation are discussed.This study sheds light on the molecular basis of unconventional membrane dynamics during autophagy,which is gov-erned by the Atg proteins.RESULTSLipidation of Atg8Causes Clustering of LiposomesIn VitroAs reported previously(Ichimura et al.,2004),when puri-fied Atg8G116(hereafter,referred to as Atg8),Atg7,and Atg3were incubated with liposomes containing PE in the presence of ATP,Atg8-PE was efficiently formed (Figure1A,lanes1–6).Intriguingly,the reaction mixture became turbid during the incubation(Figure1B),which under a light microscope,was found to be a result of grad-ually forming aggregates(Figure1C).Both the degree of turbidity and the size of the aggregates appeared to corre-late with the amount of Atg8-PE produced in the mixture. Size-distribution analyses using dynamic light scattering (DLS)clearly showed that the aggregates formed in an Atg8-PE dose-dependent manner(Figure1D).These aggregates disappeared when the samples were treated with the detergent CHAPS(Figure1E,+CHAPS).In addi-tion,if a small amount of PE modified with thefluorescent dye7-nitro-2,1,3-benzoxadiazol-4-yl(NBD)was included in the liposome preparation,the aggregates became uniformlyfluorescent(Figure1E,NBD-PE).These results suggest that the aggregates generated during the produc-tion of Atg8-PE were clusters of liposomes.When the proteins were denatured with urea,the clus-ters of liposomes dissociated,although Atg8remained conjugated to PE(Figure1E,+urea and Figure1F,lane 2),indicating that the liposomes aggregated due to some function of the Atg8protein rather than an artifact caused by Atg8-PE as the lipid with the extraordinarily large head group.When the aggregates were sedimented by centrifugation,Atg8-PE co-precipitated with the lipo-somes(Figure1G,lane2),whereas Atg7,Atg3,and unconjugated Atg8did not(Figure1G,lane3).The sedimented liposomes containing Atg8-PE remained clustered even if they were briefly sonicated(Figure1H, ppt.).These results suggested that Atg8-PE molecules function to tether together membranes to which they are anchored.Atg8-PE Also Mediates Liposome FusionWe also examined if membrane fusion occurred between the liposomes connected by Atg8-PE.To this end,we took advantage of a well-characterized lipid mixing assay (Struck et al.,1981).This method is based on energy transfer from NBD to lissamine rhodamine B(Rho),each of which is conjugated to PE.Because the amino group of the ethanolamine moiety is modified with the dyes, these lipids cannot be conjugated with Atg8.If both of the conjugated dyes are present at appropriate concen-trations in the same liposome,thefluorescence of NBD is effectively quenched by Rho(Figure2A,compare col-umns1and4).If a‘‘NBD+Rho’’liposome is fused with a‘‘nonlabeled’’liposome,which results in an increase of the average distance between the two dyes on the membrane,the NBDfluorescence will be dequenched.A mixture of the nonlabeled and NBD+Rho liposomes were subjected to the conjugation reaction.The resulting liposome clusters were dissociated by proteinase K treat-ment,followed byfluorescence measurements.Remark-ably,a significant ATP-dependent increase of thefluores-cence was observed(ATP is required for the production of Atg8-PE;Figure2B,column6).This increasedfluores-cence was not observed with samples of nonlabeled lipo-somes alone,NBD+Rho liposomes alone,or a mixture of nonlabeled liposomes and liposomes containing NBD-PE but not Rho-PE(Figure2B,columns1-3).These results suggest that membrane fusion occurred between the lipo-somes tethered together by Atg8-PE.The increasedfluo-rescence was only observed if the reaction mixture was treated with proteinase K(Figure2B,columns4and6). This appeared to be due to the presence of Atg7and/or Atg3rather than Atg8or some effect of the clustering, because the NBDfluorescence was not increased by the addition of Atg4(Figure2B,column5),which detached Atg8from the membranes and dissociated the clusters of liposomes(see below).Instead,decreasing the con-centrations of the conjugation enzymes allowed the dequenching of the NBDfluorescence to be detected without proteinase K digestion(Figure2B,column7). The fusion of the liposomes was examined with various amounts of Atg8(Figure2C).The level of fusion increased Atg8dose-dependently and reached maximum at2m M (Figure2C).In contrast,a larger amount of Atg8produced an inhibitory effect(data not shown).This suggested that formation of the large aggregates resulted from excessive tethering by Atg8-PE,which no longer lead to fusion. We also carried out time-course experiments to roughly estimate the fusion rate using the lower concentrations of the conjugation enzymes(Figure2D),which eliminated the need for the proteinase K treatment(Figure2B).It should be noted that the incubation time includes the times re-quired for the formation of Atg8-PE and the subsequent tethering and fusion reactions.Under these conditions, the band of Atg8-PE could be seen on an SDS-PAGE gel after a10min incubation,and the reaction was completed within30min(Figure S1in the Supplemental Data available with this article online).It appeared that166Cell130,165–178,July13,2007ª2007Elsevier Inc.Figure1.Membrane Tethering Function of Atg8-PE In Vitro(A–C)Purified Atg8(10m M),Atg7(1m M),and Atg3(1m M)were incubated with liposomes(350m M lipids)composed of55mol%DOPE,30mol% POPC,and15mol%blPI in the presence(lanes1–6)or absence(lanes7–12)of1mM ATP at30 C for the indicated time periods,followed by urea-SDS-PAGE and CBB-staining(A),measurement of the absorbance at600nm(B),or observation under a light microscope(Nomarski images)(C).(D)Conjugation reactions with the various amounts of Atg8were performed as described in(A).After incubation for60min,the size distribution of the aggregates was examined using DLS measurements.d.nm,apparent diameter(nm).(E and F)The conjugation reactions were carried out as described in(A).They were further incubated at30 C for30min in the presence of either 6M urea or1%CHAPS and were then subjected to microscopy(E)or urea-SDS-PAGE and CBB-staining(F).The reaction was also performed with liposomes containing1mol%NBD-labeled DOPE(thus containing54mol%unlabeled DOPE),followed byfluorescence microscopy.Afluo-rescence image with afilter for YFP(NBD-PE,FL)and a Nomarski image(NBD-PE,DIC)are shown.(G and H)Atg8(30m M),Atg7(2m M),and Atg3(2m M)were incubated with liposomes(350m M lipids)consisting of70mol%DOPE and30mol% POPC in the presence of1mM ATP at30 C for45min(total).The mixture was microcentrifuged at15,000rpm for10min to generate the pellet (ppt.)and the supernatant(sup.)fractions.The fractions were briefly sonicated and were analyzed by urea-SDS-PAGE(G)or observed under a light microscope(H).In this experiment,blPI was omitted to prevent Atg7and Atg3from tightly binding to the liposome.We showed that Atg8could also cause hemifusion of liposomes with this lipid composition.Cell130,165–178,July13,2007ª2007Elsevier Inc.167the liposomes began to fuse shortly after the formation of Atg8-PE.The fusion reaction proceeded concurrently with the conjugation reaction and continued for 30min after the completion of the Atg8-PE production (Figure 2D,filled circles).Small liposomes <100nm in diameter tend to sponta-neously fuse (Chen et al.,2006),and the liposomes we used in the above experiments were 70nm in diameter (Figure 1D).However,we also showed that Atg8-PEcaused a significant level of fusion between larger lipo-somes in spite of their stability against spontaneous fusion (Figure S1).Taken together,these results suggest that not only tethering but also fusion of the liposomes is mediated by Atg8-PE.The Atg8-Mediated Membrane Fusion Is Hemifusion Recent in vitro studies on membrane fusion mediated by SNARE proteins and a class of viral proteinsrevealedFigure 2.Membrane Hemifusion Occurs between Liposomes Tethered by Atg8-PE(A and B)Nonlabeled (55mol%DOPE,30mol%POPC,and 15mol%blPI),NBD-labeled (55mol%DOPE,29mol%POPC,15mol%blPI,and 1mol%NBD-DOPE),and NBD+Rho-labeled (55mol%DOPE,27.5mol%POPC,15mol%blPI,1mol%NBD-DOPE,and 1.5mol%Rho-DOPE)liposomes were mixed in the differ-ent combinations and ratios indicated.Their relative intensities of the NBD fluorescence ob-served are shown (the value obtained with a 4:1mixture of the nonlabeled and NBD+Rho lipo-somes was defined as 1)(A).These mixtures of liposomes were incubated with Atg8(4m M),Atg7(0.5or 1.0m M),and Atg3(0.5or 1.0m M)in the presence (filled columns)or absence (open columns)of 1mM ATP for 60min,and were then treated with 1unit/ml apyrase.The mixtures were further incubated for 30min with the buffer (columns 4and 7),1m M Atg4(columns 5and 8),or 0.2mg/ml proteinase K (columns 1-3,6and 9),followed by measure-ment of the NBD fluorescence.The experi-ments were repeated three times and the average fluorescence values divided by those obtained from the original liposome samples (F/F 0)are presented with error bars for the stan-dard deviations (B).(C)A 4:1mixture of the nonlabeled and NBD+Rho liposomes was incubated with various amounts of Atg8,1.0m M Atg7,and 1.0m M Atg3in the presence (open circles)or absence (filled circles)of ATP,and the samples were then treated with proteinase K,followed by measuring the NBD fluorescence.(D)The conjugation reactions were performed with the mixed liposomes used for the lipid mixing assay,0.5m M Atg7,and 0.5m M Atg3in the presence or absence of Atg8(4m M)and ATP.After incubation for the indicated time periods,an aliquot of the samples was immediately subjected to the fluorescence measurements.The values that were obtained by subtracting the signals observed in the absence of ATP from those observed in the presence of ATP are presented.(E)The lipid mixing assay was performed with 4m M Atg8,1m M Atg7,and 1m M Atg3in the presence or absence of ATP (white bars in columns 3and 2,respectively)as described in(C).For PEG-induced fusion reactions,the mixed liposomes were incubated at 37C for 30min in the presence or absence of 12.5%PEG 3350(white bars in columns 5and 4,respectively).These samples as well as the original liposomes (column 1)were then incubated with 20mM sodium dithionite on ice for 20min in the presence (black bars)or absence (gray bars)of 0.5%Triton X-100,followed by the NBD fluorescence measurement.168Cell 130,165–178,July 13,2007ª2007Elsevier Inc.that fusion proceeds through an intermediate state called hemifusion,in which outer(contacting)leaflets of two apposed lipid bilayers merge,while inner(distal)leaflets remain intact(Chernomordik and Kozlov,2005).It was also reported that fusion can be arrested or delayed at the hemifusion state under some conditions.Therefore, we investigated whether the liposome fusion caused by Atg8in vitro was complete fusion(the merger of both inner and outer leaflets)or hemifusion(Figure2E).This can be examined using the membrane impermeable reductant sodium dithionite that selectively abolishes thefluores-cence of NBD conjugated to the lipid head group in the outer leaflet(Meers et al.,2000).Accordingly,when so-dium dithionite was added to the original liposomes,the background level of the NBDfluorescence was decreased by about50%,whereas it was hardly detected in the pres-ence of the detergent(Figure2E,column1).Strikingly,the NBDfluorescence increased by the Atg8-mediated fusion was totally eliminated by addition of sodium dithionite to the same level as those observed in the original lipo-somes and the reaction mixture incubated without ATP (Figure2F,columns1-3).Whereas,we confirmed that in liposome fusion induced by polyethylene glycol(PEG), which causes complete fusion(Akiyama and Ito,2003), about half of the increasedfluorescence was retained af-ter sodium dithionite treatment(Figure2F,columns4and 5).Taken together,membrane fusion mediated by Atg8 in vitro was suggested to be hemifusion.To obtain direct evidence of hemifusion,we analyzed the morphology of liposomes by electron microscopy (Figures3A–3E),in which liposomal membranes were observed as double white lines that correspond to the outer and inner leaflets.When the clusters of liposomes formed by Atg8-PE were analyzed,tight junctions between the liposomes were observed(Figures3B and 3C,arrowheads).Consistent with the biochemical results suggesting that complete fusion does not occur,the size of the individual liposomes did not appear to significantly increase(compare Figures3A and3B).Instead,hallmarks of hemifusion,trifurcated structures formed by one contin-uous outer leaflet and two separate inner leaflets,could be observed at the junction between the liposomes(Figures 3C–3E,arrows).These results strongly support our con-clusion that Atg8-PE causes hemifusion of liposomes. Atg8Forms a Multimer in Responseto the Conjugation with PEWe also performed immunoelectron microscopy of the liposomes clustered by Atg8-PE(Figures3F–3I).Intrigu-ingly,Atg8-PE tended to be enriched at the junction be-tween the liposomes(Figures3G–3J).While,if the mixture incubated without ATP was similarly analyzed,the signal was rarely observed on the liposome(Figure3F;the gold particles observed should represent unconjugated Atg8 adsorbed onto the grid).These results indicate that Atg8-PE is directly involved in the tethering and hemifu-sion of liposomes.We observed that‘‘naked’’liposomes do not associate with liposomes carrying Atg8-PE(data not shown), suggesting that tethering should be achieved due to interactions between Atg8-PE molecules on different membranes.We therefore examined the intermolecular interaction of Atg8-PE by crosslinking experiments(Fig-ure4).The reaction mixture containing Atg8-PE or unconjugated Atg8was incubated with the lysine-to-lysine reactive crosslinker DSS.We found that a crosslink adduct with a molecular weight of 24kDa on a SDS-PAGE gel specifically appeared in the sample containing Atg8-PE(Figure4A,lane5).Considering the molecular weights of the proteins included,this adduct should represent an Atg8-PE homodimer.Immunoblotting anal-yses with anti-Atg8revealed that two additional crosslink adducts of about37and100kDa were also specifically produced in the Atg8-PE-containing sample(Figure4B, lanes2–4and Figure S2).These products were immuno-stained neither with anti-Atg7nor anti-Atg3(data not shown),suggesting that they represent a trimer and a larger multimer of Atg8-PE,respectively,and thus that Atg8multimerizes in response to PE conjugation. We also showed that this multimerization correlates with the membrane tethering ability of Atg8(see below), indicating that interactions between Atg8-PE molecules on different membranes are responsible for the tethering of the membranes.The Membrane-Tethering and HemifusionFunctions of Atg8Are Modulatedby the Deconjugation Enzyme Atg4Our results suggest that the membrane-tethering and hemifusion functions of Atg8are evoked by the conjuga-tion with PE,whereas Atg4functions as a deconjugase that cleaves the linkage between Atg8and PE(Kirisako et al.,2000).We reconstituted this reaction in vitro.After producing Atg8-PE using the conjugation reaction,the re-action was terminated by adding apyrase to deplete the remaining ATP.When purified Atg4was then added, Atg8-PE was rapidly and almost completely deconjugated (Figure4C,lanes1-6).In contrast,when the Atg4was pretreated with the cysteine protease inhibitor N-ethylma-leimide(NEM),the deconjugation reaction did not occur (Figure4C,lanes7–12).These results clearly show that Atg4is sufficient for the deconjugation of Atg8-PE.Upon deconjugation,the liposome aggregates immediately dissociated(Figure4D).In addition,we found that multi-merization of Atg8is also reversible;the crosslink adducts corresponding to the Atg8-PE dimer(Figure4A,lane6)as well as the trimer and the multimer(data not shown)were hardly formed when DSS was added after the deconjuga-tion reaction.We also showed that the presence of Atg4in the conjugation reaction retarded the accumulation of Atg8-PE and accordingly interfered with the tethering and hemifusion of the liposomes(data not shown).It was indicated that membrane tethering and hemifusion by Atg8can be regulated by the balance between the conjugation and deconjugation reactions.Cell130,165–178,July13,2007ª2007Elsevier Inc.169Identification of Mutations that Impair the Postconjugational Function of Atg8In VivoIf the function of Atg8-PE we observed in vitro was involved in autophagosome formation in vivo,Atg8mutants defi-cient for this function should result in defective autophagy.To examine this idea,we performed structure-based and systematic mutational analyses of Atg8(Figure 5).The structures of mammalian homologs revealed that Atg8family proteins consist of two domains:an N-terminal heli-cal domain (NHD)and a C-terminal ubiquitin-like domain (ULD)(Paz et al.,2000;Coyle et al.,2002;Sugawara et al.,2004;Figures 5E–5H).Among the highly conserved residues in the ULD,we selected those with side chains that were exposed on the domain surface (Figure 5A),and individually replaced them with alanine,except that serine was substituted for Ala75.Consequently,we didnot mutate residues suggested to be important for interac-tions with the conjugation enzymes,because these resi-dues are conserved only for their hydrophobic nature (Sugawara et al.,2004).The Atg8variants were expressed from centromeric plasmids in D atg8yeast cells,and their autophagic activities were biochemically assessed (see Supplemental Experimental Procedures ).In nutrient-rich media,the autophagic activity was low in all of the mutant cells as well as in the wild-type cells (data not shown).In contrast,in nitrogen starvation conditions,which strongly induced autophagy,a number of mutants were found to have defective autophagic phenotypes (Figure 5B).Ala-nine replacement of seven residues,Ile32,Lys48,Leu50,Arg65,Asp102,Phe104,and Tyr106,significantly impaired the autophagic activity to 30%–60%of that of the wild-type (Figure 5B).Immunoblotting analyses showedthatFigure 3.Electron Microscopic Analyses of the Liposomes Tethered and Hemi-fused by Atg8-PEConjugation reactions were performed with 4m M Atg8,1m M Atg7,and 1m M Atg3in the presence (B–E and G–I)or absence (A and F)of ATP for 60min and subjected to phospho-tungstic acid-staining and electron micros-copy (A–E).The junctions between the lipo-somes and the structures suggested to represent hemifusion are indicated with arrow-heads and arrows,respectively.The same samples were also subjected to immunostain-ing using purified anti-Atg8-IN-13and anti-rab-bit IgG conjugated with 5nm gold particles,fol-lowed by phosphotungstic acid-staining and electron microscopic observation (F–I).To as-sess the enrichment of Atg8-PE at the junction of the liposomes (J),images of two contacting liposomes as shown in G and H were randomly picked up (n =41).The lengths of contacting (CR)and noncontacting regions (non-CR)of the liposomal membranes were measured (white bars),thereby the number of gold parti-cles on each region (gray bars)was divided,in which the length of the contacting region was doubled,to calculate the linear density (black bars).The average values are presented with error bars for the standard deviations.170Cell 130,165–178,July 13,2007ª2007Elsevier Inc.a substantial amount of each of the Atg8mutant proteins accumulated in the cells (Figure 5D),although there were some differences in their mobilities in SDS-PAGE analysis;for instance the PE-conjugated and unconjugated forms of the D102A mutant exhibited almost the same mobility.None of the mutations significantly affected the formation of Atg8-PE (Figure 5D),suggesting that the mutations impaired a function of Atg8that was exerted after the conjugation with PE.Notably,these mutants accumulated different levels of unconjugated Atg8under the starvation conditions (Figure 5D,starvation),which allowed us to classify them into three groups.For the class I mutants K48A and L50A,the levels of the unconjugated forms were similar to that of the wild-type (Figure 5D,denoted in purple).On the other hand,compared to the wild-type,lower levels of the unconjugated forms were detected in the class II mutants I32A,D102A,F104A and Y106A (Figure 5D,denoted in red),whereas a larger amount of the unconju-gated class III mutant R65A accumulated (Figure 5D,denoted in orange).We then mapped the mutated resi-dues onto the three-dimensional structure of LC3(Suga-wara et al.,2004),which revealed that class of the mutant corresponded to the location of the mutation.All the class II residues were clustered in a specific region on the ULD (hereafter,referred to as the class II region),and the two neighboring class I residues were located close to the class II region (Figure 5E).In contrast,the class III residue was located away from the other mutated residues (Fig-ures 5G and 5H).The NHD of Atg8contains two helices:a 1and a 2(Figure 5A).We constructed two mutants,one with a dele-tion of a 1(D N8)and a second bearing deletions of both helices (D N24).It was shown that the NHD is involved in autophagy partially but significantly;the D N8and D N24mutations decreased the autophagic activity by about 30and 40%,respectively (Figure 5C).We also showed that the deletions did not affect the stability of the proteins or the formation of the PE conjugates (Figure S3).Effects of the Atg8Mutations on the Membrane-Tethering FunctionWe next examined whether the mutations affected the liposome-clustering ability of Atg8in vitro (Figure 6).Figure 4.The Membrane-Tethering Function and Multimerization of Atg8Are Reversibly Regulated in Response to Conjugation with PE(A)Conjugation reactions were performed as described in Figure 3in the presence (lanes 2,3,5,and 6)or absence (lanes 1and 4)of ATP.They were mixed with 1unit/ml apyrase,and then incubated with (lanes 3and 6)or with-out (lanes 1,2,4,and 5)purified Atg4(0.5m M)at 30 C for 30min.These samples were further incubated with (lanes 4–6)or without (lanes 1–3)100m M DSS for 30min,and then analyzed by urea-SDS-PAGE and CBB-staining.(B)The reaction mixture including ATP was in-cubated with different concentrations of DSS as indicated,followed by urea-SDS-PAGE and immunoblotting with anti-Atg8-IN13.We also identified a crosslink product that reacted with anti-Atg3(Atg8xAtg3).(C and D)The conjugation reactions performed as described in Figure 1A were mixed with 1unit/ml apyrase.Atg4(0.5m M)pretreated with (lanes 7–12)or without (lanes 1–6)10mM NEM was then added,and the samples were incubated for the indicated time periods and subjected to urea-SDS-PAGE and CBB-stain-ing (C).The same samples were also observed under a light microscope (D).Cell 130,165–178,July 13,2007ª2007Elsevier Inc.171。

PLEASEREAD THIS OWNER'S MANUAL THOROUGHLY BEFORE OPERATING AND KEEP IT HANDY FOR REFERENCE AT ALL TIMES.OWNER'S MANUALREFRIGERATOR-FREEZERPOR FAVOR,LEIA TODO ESTE GUIA DO USUçRIO COM ATEN ÌO,ANTES DE OPERAR EMANTENHA-O A MÌO PARA CONSULTç-LO SEMPRE QUE PRECISAR.Guia do Usu rioFRIGOR FICO-CONGELADORLEA ESTE MANUAL DE INSTRUCCIONES DETENIDAMENTE Y GUçRDELO COMO REFERENCIA PARA EL FUTURO.MANUAL DE INSTRUCCIONESFRIGORIFICO -CONGELADORINSTALLATION (4)FEATURE CHART (5)OPERATION (6)StartingTemperature ControlIce MakingChilled CompartmentDefrostingDeodorizer (Optional)SUGGESTIONS ON FOOD STORAGE (9)REVERSIBLE DOOR (10)PrecautionHow to reverse the doorCLEANING (12)GENERAL INFORMATION (12)LAMP REPLACEMENT (13)IMPORTANT WARNINGS (3)BEFORE CALLING FOR SERVICE .........13CONTENTSVitamin Kit (Optional)THIS REFRIGERATOR IS MANUFACTURED WITH GREAT CARE AND UTILIZES THE LATEST IN TECHNOLOGY.WE ARE CONFIDENT THAT YOU WILL BE FULLY SATISFIED WITH ITS PERFORMANCE AND RELIABILITY.BEFORE USING YOUR REFRIGERATOR, PLEASE READ THIS BOOKLET CAREFULLY. IT PROVIDES EXACT INSTRUCTIONS FOR INSTALLATION, OPERATION, AND MAINTENANCE, AND ALSO SUPPLIES SOME USEFUL HINTS.DO NOT USE AN EXTENSION CORD Use this appliance on a dedicated circuit; that is, an outlet that is the only outlet on the circuit. Overloading a circuit causes low voltage, malfunction, possible damage to the equipment, and poor cooling capacity.Check your local electric code or consult a qualified electrician if you have questions. ACCESSIBILITY SUPPLY PLUGThe refrigerator-freezer should be so positioned that the supply plug is accessible for quick disconnection if an accident happens. SUPPLY CORD REPLACEMENTIf the supply cord is damaged, it must be replaced by the manufacturer or replacement or a similarly qualified person in order to avoid a hazard.ABOUT GROUNDING (EARTHING)In the event of an electric short circuit, grounding (earthing) reduces the risk of electric shock by providing an escape wire for the electric current. In order to prevent possible electric shock, this appliance must be grounded.Improper use of the grounding plug can result in an electric shock. Consult a qualified electrician or service person if the grounding instructions are not completely understood, or if you have doubts on whether the appliance is properly grounded.DO NOT MODIFY OR EXTEND THEIt will cause electric shockor fire.VERY DANGEROUS ATTRACTION An empty refrigerator can be a dangerous attraction to children. Remove either gaskets, latches, lids or the entire door from your unused appliance,or take some other action to make it harmless.This appliance must be grounded (earthed).DON'T WAIT! DO IT NOW!Do not store inflammable materials, explosives, or chemicals in the refrigerator.Disposal of the old applianceThis appliance contains fluid (refrigerant, lubricant) and is made of parts and materials which are reusable and/or recyclable.All the important materials should be sent to the collection center of waste material and can be reused after rework (recycling). For take back, please contact with the local agency.SELECT A GOOD LOCATION1. Place your refrigerator where it is easy to use.2. Avoid placing the refrigerator near heat sources, direct sunlight or moisture.3. To ensure proper air circulat ion around thefridge-freezer, please maintain sufficient space on both the sides as well as top and maintain at least 2 inches (5 cm) from the rear wall.4. To avoid vibrations, the refrigerator must be level. If necessary, adjust the leveling screw(s) to compensate for unevenness of the floor.To close the doors easily, the front should be slightly higher than the rear. Leveling screw(s) can be turned easily by tipping the cabinet slightly. Turn the leveling screw(s) clockwise to raise the refrigerator, counterclockwise to lower it.NEXT1. Wipe off all dust accumulated during shippingand clean your refrigerator thoroughly.2. Install accessories such as the ice cube box,evaporating tray cover, etc., in their properplaces. They are packed together to prevent possible damage during shipping.3. Connect the power supply cord (or plug) to theoutlet. Don't double up with other appliances on the same outlet.4. Prior to use, let the refrigerator run for 2-3hours. Check the flow of cold air in the freezer compartment to ensure proper cooling hastaken place.Your refrigerator is now ready for use.DISPOSAL OF YOUR OLD APPLIANCE1. When this crossed-out wheeled bin symbol is attached to aproduct it means the product is covered by the European Directive2002/96/EC.2. All electrical and electronic products should be disposed of separatelyfrom the municipal waste stream via designated collection facilitiesappointed by the government or the local authorities.3. The correct disposal of your old appliance will help prevent potentialnegative consequences for the environment and human health.4. For more detailed information about disposal of your old appliance,please contact your city office, waste disposal service or the shopwhere you purchased the product.• Information of fluorinated greenhouse gasesused as refrigerant of this refrigerator.All parts shown may not be included with every model.N O T EFREEZERCOMPARTMENTREFRIGERATOR COMPARTMENTType Ice TrayControl Knob Control MicomLampVegetable Drawer fresh and crisp.Freezer Door RackEgg Storage RackRefrigerator Door RackLeveling ScrewsCap Hinge (Optional)Removable Rack ShelfShelvesSTARTINGWhen your refrigerator is first installed, allow it 2-3 hours to stabilize at normal operating temperatures prior to filling it with fresh or frozen foods.If operation is interrupted, wait 5 minutes before restarting.The default setting of the temperature control knob for the freezer compartment is the MID point.For colder temperatures, adjust the control knob to MAX , and for warmer temperatures, adjust the control knob to MINposition.TEMPERATURE CONTROLREFRIGERATORFREEZERThe default setting of the temperature control button for the refrigerator compartment is NORMAL.You can set the refrigerator temperature using the Refrigerator Temperature Control .Whenever pressing the button, the LED emit light.The temperature of the compartment is highered as the LED luminesce from MIN to MAX .You can select the desired set point in five (5) steps between minimum and maximum.For the economic electricity, set the LED to theMINposition.�����������������������������������������������������������������������������ICE MAKINGDetermine which type ice-making system you have.General TypeG To make ice cubes, fill the ice tray with water andplace it on the ice cube box. Then insert the icecube box in the freezer compartment.G To remove ice cubes, hold the tray at its ends andtwist gently.Twisting Ice Serve TypeG To make ice cubes, fill the ice tray with water andinsert in its position.G To remove ice cubes, hold the knob of the tray andturn gently. Then, ice cubes drop in the ice cubebox.G You can remove the Twisting Ice Serve to make thefreezer compartment larger.G You should remove the ice Trays and Ice Cube Boxfirst, then pull the frame out toword the right side.FrameIce T raysIce Cube BoxIce T rays Ice Cube BoxCHILLED COMPARTMENTWhen the door is opened, the warmer air doesn't influence the fresh meat. You can keep food fresher in it.DEFROSTINGG Defrosting takes place automatically.GThe defrosted water flows down to the evaporating tray which is located in the lower back side of the refrigerator and is evaporated automatically.DEODORIZER (OPTIONAL)G By using a catalyst, deodorizing performance is guaranteed.GUnpleasant odor of food in the fresh food compartment is deodorized with no harm to you and the food.How to useGAs the catalyst is located in cooling air outlet for circulating air in fresh food compartment, there is no need to touch it.GDo not puncture the cooling air outlet with a sharp tip because the deodorizing catalyst may be damaged.GWhen storing food with a strong odor, wrap it or store it in a container with a lid because the odor may be absorbed by other foods.VITAMIN KIT (OPTIONAL)It contains anti-oxydant that is able to avoidoxidation process in order to make the fruits and vegetables fresh for longer time.STORING FOODG Store fresh food in the refrigerator compartment. How food is frozen and thawed is an important factorin maintaining its freshness and flavor.G Do not store food which goes bad easily at low temperatures, such as bananas, pineapples, and melons.G Allow hot food to cool prior to storing. Placing hot food in the refrigerator could spoil other food and lead to higher electric bills!G When storing, cover food with vinyl wrap or store in a container with a lid. This prevents moisture from evaporating and helps food to keep its taste and nutrients.G Do not block air vents with food. Smooth circulation of chilled air keeps refrigerator temperatures even.G Do not open the door frequently. Opening the door lets warm air enter the refrigerator, causing temperatures to rise.FREEZER COMPARTMENTG Do not store bottles in the freezer compartment - they may break when frozen.G Do not refreeze food that has been thawed. This deteriorates taste and nutrition.G When storing frozen food like ice cream for a long period, place it on the freezer shelf,not in the door rack.G Avoid storing any food close to the bottom area of the freezer shelf to keep efficient air circulation. REFRIGERATOR COMPARTMENTG Avoid placing moist food deep inside refrigerator shelves, it could freeze upon direct contactwith chilled air.G Always clean food prior to refrigeration. Vegetables and fruits should be washed and wiped,and packed food should be wiped clean to prevent adjacent food from spoiling.G When storing eggs in their storage rack, ensure that they are fresh. Always store them in a upright position, which keeps them fresh longer.PRECAUTION1. The door can be reversed if required by location or user preference.2. Before reversing the door, remove all foods and accessories (shelves, trays, bins, etc.) Which are not attached to the refrigerator.3. Use a phillips driver, bolt driver, torque wrench, or spanner to remove or attach the bolt.4. Be careful not to drop the refrigerator when assembling or disassembleling the lower hinge and the adjustable screw assembly.5. Don’t lay the refrigerator down while working on it, as this will cause a malfunction and damage.6. Be careful not to drop the doors when removing or replacing them.HOW TO REVERSE THE DOOR(when converting from the left-opening type to right opening type)- Converting Door is OptionalRemove the cap and upper hingeRemove the Freezer DoorRemove the center hinge and the Refrigerator Door Remove the lower hinge11Reverse the pin position of the upper hinge12Assemble the upper hinge and replace the cap567Move the cap and bracket to the opposite side of the refrigerator door8Move the cap to the opposite side andIt is important that your refrigerator be kept clean to prevent undesirable odors. Spilled food should be wiped up immediately, since it may acidify and stain plastic surfaces if allowed to settle.Never use metallic scouring pads, brushes, coarse abrasive cleaners, or strong alkaline solutions on any surface.Before you clean, remember that damp objects will stick or adhere to extremely cold surfaces. Do not touch frozen surfaces with wet or damp hands. EXTERIOR -Use a lukewarm solution of mild soap or detergent to clean the durable finish of your refrigerator. Wipe with a clean, damp cloth and then dry.INTERIOR -Regular cleaning of the interior and interior parts is recommended. If you have the No Frost model which defrosts automatically, leave both doors open during the entire cleaning process. Disconnect the power supply, and remove food and all compartment shelves, storage trays etc.Wash all compartments with a baking soda solution. Rinse and dry.INTERIOR PARTS -Wash compartment shelves, door racks, storage trays, and magnetic door seals etc. with the baking soda solution or a mild detergent and warm water. Rinse and dry.VACATION TIMEDuring average length vacations, you will probably find it best to leave the refrigerator in operation. Place freezable items in freezer for longer life. When you plan to be away for an extended period, remove all food, disconnect the power cord, clean the interior thoroughly, and leave each door OPENto prevent odor formation.POWER FAILUREMost power failures are corrected in an hour or two and will not affect your refrigerator temperatures. However, you should minimize the number of door openings while the power is off. During power failures of longer duration, place a block of dry iceon top of your frozen packages.DRY ICE WARNINGWhen using dry ice, provide adequate ventilation. Dry ice is frozen carbon dioxide (CO2).When it vaporizes, it can displace oxygen, causing dizziness, light-headedness, unconsciousness, and death by suffocation.Open a window and do not breathe the vapors.IF YOU MOVEIf you move, empty the refrigerator (freezer) completely and wash a mild solution of baking soda and water. (2 TBS soda to 1 quart water) Be sure the soda is completely dissolved to avoid scratching the inside of the refrigerator (freezer). Secure any loose items, such as racks, bins, ice trays, etc. Do not try to move a loaded refrigerator. ANTI-CONDENSATION PIPEThe outside wall of the refrigerator cabinet may sometimes get warm,especially just afterinstallation.Don't be alarmed.This is due to theanti-condensation pipe,which pumps hot refrigerantto prevent sweating on theouter cabinet wall.WARNINGAlways remove power cord from the wall outlet prior to cleaning in the vicinity of electricalparts (lamps, switches, controls, etc.).Wipe up excess moisture with a sponge orcloth to prevent water or liquid from getting into any electrical part and causing a electricshock. Do not use flammable or toxic cleaning liquids.1. Unplug the power cord from the outlet.2. Remove refrigerator shelves.3. To remove the lamp cover, insert a slotted driver at the one of under hole of the lamp cover and pull it out forwards.4. Turn the lamp counterclockwise.5. Assemble in reverse order of disassembly.Replacement bulb must be the same specification as original.SERVICE CALLS CAN OFTEN BE AVOIDED!IF YOU FEEL YOUR REFRIGERATOR IS NOT OPERATING PROPERLY, CHECK THESE POSSIBLECAUSES FIRST :。

纯化高转染级别的质粒DNA 【工作台流程】BENCH PROTOCOL准备工作:1.将RNase A 溶液加入P1缓冲液(Buffer P1)2.加40ml96－100％乙醇(ethanol)于试剂盒的去内毒素水中(endotoxin-fre water)3.可选: 加 LyseBlue 试剂于 Buffer P14.检查 Buffer P2是否有SDS沉淀precipitation 有沉淀可在37摄氏度下暖化溶解5.Buffer P3 置于4摄氏度中预冷pre-chill6.细菌扩增培养1.补充:2.准备4-6个50ml新的或灭菌的离心管用于冷冻离心机下离心过夜菌液, 2个用于接下来的菌块重悬和与buffer混合3.1个无内毒素的30ml聚丙烯（不推荐聚碳酸酯, 因为不耐乙醇）管子（最好用圆底的离心管以利于高速离心）用于收集洗提的质粒DNA4.5个1.5ml的EP管用于收集抽提过程各步骤的产物用于实验后分析和质量控制5.准备1个冰浴盒用于步骤66.准备室温下的异丙醇10.5ml7.4摄氏度冰冻离心机和分光光度仪,琼脂糖凝胶电泳步骤: peocedureA.细菌培养、收获和裂解bacterial culture,harvest & lysis1.100ml高拷贝high-copy或250ml低拷贝low-copy过夜overnight LB培养菌液于冰冻离心机4摄氏度；6000×g；15min从新鲜的抗生素平板上挑取一个单菌落, 2-5ml抗生素LB液体初始培养基30摄氏度孵化约8小时（振荡300rpm, 注意孵化容器容积至少4倍于培养液）抗生素LB液体培养基稀释初始培养液到1:500-1000。

(高拷贝质粒100-200ul初始培养液加于100ml培养基；低拷贝质粒250-500ul初始培养液加于250ml培养基)37摄氏度300rpm振荡12-16小时。

(孵化容器容积至少4倍于培养液, 培养至细菌密度约3-4×109/ml, 即离心后菌块湿重约3g/L培养基)培养基配方：10g蛋白胨+5g酵母+10g氯化钠溶于800ml蒸馏水, 用1 N NaOH 调pH值至7.0, 用蒸馏水调整至1L。

Platts Crude Alert A page reference guide for PCR usersPlatts Crude Alert (PCR)Platts Crude Alert (PCR) gives traders, risk managers, analysts and energy executives a comprehensive set of information on the global and regional crude oil markets. From up-to-the-minute information on real-time deals, breaking news, and market analysis to the latest global price assessments, PCR provides timely and relevant insight so you stay on top of the markets and make critical business decisions with speed and clarity.The PCR quick guide is designed to help you discover and navigate through the vast content available to you easily and more efficiently.It includes:–Page numbers to assessments and commentary by region for key content.–Contact details of Platts’ global editorial oil team.This document is updated on a regular basis so go to /platts/en/our-methodology/symbol-page-directories to make sure you are using the most recent version.Platts Flash FeatureA number of assessments and all key oil benchmarks are flashed on Platts Crude Alert as soon as they are published, in realtime. These flashes appear on PCR108.All Platts News and Analysis in onelocation — page PCR100.The areas Platts market reporters focus on each day are as varied as the global oil industry: from the hydrocarbon discoveries and dusters in the upstream, to planned and unplanned outages at refineries, pipelines and terminals; from the strategies and actions of global and regional companies to the intricacies of policy and regulation in both oil producing and consuming nations.PCR100 gives you all the latest Platts news and analysis in one place*. You’ll get:– A real-time snapshot of what and who is moving global markets on any given day.–Analytical coverage on what lies behindmarket moving events.*Platts updates page PCR101 as new content becomes available. At the time of this printing, this is the content outlined in the content master page index. Questionsorfeedback?***************************Platts Subscriber Notes Page IndexPCR1500Platts Oil Price Corrections Index PagePCR3200Platts PCR Master Page IndexOn some vendor systems the add-on services will use PCR as the page prefix. To access PCR pages on Bloomberg, use PGA as the page prefix.Crude Oil Key PagesIntraday Brent/Dubai Cash Indications – Asia PCR2201 Daily AssessmentsEuropeAsiaAsiaMonthly AveragesEuropeAsiaCommentaryEuropeCrude Oil Key Pages — Commentary Continued AsiaBOTEsAsiaMiscellaneousSwaps Key PagesAssessmentsSpecial Key PagesDME Settlement PCR2710OPEC PCR2027Singapore Cracking PCR2810Singapore Cracking PCR2811Singapore PCR2812To download the most up-to-date and complete Platts Crude Alert page directory, visit symbolandpagedirectories/pagedirectoriesPlatts ContactsLondonVera BleiGlobal Head of Oil+ 44-02-7176-8659********************** LondonBeth EvansGlobal Director of Energy News +44-20-7176-6117*********************** ICE IM: bethevansplatts LondonJoel HanleySenior Director, Crude+44-(0)20-7176-6142************************ICE IM: platts_emea_oildirectorHoustonRichard SwannSenior Director, Refined Products+1-713-658-3273**************************ICE IM: platts_americas_oildirectorSingaporeCalvin LeeEditorial Director, Asia and Middle East Oil+65-6530-6429***********************ICE IM: platts_asia_oildirectorEuropeCrude OilLondonAndrew CritchlowHead Of Energy News, EMEA+44-20-7176-0146North Sea+44-20-7176-6059ICE IM: platts_americas_oildirector platts_crude_northsea West AfricaOffice: +44-20-7176-6112ICE IM: platts_americas_oildirectorplatts_crude_westafricaUrals/Mediterranean+44-20-7176-6683ICE IM: platts_americas_oildirectorplatts_crude_medRussian DomesticCrude and Products+44-20-7176-6176ICE IM: platts_americas_oildirectorplatts_russia_crudeLondonAndrew CritchlowHead Of Energy News, EMEA+44 20 7176 0146AsiaSingaporeMriganka Jaipuriyar Assoc. 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All rights reserved./platts OLFS0721。

VX-1700HOJA DE ESPECIFICACIONES – AMÉRICA L ATINA Comunicaciones de largo alcanceEl radio VX-1700 multiuso está diseñado para operar como radio móvil o como estación base para comunicaciones móviles fijas de largo alcance. Los modos de operación incluyen LSB/USB (J3E y J2B), AM (A3E) y CW (A1A), convirtiendo al radio VX-1700 en la solución ideal para una amplia variedad de aplicaciones. Popular para el uso por parte de ciudades y gobiernos locales para apoyar la gestión de seguridad pública y respuesta a situaciones de desastre.Amplia capacidad de canalesEl radio VX-1700 puede almacenar hasta 200 canales dispuestos en cinco grupos con la flexibilidad necesaria para tener cualquier número de canales por grupo. Cada canal puede ser programado con una descripción alfanumérica de 6 caracteres para una gestión de llamadas rápida y fácil. Opción de establecimiento automático de enlaces (ALE) Con la opción ALE-1 instalada, el radio VX-1700 selecciona automática- mente el canal que tenga el mejor análisis de calidad de enlace (LQA) entre los canales programados.Opciones flexibles de llamadasIncluye 6 modos de llamada integrados para apoyar varios tipos de comunicaciones:L lamada selectiva (SELCALL): efectúe llamadas a un individuo o grupo usando el número de ID asignado para cada transceptor de llamada privadaL lamada de verificación de señal (Beacon Request Call): verifique la calidad de la señal entre transceptores antes de efectuar una llamada selectiva para cerciorarse de que la misma sea viableL lamada telefónica (TELCALL): efectúe una llamada a través del servicio de interconexión telefónica para expandir el contacto con individuos por teléfonoL lamada de mensajería: envíe mensajes de texto (hasta 64 caracteres) a otro transceptor para expandir las opciones de comunicaciónL lamada de solicitud de posición: monitoree la información de posición de otro transceptor cuando use una unidad móvilL lamada de envío de posición: transmita la información de posición a otro transceptor para notificar a otros usuarios su ubicación actual Operación de vigilancia doblePermite operar el radio VX-1700 en un canal mientras se vigila periódicamente el canal designado en la memoria para cerciorarse de que no se pierda ninguna llamada. Es ideal para situaciones de coordinación de emergencias cuando es imprescindible que una llamada llegue al despachador.VX-1700La diferencia Vertex Standard Nuestro principal objetivo es alcanzar un máximo nivel de satisfacción del cliente ofreciendo productos y servicios que excedan sus expectativas. Los radios Vertex Standard son diseñados para durar y son fabricados con total precisión para ofrecer el máximo valor posible y un desempeño óptimo proporcionando una conexión sin riesgos.Otras funciones4 teclas programables Silenciador de ruido Semi-interrupción CWFunción de tono lateral CWFunciones de Bloqueo de Canal Ocupado (BCLO), Bloqueo de Tono Ocupado (BTLO) y Limitador deTiempo de Transmisión (TOT) Operación activada por voz (VOX)AccesoriosMH-31A8J: Micrófono manual dinámico MD-12A8J: Micrófono de escritorioMD-100A8X: Micrófono de escritorio para control de canalesFP-1030A: Fuente de alimentación externa MLS-100: Parlante externo, 12 WMLS-200: Parlante externo a prueba de agua, 12 W ALE-1: Unidad de establecimiento automático de enlaces FC-30: Sintonizador de antena(líneas coaxiales 1,8 MHz - 30 MHz)FC-40: Sintonizador de antena (antenas alámbricas y de estilo látigo)YA-30: Antena HF de amplio alcance de 23,4 m (dipolo) YA-31: Antena HF de amplio alcance de 15 m (dipolo o cable) Y A-007FG: Antena móvei HF de alta frecuencia multibanda (7 MHz a 30 MHz requiere FC-40) M MB-89: Soporte móvil de un solo toqueLas especificaciones están sujetas a cambios sin aviso previo ni obligación. Diseño e ingeniería japonesa. Vertex Standard LMR, Inc. Vertex Standard es marca comercial de Vertex Standard LMR, Inc. Todas las demás marcas comerciales pertenecen a sus respectivos propietarios. © 2014 Vertex Standard LMR, Inc. Todos los derechos reservados. LSS1700_ES_05/2014Especificaciones generalesRango de frecuencia RX TX30 kHz – 30.0000 MHz 1.600 – 30.0000 MHzCantidad de canales 200Tipo de emisiónA1A(CW); J3E(LSB/USB); A3E(AM); J2B (USB/LSB)Requisitos de alimentaciónDC 13.8 V ±15%, Conexión a tierra negativaPasos del sintetizador de frecuencias 10 Hz, 100 Hz, 1 kHz Estabilidad de frecuencia ± 1 ppm (-10º C a +55º Típica)Consumo de corrienteEn espera: 25 mA; RX, sin señal: 1.0 A; RX: 1.5 ATX: 24 A (salida de 125 W)Rango de temperatura de operación -10º C a +55º C Impedancia de antena 50 Ohms Dimensiones (Al x An x P)99 x 241 x 285 mmPeso (aprox.)4,3 kgEspecificaciones de receptorFrecuencia intermedia 1ra: 45.274 MHz, 2da: 24 kHzSensibilidad(A1A/J2B/J3E/A3E: S/N 10 dB) 0.5 – 1.6 MHz: 1.41 μV (A1A/J2B/J3E); 8 μV (A3E) 1.6 – 30 MHz: 0.16 μV (A1A/J2B/J3E); 1 μV (A3E)Sensibilidad del silenciador (A1A/J2B/J3E)0.5 – 1.6 MHz: 2.5 μV 1.6 – 30 MHz: 2 μV Rechazo de imagen y frecuencia intermedia (IF)Superior a 80 dBSelectividad A1A(W), J2B(W), J3E: > 2.2 kHz a -6 dB; < 4.5 kHz a -60 dB A1A(N), J2B(N): > 500 Hz a -6 dB; < 2.0 kHz a - 60 dBA3E: > 6 kHz a - 6 dB; < 20 kHz a -60 dBSalida de audio 2.2 W en 8 Ohms a 10% THD Impedancia de audio 4 – 16 Ohms (8 Ohms nominales)Radiación conducidaMenos de 4000 μWEspecificaciones de transmisorPotencia de salida 125 W (A1A, J2B, J3E a 1.6000 – 3.9999 MHz)*100 W (A1A, J2B, J3E a 4.0000 – 30.000 MHz)Portadora AM de 31 W (A3E a 1.6000 – 3.9999 MHz)Portadora AM de 25 W (A3E a 4.0000 – 30.000 MHz)Ciclo de trabajo RX: TX = 4 min.: 1 min.ModulaciónJ3E: modulador tipo PSN A3E: Nivel bajo (etapa temprana)Radiación de espurias Superior a 56 dB (armónicos) Supresión de portadora J3E Superior a 50 dB por debajo de salida pico Supresión de banda lateral indeseada Superior a 60 dB por debajo de salida picoEmisiones espurias: 56 dBRespuesta de audio (J3E)No más de -6 dB desde 400 Hz – 2500 HzAncho de banda ocupado A1A: menos de 0.5 kHz; J3E: menos de 3.0 kHz; A3E: menos de 6.0 kHzImpedancia del micrófono200 – 10 k Ohms, (600 Ohms Nominal)*100 W al usar FC-30。

BD OptiBuild™Technical Data SheetBB700 Rat Anti-Mouse CD8bProduct InformationMaterial Number:742198Size:50 µgClone:H35-17.2Alternative Name:Ly-3; Lyt-3; Lymphocyte antigen 3; Ly-C; CD8b1Reactivity:Tested in Development:MouseIsotype:Rat IgG2b, κImmunogen:5-day MLR, C57BL/6 anti-BALB/cApplication:Flow cytometry(Qualified)Concentration:0.2 mg/mlEntrez Gene ID:12526Storage Buffer:Aqueous buffered solution containing ≤0.09% sodium azide. Regulatory Status:RUODescriptionThe H35-17.2 monoclonal antibody specifically binds to both alloantigeneic forms of the β chain of the CD8differentiation antigen (Ly-3 or Lyt- 3). The CD8 α and α' chains (CD8a) form heterodimers with the CD8 β chain (CD8b, Ly-3, or Lyt-3) on the surface of most thymocytes. A subpopulation of mature T lymphocytes (i.e., MHC class I-restrictedT cells, including most T suppressor/cytotoxic cells) expresses almost exclusively the CD8 αβ heterodimer (the α' chain is absent). Subsets of γδ TCR-bearing T cells, intestinal intraepithelial lymphocytes, and dendritic cells express CD8a without CD8b. It has been suggested that the expression of the CD8a/CD8b heterodimer is restricted to T lymphocytes which matured in the thymus or in an extrathymic environment that had been influenced by thymus- initiated neuroendocrine signals. CD8 is an antigen coreceptor on the T-cell surface which interacts with MHC class I molecules on antigen-presenting cells. It participates in T-cell activation through its association with the T-cell receptor complex and protein tyrosine kinase lck (p56lck). The H35-17.2 mAb blocks T-cell-mediated cytolysis of allogeneic lymphoma cells.The antibody was conjugated to BD Horizon™ BB700, which is part of the BD Horizon Brilliant™ Blue family of dyes. It is a polymer-based tandem dye developed exclusively by BD Biosciences. With an excitation max of 485 nm and an emission max of 693 nm, BD Horizon BB700 can be excited by the 488 nm laser and detected in a standard PerCP-Cy™5.5 set (eg, 695/40-nm filter). This dye provides a much brighter alternative to PerCP-Cy5.5 with less cross laser excitation off the 405 nm and 355 nm lasers.Preparation and StorageStore undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody waspurified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with BD Horizon BB700 under optimal conditions that minimize unconjugated dye and antibody.Recommended Assay ProcedureFor optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794 or 566349).When setting up compensation, it is recommended to compare spillover values obtained from cells and BD™ CompBeads to ensure that beads will provide sufficiently accurate spillover values.For optimal results, it is recommended to perform two washes after staining with antibodies. Cells may be prepared, stained with antibodies and washed twice with wash buffer per established protocols for immunofluorescent staining prior to acquisition on a flow cytometer. Performing fewer than the recommended wash steps may lead to increased spread of the negative population.Suggested Companion ProductsCatalog Number Name Size Clone553141Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™)0.1 mg 2.4G2 554656Stain Buffer (FBS)500 mL554657Stain Buffer (BSA)500 mL563794Brilliant Stain Buffer100 Tests555899Lysing Buffer100 mLProduct Notices1.This antibody was developed for use in flow cytometry.2.The production process underwent stringent testing and validation to assure that it generates a high-qualityconjugate with consistent performance and specific binding activity. However, verification testing has not been performed on all conjugate lots.3.Researchers should determine the optimal concentration of this reagent for their individual applications.4.An isotype control should be used at the same concentration as the antibody of interest.5.Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in runningwater before discarding to avoid accumulation of potentially explosive deposits in plumbing.6.For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at/colors.7.Please refer to /us/s/resources for technical protocols.8.BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444;8,575,303; 8,354,239.9.BD Horizon Brilliant Blue 700 is covered by one or more of the following US patents: 8,455,613 and 8,575,303.10.Cy is a trademark of GE Healthcare.ReferencesGolstein P, Goridis C, Schmitt-Verhulst AM . Lymphoid cell surface interaction structures detected using cytolysis-inhibiting monoclonal antibodies. Immunol Rev. 1982; 68:5-42. (Immunogen: Cytotoxicity, Immunoprecipitation, Inhibition). Lefrancois L. Phenotypic complexity of intraepithelial lymphocytes of the small intestine. J Immunol. 1991;147(6):1746-1751. (Biology).Ledbetter JA, Seaman WE, Tsu TT, Herzenberg LA. Lyt-2 and Lyt-3 antigens are on two different polypeptide subunits linked by disulfide bonds. Relationship of subunits to T cell cytolytic activity. J Exp Med. 1981; 153:1503-1516. (Biology). Walker ID, Murray BJ, Hogarth PM, Kelso A, McKenzie IF. Comparison of thymic and peripheral T cell Ly-2/3 antigens. Eur J Immunol. 1984; 14(10):906-910. (Biology).Nakayama K, Nakayama K, Negishi I, et al. Requirement for CD8 beta chain in positive selection of CD8-lineage T cells. Science. 1994; 263(5150):1131-1133. (Biology).MacDonald HR, Schreyer M, Howe RC, Bron C. Selective expression of CD8 alpha (Ly-2) subunit on activated thymic gamma/delta cells. Eur J Immunol. 1990; 20(4):927-930. (Biology).Rocha B, Vassalli P, Guy-Grand D. The extrathymic T-cell development pathway. Immunol Today. 1992; 14(3):140-141. (Biology).Murosaki S, Yoshikai Y, Ishida A, et al. Failure of T cell receptor V beta negative selection in murine intestinal intra-epithelial lymphocytes. Int Immunol. 1991; 3(10):1005-1013. (Biology).Wang J, Klein JR. Thymus-neuroendocrine interactions in extrathymic T cell development. Science. 1994;265(5180):1860-1862. (Biology).Sydora BC, Brossay L, Hagenbaugh A, Kronenberg M, Cheroutre H. TAP-independent selection of CD8+ intestinal intraepithelial lymphocytes. J Immunol. 1996; 156(11):4209-4216. (Biology).Vremec D, Zorbas M, Scollay R, et al. The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells. J Exp Med. 1992; 176(1):47-58. (Biology).Wu L, Vremec D, Ardavin C, et al. Mouse thymus dendritic cells: kinetics of development and changes in surface markers during maturation. Eur J Immunol. 1995; 25(2):418-425. (Biology).Süss G, Shortman K. A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis. J Exp Med. 1996; 183(4):1789-1796. (Biology).Fujiura Y, Kawaguchi M, Kondo Y, et al. Development of CD8 alpha alpha+ intestinal intraepithelial T cells in beta 2-microglobulin- and/or TAP1-deficient mice. J Immunol. 1996; 156(8):2710-2715. (Biology).Bierer BE, Sleckman BP, Ratnofsky SE, Burakoff SJ. The biologic roles of CD2, CD4, and CD8 in T-cell activation. Annu Rev Immunol. 1989; 7:579-599. (Biology).Janeway CA Jr. The T cell receptor as a multicomponent signalling machine: CD4/CD8 coreceptors and CD45 in T cell activation. Annu Rev Immunol. 1992; 10:645-674. (Biology).Zamoyska R. The CD8 coreceptor revisited: one chain good, two chains better. Immunity. 1994; 1(4):243-246. (Biology). LeFrancois L. Extrathymic differentiation of intraepithelial lymphocytes: generation of a separate and unequal T-cell repertoire. Immunol Today. 1991; 12(12):436-438. (Biology).O'Rourke AM, Mescher MF. The roles of CD8 in cytotoxic T lymphocyte function. Immunol Today. 1993; 14(4):183-188. (Biology).Ledbetter JA, Rouse RV, Micklem HS, Herzenberg LA. T cell subsets defined by expression of Lyt-1,2,3 and Thy-1 antigens.Two-parameter immunofluorescence and cytotoxicity analysis with monoclonal antibodies modifies current views. J ExpMed. 1980; 152(2):280-295. (Biology).BD BiosciencesUnited States Canada Europe Japan Asia Pacific Latin America/Caribbearn877.232.8995888.268.543032.53.720.5500120.8555.9065.6861.06330800.771.7157For country contact information, visit /contactConditions: The information disclosed herein is not to be construed as a recommendation to use the above product in violation of any patents. BD Biosciences will not be held responsible for a patent infringement or other v ©2020 BD. All rights reserved. Unless otherwise noted, BD, the BD Logo and all other trademarks are the property of Becton, Dickinson and Company or its affiliates.。

For Research Use Only. Not for use in diagnostic procedures.TrueCut ™ Cas9 ProteinsCatalog Nos.A36496, A36497, A36498, A36499, A50574, A50575, A50576, A50577WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofi/support .Product descriptionInvitrogen ™ TrueCut ™ Cas9 Proteins are used for genome editing applications with CRISPR technology. Cas9 protein forms a very stable ribonucleoprotein (RNP) complex with the guide RNA (gRNA) component of the CRISPR-Cas9 system. Incorporation of nuclear localization signals (NLS) aid its delivery to the nucleus, increasing the rate of genomic DNA cleavage. It is cleared rapidly, minimizing the chance for off-target cleavage when compared to plasmid systems (Liang et al ., 2015). The Cas9 nuclease hasbeen tested in a wide variety of suspension and adherent cell lines and has shown superior genomic cleavage efficiencies and cell survivability compared to plasmid-based CRISPR systems.Two types of TrueCut ™ Cas9 Proteins are available for selection, depending upon the requirements of your particular experiment:• TrueCut ™ Cas9 Protein v2 is a recombinant Streptococcus pyogenes Cas9 (wt) protein that is the preferred choice for most CRISPR genome editing procedures where the highest level of editing efficiency is required. • TrueCut ™ HiFi Cas9 Protein is an engineered high fidelity Cas9 protein which is ideal for experiments that are sensitive to off-target events, while still maintaining a high level of editing efficiency.Table 1.Contents and storagePub. No. MAN0017066Rev.D.0Storage and handling• Store TrueCut ™ Cas9 Protein v2 and TrueCut ™ HiFi Cas9 Protein at –20°C until required for use. • Maintain RNAse-free conditions by using RNAse-free reagents, tubes, and barrier pipette tips while setting up your experiments.Before you beginMaterials required but notprovided• TrueGuide™ Synthetic gRNAs (see /trueguide)orGeneArt™ Precision gRNA Synthesis Kit (Cat. No. A29377)• Lipofectamine™ CRISPRMAX™ Cas9 Transfection Kit (Cat. Nos. CMAX00001,CMAX00003, CMAX00008, CMAX00015, CMAX00030) (for most cell lines)orNeon™ Transfection System (Cat. Nos. MPK5000, MPK1025, MPK1096) (for highesttransfection efficiency in challenging cell types including suspension cell lines)• GeneArt™ Genomic Cleavage Detection Kit (Cat. No. A24372)• Opti-MEM™ I Reduced Serum Medium (Cat. No. 31985-062)• 1X TE buffer, pH 8.0 (Cat. No. AM9849) and nuclease-free water (Cat. No. AM9914G)Prepare working stock ofTrueGuide™ Synthetic gRNA If TrueGuide™ Synthetic gRNA is being used, resuspend the gRNA (sgRNA, crRNA, ortracrRNA) in1X TE buffer to prepare 100 μM (100 pmol/μL) stock solutions.Before opening, centrifuge each TrueGuide™ Synthetic gRNA tube at low speed (maximum1.RCF 4,000 × g) to collect the contents at the bottom of the tube, then remove the cap from thetube carefully.Using a pipette and sterile tips, add the required volume of 1X TE buffer to prepare 100 μM2.(100 pmol/μL) stock solutions.3.Vortex the tube to resuspend the oligos, briefly centrifuge to collect the contents at thebottom of the tube, then incubate at room temperature for 15–30 minutes to allow the gRNAoligos to dissolve.Vortex the tube again to ensure that all the contents of the tube are resuspended, then briefly4.centrifuge to collect the contents at the bottom of the tube.(Optional) Check the concentration of the resuspended oligos using the NanoDrop™5.Spectrophotometer (or equivalent) or a UV-base plate reader.6.(Optional) Aliquot the working stock into one or more tubes for storage.Use working stocks immediately or freeze at –20°C until needed for use.7.(Optional) Generate gRNA byin vitro transcription If using in vitro transcribed gRNA with TrueCut™ Cas9 Protein v2 or TrueCut™ HiFi Cas9Protein in CRISPR-Cas9-mediated genome editing, the GeneArt™ Precision gRNA SynthesisKit is recommended for preparation of the gRNA. For detailed instructions on how togenerate full length gRNA, see the GeneArt™ Precision gRNA Synthesis Kit User Guide (Pub.No. MAN0014538), at .Transfection guidelinesGeneral CRISPR/gRNAtransfection guidelines• The efficiency with which mammalian cells are transfected with gRNA varies accordingto cell type and the transfection reagent used. See Table 2 (page 3) for delivery reagentrecommendations.• For gene editing (including gene knockout) editing efficiency is highest with a 1:1 molarratio of gRNA to TrueCut™ Cas9 Protein v2 or TrueCut™ HiFi Cas9 Protein. In some celltypes such as iPSC and THP1, we have used up to 2 μg TrueCut™ Cas9 Protein v2 and400 ng gRNA per well in 24-well format.• For HDR knock-in editing, a 1.5:1 molar ratio of donor ssODN to gRNA or TrueCut™ Cas9Protein v2 or TrueCut™ HiFi Cas9 Protein is recommend for highest knock-in efficiency.The donor can be added directly to RNPs (a premixed gRNA-Cas9 protein). If using adsDNA donor, further optimization may be necessary to determine the appropriate donoramount, since the toxicity level is dependent on the length and format of the donor DNAand cell type.• The optimal cell density for transfection varies depending on cell size and growthcharacteristics. In general, use cells at 30–70% confluence on the day of transfectionwith lipid-mediated delivery, or 70–90% confluence for electroporation using the Neon™Transfection System.• After the optimal cell number and dosage of Cas9/gRNA and/or donor that providesmaximal gene editing efficiency is determined for a given cell type, do not varyconditions across experiments to ensure consistency.For an overview of the factors that influence transfection efficiency, see the “TransfectionBasics” chapter of the Gibco™ Cell Culture Basic Handbook, available at /cellculturebasics.• Use the TrueGuide™ Positive Controls (human AVVS1, CDK4, HPRT1, or mouse Rosa 26)and negative control gRNA (non-coding) to determine gRNA amount and transfectionconditions that give the optimal gene editing efficiency with highest cell viability. TheTrueGuide™ Positive and Negative sgRNA and crRNA Controls are available separatelyfrom Thermo Fisher Scientific. For more information, refer to /trueguide.• The cell number and other recommendations provided in the following proceduresare starting point guidelines based on the cell types we have tested. For multiplewells, prepare a master mix of components to minimize pipetting error, then dispense theappropriate volumes into each reaction well. When making a master mix for replicate wells,we recommend preparing extra volume to account for any pipetting variations.Recommended deliveryoptions• Choosing the right delivery reagent is critical for transfection and gene editing efficiency.See our recommendations in Table 2. For more information on transfection reagents, see/transfection.• For cell line specific transfection conditions using the Lipofectamine™ CRISPRMAX™Transfection Reagent or the Neon™ Transfection System, see the Appendix (page 13).• For best results, perform electroporation and transfection of cells using both TrueCut™Cas9 Proteins and TrueGuide™ Synthetic gRNA.HiFi Cas9 Protein.Table 2. Recommended delivery options for TrueCut™ Cas9 Protein v2 and TrueCut™Guidelines for verification of editing efficiencyVerification of gene editingefficiency• Before proceeding with downstream applications, verify the gene editing efficiency of thecontrol target and select the condition that shows the highest level of editing efficiency forfuture screening experiments.• To estimate the CRISPR-Cas9-mediated editing efficiency in a pooled cell population,use the GeneArt™ Genomic Cleavage Detection Kit (Cat. No. A24372), or performIon Torrent™ next generation sequencing or a Sanger sequencing-based analysis.• While the genomic cleavage detection (GCD) assay provides a rapid method forevaluating the efficiency of indel formation following an editing experiment, nextgeneration sequencing (NGS) of the amplicons from the edited population or Sangersequencing of amplicons cloned into plasmids give a more accurate estimate of thepercent editing efficiency and indel types for knockout and HDR knock-in editing.GeneArt™ Genomic CleavageDetection (GCD) Assay• After transfections, use the GeneArt™ Genomic Cleavage Detection Kit (Cat. No. A24372)to estimate the CRISPR-Cas9-mediated cleavage efficiency in a pooled cell population.• You can design and order target-specific primer sets for the GCD assay through ourTrueDesign Genome Editor, available at /crisprdesign.• To perform the GCD assay for the positive control, you need the primers listed in Table 3.We recommend using Invitrogen™ Custom DNA Value or Standard Oligos, available from/oligos, for target specific primer sets needed for the GCD assay.• You can set up the GCD assay in a 96-well plate format and analyze multiple gRNA-treated samples in parallel on a 2% E-Gel™ 48 agarose gel (48-well).• For more information and detailed protocols, see the GeneArt™ Genomic Cleavage DetectionKit User Guide (Pub. No. MAN0009849), available for download at /GCDManual.Table 3. Target sequences for the positive and negative control (non-targeting) TrueGuide™ Synthetic gRNA sequences.Guidelines for clone isolation and validationAfter you have determined the cleavage efficiency of the pooled cell population, isolate single cell clones for further validation and banking. You can isolate single cell clones from the selected pool using limiting dilution cloning (LDC) in 96-well plates or by single cell sorting using a flow cytometer.Limiting dilution cloning(LDC)• Based on the editing efficiency and estimated cell viability, you can estimate the number of single clones needed to obtain a desired knock-out (KO) clonal cell line. For example, if you desire a homozygous KO with mutations in both copies of a gene and the resulting GeneArt ™ cleavage detection efficiency was 50%, then the probability of having both alleles knocked out in any cell is 25% (0.5 × 0.5 = 0.25).If the probability of an indel leading to frame shift is 2/3, then the chance of having a homozygous KO is ~11% per cell [(0.5 × 0.5) × (0.66 × 0.66) = 0.11].• We recommend performing limiting dilution by targeting 0.8 cells/well, which requires you to resuspend the transfected cells (post-counting) at a density of8 cells/mL in complete growth medium, then transferring 100 µL of this to each well of a 96-well plate. If you plate at least ten 96-well plates in this manner and expect only 20% of cells tosurvive, then the probability of having homozygous KO clones in the 192 surviving cells will be 19–21 cells (192 × 11%).• Note that single cell clone survivability varies by cell type. Some cells that do not like to remain as single cells need to be plated at a low density to get well separated colonies, which will then have to be manually picked for further screening.Example LDC procedureusing 293FT cells1. Wash the transfected cells in each well of the 24-well plate with 500 µL of PBS. Carefully aspirate the PBS and discard.2. Add 500 µL of TrypLE ™ cell dissociation reagent to the cells and incubate for2–5 minutes at 37°C.3. Add 500 µL of complete growth medium to the cells to neutralize the dissociation reagent.Pipette the cells up and down several times to break up the cell aggregates. Make sure that the cells are well separated and are not clumped together. 4. Centrifuge the cells at 300 × g for 5 minutes to pellet.5. Aspirate the supernatant, resuspend the cells in an appropriate volume of pre-warmed(37°C) growth medium, then perform a cell count. 6. Dilute the cells to a density of 8 cells/mL of complete growth medium. Prepare a total ofSequence analysis• For next generation sequencing (NGS) based editing efficiency analysis, you canspecifically amplify the edited region and barcode amplicons by pooling all amplicons in a single tube and performing sequencing using various NGS platforms such as the Ion Torrent ™ Targeted Amplicon-seq Validation (TAV). For more information on NGS analysis, refer to Ion Torrent ™ targeted sequencing solutions at /ionapliseqsolutions .• For Sanger sequencing-based editing efficiency analysis, refer to our application note referenced at /sangercrispr .• Use the SeqScreener Gene Edit Confirmation App on Thermo Fisher ™ Connect todetermine the spectrum and frequency of targeted mutations (see Pub. No. MAN0019454 at . for details).50 mL of cell suspension at this cell density and transfer to a sterile reservoir.Note: You can also perform a serial dilution to get a better estimate of cell density.Using a multichannel pipettor, transfer 100 µL of the cell suspension into each well of 96-well7.tissue culture plates until the desired number of plates is seeded. Make sure to mix the cellsin between seeding the plates to avoid the formation of cell aggregates.Note: In general, we seed ten 96-well plates to achieve a large number of clones. Numberof plates to seed depends on the editing efficiency of pooled cell population and viability ofcells post single cell isolation.Incubate the plates in a 37°C, 5% CO2 incubator.8.Scan the plates for single cell colonies as soon as small aggregates of cells are visible under a9.4X microscope (usually after first week, depending on the growth rate of the cell line).Continue incubating the plates for an additional 2–3 weeks to expand the clonal populations10.for further analysis and characterization.Example single cell sortingprocedure in a 96-well plateusing flow cytometer Single cells can be sorted into a 96-well plate format using a flow cytometer with singlecell sorting capability. After sorting and expanding the single cell clones, analyze andcharacterize the clonal populations using suitable assays.1.Wash the transfected 293FT cells in each well of the 24-well plate with 500 µL of PBS.Carefully aspirate the PBS and discard.Add 500 µL of TrypLE™ cell dissociation reagent and incubate for 2–5 minutes at 37°C.2.Add 500 µL of complete growth medium to the cells to neutralize the dissociation reagent.3.Pipette the cells up and down several times to break up the cell aggregates. Make sure thatthe cells are well separated and are not clumped together.Centrifuge the cells at 300 × g for 5 minutes to pellet.4.5.Aspirate the supernatant, then wash the cell pellet once with 500 μL of PBS.Resuspend 1 × 106 cells in 1 mL of FACS buffer, then add propidium iodide (PI) to the cells at6.a final concentration of 1 µg/mL. Keep the resuspended cells on ice.Filter the cells using suitable filters before analyzing them on a flow cytometer with single7.cell sorting capability.Sort PI-negative cells into a 96-well plate containing 100 μL of complete growth medium. If8.desired, you can use 1X antibiotics with the complete growth medium.Incubate the plates in a 37°C, 5% CO2 incubator.9.Scan the plates for single cell colonies as soon as small aggregates of cells are visible under10.a 4X microscope. Colonies should be large enough to see as soon as 7–14 days (usually afterfirst week, depending on the growth rate of the cell line). You can perform image analysis toensure that the colonies are derived from single cells.11.After image analysis, continue incubating the plates for an additional 2–3 weeks to expandthe clonal populations for further analysis and characterization.Characterize edited clones You can analyze the single cell clones for purity and the desired genotype (homozygous orheterozygous allele) by various molecular biology methods such as genotyping PCR, qPCR,next generation sequencing, or western blotting.Supporting tools At Thermo Fisher Scientific, you can find a wide variety of tools to meet your gene editingand validation needs, including Invitrogen™ LentiArray CRISPR and Silencer™ Select RNAilibraries for screening, primers for targeted amplicon sequencing, antibody collection forknock-out validation, and ORF collections and GeneArt™ gene synthesis service for cDNAexpression clones that can be used for rescue experiment reagents.thermofisher .com/support | thermofisher .com/askaquestion thermofisher .comLimited product warrantyLife Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies’ General Terms and Conditions of Sale found on Life Technologies’ website at /us/en/home/global/terms-and-conditions.html . If you have any questions, please contact Life Technologies at www /support .Manufacturer: Thermo Fisher Scientific Baltics UAB | V. A. Graiciuno 8 | LT-02241 Vilnius, LithuaniaThe information in this guide is subject to change without notice.DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.Revision history:Pub. No. MAN0017066Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.©2021 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified .。

vigs载体构建的基本流程1.首先准备vigs载体构建所需的质粒。

Firstly, prepare the plasmid required for vigs vector construction.2.将目标基因插入到质粒的多克隆位点中。

Insert the target gene into the multicloning sites of the plasmid.3.通过PCR扩增目标基因。

Amplify the target gene by PCR.4.将PCR产物纯化和酶切。

Purify and digest the PCR product.5.选择合适的酶切位点和vigs载体进行连接。

Chooseappropriate restriction enzyme cutting sites and connect them with the vigs vector.6.进行连接反应。

Perform ligation reaction.7.转化大肠杆菌，分离获得重组质粒。

Transform E. coli and isolate the recombinant plasmid.8.进行PCR验证所得质粒。

Perform PCR verification of the obtained plasmid.9.测序确认目标基因已正确插入。

Sequencing to confirm the correct insertion of the target gene.10.提取质粒，获得高质量的vigs载体。

Extract the plasmid to obtain high-quality vigs vector.11.通过限制性内切酶消化鉴定重组质粒。

Identify the recombinant plasmid by restriction endonuclease digestion.12.进行质粒的DNA测序。

法国VL凝胶成像及分析系统操作指南
一、图像摄取：
1．启动电脑系统，打开灯箱开关；
2．双击显示屏上的Bio-cap图标，进入图像摄取主画面；
3．放入要摄取图像的样品（胶片、电泳胶等），按下步分别摄像；
A、核酸凝胶样品（要用紫外光板的），先移动CCD至暗箱到右边，单击画
面的“integration time”钮，调整爆光时间，CCD上的光圈、焦距并配合主画面上的“adjustment”钮下的对比度及亮度调节钮，至出现清晰画面为止；
B、除核酸凝胶样品外的所有样品（要用白光板的），移动CCD至暗箱至左
边，单击画面上的“read time”钮，调整CCD上的光圈，焦距及主画面“adjustment”钮下的对比度及亮度调节钮至清晰画面出现为止，必要时，可打开暗箱的开关以增加照明度；
4．单击“Freeze”钮，固定所摄到的图像，同时可开闭灯箱开关；
5．单击“保存”。

并输入图像编号，按“确定“钮，图像存入文件夹即可完成图像保存工作。

6．如要打印图像，先打开热敏打印机开关，单击“热敏打印机”钮，图像即可输出。

图像分析：
A、启动电脑系统；
B、双击显示屏上的“Bio-capt”，“Bio-1D++”或“Bio-2D”图标，进入主画
面；
C、打开文档，从“Vlimage”中调出所要分析的图像，按需要分析的内相应
分析（可借用“？！”钮启动帮助功能工作）
D、分析工作完成后，关闭主画面即可退出图像分析程序。

二、注意事项：
1．保持内室环境清洁，干燥；
2．保持灯箱光板干净（特别是用了核酸电泳胶后）；
3．光板上不要用硬、尖物拖、划以防止出现划痕影响图像观察。

phys. stat. sol. (a) 203, No. 13, 3207–3225 (2006) / DOI 10.1002/pssa.200671403© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Review ArticleSingle defect centres in diamond: A reviewF. Jelezko and J. Wrachtrup *3. Physikalisches Institut, Universität Stuttgart, 70550 Stuttgart, GermanyReceived 9 February 2006, revised 28 July 2006, accepted 9 August 2006Published online 11 October 2006PACS 03.67.Pp, 71.55.–r, 76.30.Mi, 76.70.–rThe nitrogen vacancy and some nickel related defects in diamond can be observed as single quantum sys-tems in diamond by their fluorescence. The fabrication of single colour centres occurs via generation of vacancies or via controlled nitrogen implantation in the case of the nitrogen vacancy (NV) centre. The NV centre shows an electron paramagnetic ground and optically excited state. As a result electron and nuclear magnetic resonance can be carried out on single defects. Due to the localized nature of the electron spin wavefunction hyperfine coupling to nuclei more than one lattice constant away from the defect as domi-nated by dipolar interaction. As a consequence the coupling to close nuclei leads to a splitting in the spec-trum which allows for optically detected electron nuclear double resonance. The contribution discusses the physics of the NV and other defect centre from the perspective of single defect centre spectroscopy.© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim1 IntroductionThe ever increasing demand in computational power and data transmission rates has inspired researchers to investigate fundamentally new ways to process and communicate information.Among others, physicists explored the usefulness of “non-classical”, i.e. quantum mechanical systems in the world of information processing. Spectacular achievements like Shors discovery of the quantum factoring algorithm [1] or the development of quantum secure data communication gave birth to the field of quantum information processing (QIP) [2]. After an initial period where the physical nature of infor-mation was explored [3] and how information processing can be carried out by unitary transformation in quantum mechanics, researchers looked out for systems which might be of use as hardware in QIP. From the very beginning it became clear that the restrictions on the hardware of choice are severe, in particular for solid state systems. Hence in the recent past scientists working in the development of nanostructured materials and quantum physics have cooperated on different solid-state systems to define quantum me-chanical two-level system, make them robust against decoherence and addressable as individual units. While the feasibility of QIP remains to be shown, this endeavour will deepen our understanding of quan-tum mechanics and also marks a new area in material science which now also has reached diamonds as a potential host material. The usefulness of diamond is based on two properties. First defects in diamond are often characterized by low electron phonon coupling, mostly due to the low density of phonon states i.e. high Debye temperature of the material [4]. Secondly, colour centres in diamond are usually found to be very stable, even under ambient conditions. This makes them unique among all optically active solid-state systems.* Corresponding author: e-mail: wrachtrup@physik.uni-stuttgart.de3208 F. Jelezko and J. Wrachtrup: Single defect centres in diamond: A review© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim The main goal of QIP is the flexible generation of quantum states from individual two-level systems (qubits). The state of the individual qubits should be changed coherently and the interaction strength among them should be controllable. At the same time, those systems which are discussed for data com-munication must be optically active which means, that they should show a high oscillator strength for an electric dipole transition between their ground and some optically excited state. Individual ions or ion strings have been applied with great success. Here, currently up to eight ions in a string have been cooled to their ground state, addressed and manipulated individually [5]. Owing to careful construction of the ion trap, decoherence is reduced to a minimum [6]. Landmark experiments, like teleportation of quantum states among ions [7, 8] and first quantum algorithms have been shown in these systems [9, 10].In solid state physics different types of hardware are discussed for QIP. Because dephasing is fast in most situations in solids only specific systems allow for controlled generation of a quantum state with preservation of phase coherence for a sufficient time. Currently three systems are under discussion. Su-perconducting systems are either realized as flux or charge quantized individual units [11]. Their strength lies in the long coherence times and meanwhile well established control of quantum states. Main pro-gresses have been achieved with quantum dots as individual quantum systems. Initially the electronic ground as well as excited states (exciton ground state) have been used as definition of qubits [12]. Mean-while the spin of individual electrons either in a single quantum dot or coupled GaAs quantum dots has been subject to control experiments [13–15]. Because of the presence of paramagnetic nuclear spins, the electron spin is subject to decoherence or a static inhomogeneous frequency distribution. Hence, a further direction of research are Si or SiGe quantum dots where practically no paramagnetic nuclear spins play a significant role. The third system under investigation are phosphorus impurities in silicon [16]. Phospho-rus implanted in Si is an electron paramagnetic impurity with a nuclear spin I = 1/2. The coherence times are known to be long at low temperature. The electron or nuclear spins form a well controllable two-level system. Addressing of individual spins is planned via magnetic field gradients. Major obstacles with respect to nanostructuring of the system have been overcome, while the readout of single spins based on spin-to-charge conversion with consecutive detection of charge state has not been successful yet. 2 Colour centres in diamondThere are more then 100 luminescent defects in diamond. A significant fraction has been analysed in detail such that their charge and spin state is known under equilibrium conditions [17]. For this review nitrogen related defects are of particular importance. They are most abundant in diamond since nitrogen is a prominent impurity in the material. Nitrogen is a defect which either exists as a single substitutional impurity or in aggregated form. The single substitutional nitrogen has an infrared local mode of vibration Fig. 1 (online colour at: ) Schematic represen-tation of the nitrogen vacancy (NV) centre structure.phys. stat. sol. (a) 203, No. 13 (2006) 3209 © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim65070075080050010001500T =300KT =1.8K F l u o r e s c e n c e I n t e n s i t y ,C t s Wavelength,nm ZPL 637.2nmat 1344 cm –1. The centre is at a C 3v symmetry site. It is a deep electron donor, probably 1.7 eV below the conduction band edge. There is an EPR signal associated with this defect, called P1, which identifies it to be an electron paramagnetic system with S = 1/2 ground state [17]. Nitrogen aggregates are, most com-monly, pairs of neighbouring substitutional atoms, the A aggregates, and groups of four around a va-cancy, the B aggregate. All three forms of nitrogen impurities have distinct infrared spectra.Another defect often found in nitrogen rich type Ib diamond samples after irradiation damage is the nitrogen vacancy defect centre, see Fig. 1. This defect gives rise to a strong absorption at 1.945 eV (637 nm) [18]. At low temperature the absorption is marked by a narrow optical resonance line (zero phonon line) followed by prominent vibronic side bands, see Fig. 2. Electron spin resonance measure-ment have indicated that the defect has an electron paramagnetic ground state with electron spin angular momentum S = 1 [19]. The zero field splitting parameters were found to be D = 2.88 GHz and E = 0 indicating a C 3v symmetry of the electron spin wavefunction. From measurements of the hyperfine cou-pling constant to the nitrogen nuclear spin and carbon spins in the first coordination shell it was con-cluded that roughly 70% of the unpaired electron spin density is found at the three nearest neighbour carbon atoms, whereas the spin density at the nitrogen is only 2%. Obviously the electrons spend most of their time at the three carbons next to the vacancy. To explain the triplet ground state mostly a six elec-tron model is invoked which requires the defect to be negatively charged i.e. to be NV – [20]. Hole burn-ing experiments and the high radiative recombination rate (lifetime roughly 11 ns [21], quantum yield 0.7) indicate that the optically excited state is also a spin triplet. The width of the spectral holes burned into the inhomogeneous absorption profile were found to be on the order of 50 MHz [22, 23]. Detailed investigation of the excited state dephasing and hole burning have caused speculations to as whether the excited state is subject to a J an–Teller splitting [24, 25]. From group theoretical arguments it is con-cluded that the ground state is 3A and the excited state is of 3E symmetry. In the C 3v group this state thus comprises two degenerate substrates 3E x,y with an orthogonal polarization of the optical transition. Photon echo experiments have been interpreted in terms of a Jan Teller splitting of 40 cm –1 among these two states with fast relaxation among them [24]. However, no further experimental evidence is found to sup-port this conclusion. Hole burning experiments showed two mechanisms for spectral hole burning: a permanent one and a transient mechanism with a time scale on the order of ms [23]. This is either inter-preted as a spin relaxation mechanism in the ground state or a metastable state in the optical excitation-emission cycle. Indeed it proved difficult to find evidence for this metastable state and also number and energetic position relative to the triplet ground and excited state are still subject of debate. Meanwhile it seems to be clear that at least one singlet state is placed between the two triplet states. As a working hypothesis it should be assumed throughout this article that the optical excitation emission cycle is de-scribed by three electronic levels.Fig. 2 Fluorescence emission spectra of single NVcentres at room temperature and LHe temperatures.Excitation wavelength was 514 nm.3210 F. Jelezko and J. Wrachtrup: Single defect centres in diamond: A review© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 3 Optical excitation and spin polarizationGiven the fact that the NV centre has an electron spin triplet ground state with an optically allowed tran-sition to a 3E spin triplet state one might wonder about the influence of optical excitation on the electron spin properties of the defect. Indeed in initial experiments no electron spin resonance (EPR) signal of the defect was detected except when subject to irradiation in a wavelength range between 450 and 637 nm[19]. Later on it became clear that in fact there is an EPR signal even in the absence of light, yet the signal strength is considerably enhanced upon illumination [26]. EPR lines showed either absorptive or emissive line shapes depending on the spectral position. This indicates that only specific spin sub-levels are affected by optical excitation [27]. In general a S = 1 electron spin system is described by a spin Hamiltonian of the following form: e ˆˆˆH g S SDS β=+B . Here g e is the electronic g -factor (g = 2.0028 ± 0.0003); B 0 is the external magnetic field and D is the zero field splitting tensor. This ten-sor comprises the anisotropic dipolar interaction of the two electron spins forming the triplet state aver-aged over their wave function. The tensor is traceless and thus characterized by two parameters, D and E as already mentioned above. The zero field splitting causes a lifting of the degeneracy of the spin sub-levels m s = ±1,0 even in the absence of an external magnetic field. Those zero field spin wave functions T x,y,z do not diagonalize the full high-field Hamiltonian H but are related to these functions by121212=x T T T ββαα-+-=-〉〉,121211y T T T ββαα-++=+〉〉,12120|.z T T αββα+=〉〉 The expectation value of S z for all three wave functions ,,,,||x y z z x y z T S T 〈〉 is zero. Hence there is no magnetization in zero external field. There are different ways to account for the spin polarization process in an excitation scheme involving spin triplets. To first order optical excitation is a spin state conserving process. However spin–orbit (LS) coupling might allow for a spin state change in the course of optical excitation. Cross relaxation processes on the other hand might cause a strong spin polarization as it is observed in the optical excitation of various systems, like e.g. GaAs. However, optical spectroscopy and in particular hole burning data gave little evidence for non spin conserving excitation processes in the NV centre. In two laser hole burning experiments data have been interpreted by assuming different zero field splitting parameters in ground and excited state exc exc (2GHz,0,8GHz)D E ªª by an otherwise spin state preserving optical excitation process [28]. Indeed this is confirmed by later attempts to gener-ate ground state spin coherence via Raman process [29], which only proves to be possible when ground state spin sublevels are brought close to anticrossing by an external magnetic field. Another spin polaris-ing mechanism involves a further electronic state in the optical excitation and emission cycle [30, 31]. Though being weak, LS coupling might be strong enough to induce intersystem crossing to states with different spin symmetry, e.g. a singlet state. Indeed the relative position of the 1A singlet state with re-spect to the two triplet states has been subject of intense debate. Intersystem crossing is driven by LS induced mixing of singlet character into triplet states. Due to the lack of any emission from the 1A state or noticeable absorption to other states, no direct evidence for this state is at hand up to now. However, the kinetics of photo emission from single NV centres strongly suggests the presence of a metastable state in the excitation emission cycle of the state. As described below the intersystem crossing rates from the ex-cited triplet state to the singlet state are found to be drastically different, whereas the relaxation to the 3A state might not depend on the spin substate. This provides the required optical excitation dependent relaxa-tion mechanism. Bulk as well as single centre experiments show that predominantly the m s = 0 (T z ) sublevel in the spin ground state is populated. The polarization in this state is on the order of 80% or higher [27].phys. stat. sol. (a) 203, No. 13 (2006) 3211 © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim4 Spin properties of the NV centreBecause of its paramagnetic spin ground and excited state the NV centre has been the target of numerous investigations regarding its magnetooptical properties. Pioneering work has been carried out in the groups of Manson [32–36], Glasbeek [37–39] and Rand [26, 40, 41].The hyperfine and fine structure splitting of the NV ground state has been used to measure the Aut-ler–Townes splitting induced by a strong pump field in a three level system. Level anticrossing among the m s = 0 and m s = –1 allows for an accurate measurement of the hyperfine coupling constant for the nitrogen nucleus, yielding an axially symmetric hyperfine coupling tensor with A || = 2.3 MHz and A ^ = 2.1 MHz [42, 43]. The quadrupole coupling constant P = 5.04 MHz. Because of its convenient access to various transitions in the optical, microwave and radiofrequency domain the NV centre has been used as a model system to study the interaction between matter and radiation in the linear and non-linear regime. An interesting set of experiments concerns electromagnetically induced transparency in a Λ-type level scheme. The action of a strong pump pulse on one transition in this energy level scheme renders the system transparent for radiation resonant with another transitions. Experiments have been carried out in the microwave frequency domain [44] as well as for optical transitions among the 3A ground state and the 3E excited state [29]. Here two electron spin sublevels are brought into near level anticrossing such that an effective three level system is generated with one excited state spin sublevel and two allowed optical transitions. A 17% increase in transmission is detected for a suitably tuned probe beam.While relatively much work has been done on vacancy and nitrogen related impurities comparatively little is known about defects comprising heavy elements. For many years it was difficult to incorporate heavy elements as impurities into the diamond lattice. Only six elements have been identified as bonding to the diamond lattice, namely nitrogen, boron, nickel, silicon, hydrogen and cobalt. Attempts to use ion implantation techniques for incorporation of transition metal ions were unsuccessful. This might be due to the large size of the ions and the small lattice parameters of diamond together with the metastability of the diamond lattice at ambient pressure. Recent developments in crystal growth and thin film technology have made it possible to incorporate various dopants into the diamond lattice during growth. This has enabled studies of nickel defects [45, 46]. Depending on the annealing conditions Ni can form clusters with various vacancies and nitrogen atoms in nearest neighbour sites. Different Ni related centres are listed with NE as a prefix and numbers to identify individual entities. The structure and chemical compo-k 23k 12k 31k 213A 3E 1AOptical excitation 3A 3E 1A z x,yz´x´y´k 23k 31a bFig. 3 a) Three level scheme describing the optical excitation and emission cycle of single NV centres. 3A and 3E are the triplet ground and excited state. 1A is a metastable singlet state. No information is at hand presently about the number and relative position of singlet levels. The arrows and k ij denote the rates of transition among the various states. b) More detailed energy level scheme differentiating between trip-let sublevels in the 3A and 3E state.3212 F. Jelezko and J. Wrachtrup: Single defect centres in diamond: A review© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim sition of defects have mostly been identified by EPR on the basis of the hyperfine coupling to nitrogen nuclei [46]. A particularly rich hyperfine structure has been identified for the NE8 centre.Analysis of the angular dependence of the EPR spectrum for the NE8 centre showed that this centre has electronic spin S = 1/2 and a g -value typical of a d -ion with more than half filled d -shell. The NE8 centre has been found not only in HPHT synthetic diamonds but also in natural diamonds which contain the nickel-nitrogen centres NE1 to NE3 [46]. The structure of the centre is shown in Fig. 4. It comprises 4 substitutional nitrogen atoms and an interstitial Ni impurity. The EPR signature of the system has been correlated to an optical zero phonon transition at around 794 nm. The relative integral intensity of the zero phonon line and the vibronic side band at room temperature is 0.7 (Debey–Waller factor) [47]. The fluorescence emission statistics of single NE8 emitters shows a decay to a yet unidentified metastable state with a rate of 6 MHz.5 Single defect centre experimentsExperiments on single quantum systems in solids have brought about a considerable improvement in the understanding of the dynamics and energetic structure of the respective materials. In addition a number of quantum optical phenomena, especially when light–matter coupling is concerned, have been investi-gated. As opposed to atomic systems on which first experiments on single quantum systems are well established, similar experiments with impurity atoms in solids remain challenging. Single quantum sys-tems in solids usually strongly interact with their environment. This has technical as well as physical consequences. First of all single solid state quantum systems are embedded in an environment which, for example, scatters excitation light. Given a diffraction limited focal volume usually the number of matrix atoms exceed those of the quantum systems by 106–108. This puts an upper limit on the impurity content of the matrix or on the efficiency of inelastic scattering processes like e.g. Raman scattering from the matrix. Various systems like single hydrocarbon molecules, proteins, quantum dots and defect centres have been analysed [48]. Except for some experiments on surface enhanced Raman scattering the tech-nique usually relies on fluorescence emission. In this technique an excitation laser in resonance with a strongly allowed optical transition of the system is used to populate the optically excited state (e.g. the 3E state for the NV centre), see Fig. 3a. Depending on the fluorescence emission quantum yield the system either decays via fluorescence emission or non-radiatively, e.g. via inter-system-crossing to a metastable Fig. 4 (online colour at: ) Structure of the NE8 cen-tre.phys. stat. sol. (a) 203, No. 13 (2006) 3213© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimF l u o r .I n t e n s i t y ,k C t s /s Excitation power,mW.state (1A in the case of the NV). The maximum numbers of photons emitted are given when the optical transition is saturated. In this case the maximum fluorescence intensity is given as312123F max 3123()=.2k k k I k k Φ++ Here k 31 is the relaxation rate from the metastable to the ground state and k 21 is the decay rate of the opti-cally excited state, k 23 is the decay rate to the metastable state and φF marks the fluorescence quantum yield. For the NV centre I max is about 107 photon/s. I max critically depends on a number of parameters. First of all the fluorescence quantum yield limits the maximum emission. A good example to illustrate this is the GR1 centre, the neutral vacancy defect in diamond. The overall lifetime of the excited state for this defect is 1 ns at room temperature. However, the radiative lifetime is on the order of 100 ns. Hence φF is on the order of 0.01. Given the usual values for k 21 and k 31 this yields an I max which is too low to allow for detecting single GR1 centres with current technology. Figure 5 shows the saturation curve of a single NV defect. Indeed the maximum observable emission rate from the NV centre is around 105 pho-tons/s which corresponds well to the value estimated above, if we assume a detection efficiency of 0.01. Single NV centres can be observed by standard confocal fluorescence microscopy in type Ib diamond. In confocal microscopy a laser beam is focussed onto a diffraction limited spot in the diamond sample and the fluorescence is collected from that spot. Hence the focal probe volume is diffraction limited with a volume of roughly 1 µm 3. In order to be able to detect single centres it is thus important to control the density of defects. For the NV centre this is done by varying the number of vacancies created in the sam-ple by e.g. choosing an appropriate dose of electron irradiation. Hence the number of NV centres de-pends on the number of vacancies created and the number of nitrogen atoms in the sample. Figure 7 shows an image of a diamond sample where the number of defects in the sample is low enough to detect the fluorescence from single colour centres [49]. As expected the image shows diffraction limited spots. From the image alone it cannot be concluded whether the fluorescence stems from a single quantum system or from aggregates of defects. To determine the number of independent emitters in the focal vol-ume the emission statistics of the NV centre fluorescence can be used [50–52]. The fluorescence photon number statistics of a single quantum mechanical two-level system deviates from a classical Poissoniandistribution. If one records the fluorescence intensity autocorrelation function 2()()=()t I t g t ΙττΙ+2〈()〉〈〉 for short time τ one finds g 2(0) = 0 if the emission stems from a single defect centre (see Fig. 6). This is due to the fact that the defect has to be excited first before it can emit a single photon. Hence a single defect never emits two fluorescence photons simultaneously, in contrast to the case when a number of independent emitters are excited at random. If one adopts the three level scheme from Fig. 3a, rate equa-tions for temporal changes of populations in the three levels can be set up. The equations are solved by 12(2)()=1(1)e e ,k k g K K τττ-++Fig. 5 Saturation curve of the fluorescence inten-sity of a single NV centre at T = 300 K. Excitationwavelength is 514 nm. The power is measured atthe objective entrance.3214F. Jelezko and J. Wrachtrup: Single defect centres in diamond: A review © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheimg (2)(τ)τ,nswith rates 1,2=k -P = k 21 + k 12 + k 23 + k 31 and Q = k 31(k 21 + k 12) + k 23(k 31 + k 12) with23231123112= .k k k k k K k k+-- This function reproduces the dip in the correlation function g 2(τ) for τ → 0 shown in Fig. 6, which indicates that the light detected originates from a single NV. The slope of the curve around 0τ= is de-terminded by the pumping power of the laser k 12 and the decay rate k 21. For larger times τ a decay of the correlation function becomes visible. This decay marks the ISC process from the excited triplet 3E to the metastable singlet state 1A. Besides the spin quantum jumps detected at low temperature the photon sta-tistics measurements are the best indication for detection of single centres. It should be noted that the radiative decay time depends on the refractive index of the surrounding medium as 1/n medium . Because n medium of diamond is 2.4 the decay time should increase significantly if the refractive index of the sur-rounding is reduced. This is indeed observed for NV centres in diamond nanocrystals [51]. It should beFig. 7 (onl ine col our at: ) Confocal fl uorescence image of various diamond sampl es with different electron irradiation dosages.Fig. 6 Fluorescence intensity autocorrelation function of a single NV defect at room temperature.phys. stat. sol. (a) 203, No. 13 (2006) 3215 © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheimnoted, that owing to their stability single defect centres in diamond are prime candidates for single pho-ton sources under ambient conditions. Such sources are important for linear optics quantum computing and quantum cryptography. Indeed quantum key distribution has been successful with fluorescence emis-sion from single defect centres [53].A major figure of merit for single photon sources is the signal to background ratio, given (e.g.) by the amplitude of the correlation function at 0τ=. This ratio should be as high as possible to ensure that a single bit of information is encoded in a single photon only. The NV centre has a broad emission range which does not allow efficient filtering of background signals. This is in sharp contrast to the NE8 defect which shows a very narrow, only 1.2 nm wide spectrum. As a consequence the NE8 emission can be filtered out efficiently [47]. The correlation function resembles the one from the NV centre. Indeed the photophysical parameters of the NV and NE8 are similar, yet under comparable experimental conditions the NE8 shows an order of magnitude improvement in signal-to-background ratio because of the nar-rower emission range.Besides application in single photon generation, photon statistical measurements also allow to derive conclusions on photoionization and photochromism of single defects. Most notably the NV centre is speculated to exist in two charge forms, the negatively charged NV with zero phonon absorption at 637 nm and the neutral from NV 0 with absorption around 575 nm [20, 54]. Although evidence existed that both absorption lines stem from the same defect no direct charge interconversion has been shown in bulk experiments. The best example for a spectroscopically resolved charge transfer in diamond is the vacancy, which exists in two stable charge states. In order to observe the charge transfer from NV to NV 0 photon statistical measurements similar to the ones described have been carried out, except for a splitting of photons depending on the emission wavelength [55]. This two channel set up allows to detect the emission of NV 0 in one and NV in another detector arm. Figure 8 shows the experimental result. For delay time 20,()g ττ= shows a dip, indicating the sub-Poissonian statistics of the light emitted. It should -300-200-10001002000,00,40,8g (2)(τ)τ,nsPhotonsDichroicBSStartNV 0Stop NV - Fig. 8 (online colour at: ) Fluorescence cross correlation function between the NV 0 and NV emission of a single defect.。

Cell Host&MicrobeArticleHIV-1Integrase Variants Retarget Viral Integration and Are Associated with Disease Progression in a Chronic Infection CohortJonas Demeulemeester,1,2Sofie Vets,1Rik Schrijvers,1Paradise Madlala,1,3Marc De Maeyer,2Jan De Rijck,1 Thumbi Ndung’u,3,4,5,6Zeger Debyser,1,7,*and Rik Gijsbers1,7,*1Laboratory for Molecular Virology and Gene Therapy,Department of Pharmaceutical and Pharmacological Sciences2Laboratory for Biomolecular Modeling,Department of ChemistryKU Leuven–University of Leuven,3000Leuven,Belgium3HIV Pathogenesis Programme,Doris Duke Medical Research Institute4KwaZulu-Natal Research Institute for Tuberculosis and HIVNelson R.Mandela School of Medicine,University of KwaZulu-Natal,4013Durban,South Africa5Ragon Institute of MGH,MIT and Harvard,Cambridge,MA02139,USA6Max Planck Institute for Infection Biology,Chariteplatz,10117Berlin,Germany7Co-senior author*Correspondence:zeger.debyser@med.kuleuven.be(Z.D.),rik.gijsbers@med.kuleuven.be(R.G.)/10.1016/j.chom.2014.09.016SUMMARYDistinct integration patterns of different retroviruses, including HIV-1,have puzzled virologists for over 20years.A tetramer of the viral integrase(IN)assem-bles on the two viral cDNA ends,docks onto the target DNA(tDNA),and catalyzes viral genome inser-tion into the host chromatin.We identified the amino acids in HIV-1IN that directly contact tDNA bases and affect local integration site sequence selection. These residues also determine the propensity of the virus to integrate intoflexible tDNA sequences. Remarkably,natural polymorphisms IN S119G and IN R231G retarget viral integration away from gene-dense regions.Precisely these variants were associ-ated with rapid disease progression in a chronic HIV-1subtype C infection cohort.Thesefindings link integration site selection to virulence and viral evolution,but also to the host immune response and antiretroviral therapy,since HIV-1IN119is under selection by HLA alleles and integrase inhibitors. INTRODUCTIONThe retroviral life cycle is characterized by a key integration step in which a DNA copy of the viral RNA genome is inserted into the host chromatin.In the past decade,significant progress has been made in understanding this process.Massive integration site sequencing revealed that different retroviruses show distinct preferences with regard to the chromatin environment.Lentiviral integrase(IN)co-opts the cellular chromatin reader lens epithe-lium-derived growth factor/p75(LEDGF/p75;PSIP1)(Ciuffiet al.,2005)to target integration into active genes.Gammaretro-viral integration,on the other hand,is enriched near strong en-hancers and transcription start sites by an interaction between IN and bromodomain and extraterminal domain(BET)proteins (BRD2,BRD3,and BRD4;BRD2–4)(De Rijck et al.,2013;Gupta et al.,2013;Sharma et al.,2013).It is,however,the viral inta-some,a tetramer of IN assembled on the two viral cDNA ends, that catalyzes thefinal DNA cutting and joining reactions and thus determines the precise position of integration.Before strand transfer can proceed,the intasome has to dock onto target(nucleosomal)DNA(tDNA),forming the target capture complex(TCC)(Maertens et al.,2010).Molecular recognition be-tween intasome and tDNA imposes specific constraints on the implicated sequence.Already in2001,patient-derived viral INs were employed to propose that residue IN119plays a role in target site selection on naked DNA templates in vitro(Harper et al.,2001).However, a mechanistic basis for these observations was lacking.The tools for this would only become available in2010,when crystal structure of the prototype foamy virus(PFV)intasome TCC was reported(Maertens et al.,2010).This model provides insight into the retroviral integration machinery and suggests how the in-tasome recognizes and docks onto tDNA.Recently,this struc-ture was used to show that central tDNA distortion in the HIV-1 TCC is spread out over two overlapping dinucleotides surround-ing the integration site palindrome dyad axis(Serrao et al.,2014). Additionally,the authors demonstrated that residue S119in HIV-1IN is indeed analogous to A188in PFV IN,and that IN S119A and IN S119T substitutions can alter target site selection of recombi-nant IN protein into naked DNA substrates in vitro.The effect of tDNA contacting amino acids(aa)on integration site selection in a viral context,however,remains largely unstudied.In this work,we combined structural information on the PFV TCC with conservation in retroviral IN protein alignments to determine aa-tDNA base contacts.We generated HIV-1variants based on the observed variability at these positions,as-sessed replication capacities,and performed integration site sequencing to reveal their integration preferences.Altered mo-lecular recognition in the TCC underlying distinct local tDNA nucleotide biases was extrapolated to other retroviral genera. Finally,we examined the global integration profile of thesevariants and their effect on disease progression in a chronic HIV-1infection cohort.RESULTSS119and R231of HIV-1IN Directly Contact tDNA Bases In the PFV,TCC interactions between IN and tDNA are extensive.Most,however,involve the DNA phosphodiester backbone and do not directly impose sequence specificity (Maertens et al.,2010).Homology modeling of the HIV-1TCC based on the PFV structure predicts only two sites of direct aa-tDNA base contact in HIV-1IN:S119and R231.IN S119(IN A188in PFV)is located in the catalytic core domain (CCD)a 2310-helix,and its side chain directly projects into the tDNA minor groove,contacting bases at positions À2and À3on one strand and 6and 7on the com-plementary,as counted from the sites of strand transfer (Fig-ure 1A).IN R231,the HIV-1equivalent of PFV IN R329,reaches out from the C-terminal domain (CTD)b 1/b 2-loop into the tDNA ma-jor groove,where it interacts with bases 0and À1on one strand and 4and 5on the complementary (Figure 1A).This interaction is believed to contribute to tDNA deformation (Maertens et al.,2010).Although IN S119and IN R231are the only two sites of direct tDNA base recognition in HIV-1IN,modeling suggests that addi-tional contacts can be introduced.While aa side chains of IN 120-121face away from the tDNA minor groove and may affect tDNA contacts indirectly at best,IN T122(IN T191in PFV)points toward the tDNA again.HIV-1IN T122is still in close proximity to tDNA bases,and a long,flexible aa (e.g.,Lys)at this position could represent another handle on the tDNA.As HIV-1IN aa 119,122,and 231are in direct contact with tDNA bases,we reasoned that substitutions at these positions would result in novel molecular interaction patterns between the intasome and tDNA,in turn giving rise to altered local integra-tion site preferences.IN Amino Acids Contacting tDNA Bases Vary among LentivirusesIn a breadth-first approach to assess conservation at these IN positions,we aligned lentiviral (and retroviral)Pol protein se-quences obtained from UniProt (Figures 1B and S1available on-line).Inspection of the alignment in and around the CCD a 2helix shows overall conservation of this region.In agreement with the limited amount of space in the tDNA minor groove,positionsFigure 1.IN Residues Contacting tDNA Bases(A)Cartoon of the PFV intasome target capture complex.Boxes on either side zoom in on the aa-tDNA base contacts in the HIV-1intasome model.Only the IN CCD a 2helix containing residues S119and T122,and part of the CTD b 1/b 2loop harboring R231,are shown.Proximal tDNA bases are depicted and numbered starting from the sites of strand transfer on both strands.(B)The IN CCD a 2helix and CTD b 1/b 2regions from a lentiviral sequence alignment are matched with the PFV IN sequence.Numbering refers to the (Gag)-Pol polyprotein context.Secondary structures and Mg 2+cofactor binding are indicated above,and HIV-1positions 119,122,and 231below,the PFV sequence.Accession:PFV (P14350);HIV-1(P03367);HIV-2(P04584);SIV,simian immunodeficiency virus (Q8AII1);FIV,feline immunodeficiency virus (P31822);BIV,bovine immunodeficiency virus (P19560);JDV,Jembrana disease virus (Q82851);EIAV,equine infectious anemia virus (P32542);CAEV,caprine arthritis encephalitis virus (P33459);OMVV,ovine maedi visna-related virus (P16901);MVV,maedi visna virus (P03370).See also Figure S1.Cell Host &MicrobeHIV-1Integrase Variants Retarget Integrationequivalent to HIV-1IN S119are occupied by small aa (Pro,Ala,Ser,Thr).Positions corresponding to HIV-1IN 122also harbor substantial variation.Interestingly,feline immunodeficiency virus (FIV)encodes a Lys residue at this position,which may directly contact tDNA bases.More divergence is present in the CTD b 1/b 2-loop;next to substitutions,insertions and deletions are also present,compli-cating the retroviral alignment (Figures 1B and S1).In accor-dance with a possible role in tDNA binding,conservation is higher again at positions corresponding to HIV-1IN 231.Only equine infectious anemia virus (EIAV)does not encode a basic Lys or Arg tether,but a Gly instead.Amino Acids Contacting tDNA Bases Can Be Varied without Compromising Viral FitnessWe next wondered how introduction of the observed aa into HIV-1would affect the virus.Therefore,substitutions at IN positions 119,122,and 231were introduced into multiple-round HIV-1NL4-3and in the single-round firefly luciferase reporter version,HIV-1NL4-3-fLuc.At position IN 119we generated S119A/P/T sub-stitutions (Table S1).As previously observed,a long and flexible aa at position IN 122may directly contact tDNA bases.As Lys was observed at the equivalent position in FIV IN (FIV IN K124),we introduced a T122K substitution (Figures 1B and S1;Table S1).At the third tDNA base contact point,HIV-1IN 231,we investi-gated R231K and R231G substitutions (Table S1).As a control for our model and to corroborate its tethering role in HIV-1,we also included an IN R231E substitution,in analogy to the previously reported recombinant PFV IN R329E (Maertens et al.,2010).Single-round HIV-1NL4-3-fLuc viruses carrying the different IN variants were produced,and their transduction efficiency was assessed in SupT1and HeLa P4cells (Figures 2A and S2A,respectively).Viral variants IN S119A/P/T and IN T122K maintained wild-type (WT)transduction efficiency.Alterations at position IN 231were tolerated as well;a conservative IN R231K substitution resulted in WT activity,while removal of this basic tether in the IN R231G virus resulted in a trend toward lower transduction effi-ciency (88.3%±0.3%of WT).However,inversion of the charge at this position (IN R231E )severely hampered transduction (3.1%±0.7%of WT,p <0.01,t test).For comparison,we included HIV-1NL4-3-fLuc virus containing the enzymatically dead IN catalytic triad mutant D64N/D116N/E152Q.This mutant exhibited a trans-duction efficiency 3–4log lower than WT virus (data not shown).To confirm their activity,we determined the replication capac-ity of the HIV-1NL4-3IN variants in a multiple-round viral break-through assay in MT-4cells and peripheral blood mononuclear cells (PBMCs).Multiple-round replication was monitored by measuring viral p24production in the supernatant.All three IN S119mutant viruses exhibited growth kinetics comparable to WT HIV-1NL4-3corroborating the results of the fLuc assay (Fig-ures 2B and 2C).The IN R231G/K variants also replicated similarly to WT,while IN T122K virus lagged behind (Figures S2B and S2C).As expected,IN R231E virus did not replicate on either MT-4cells or PBMCs.These results indicate that substitutions of tDNA base-con-tacting aa in IN do not necessarily affect the viral transduction efficiency or fitness.Altering HIV-1Intasome tDNA Contacts Locally Retargets Viral IntegrationRetroviral INs show weak,but discernable,target sequence specificity at the site of integration.To assess whether the above-mentioned substitutions alter the local integration site preferences,we infected SupT1and HeLa P4cells with the respective HIV-1NL4-3viruses and employed 454technology to sequence proviral integration sites.A total of 36,264distinct sites were retrieved,31,615in SupT1(Table S2)and 4,649in HeLa P4cells (data not shown).The 15base pair (bp)genomic DNA sequence surrounding the integration site roughly corresponds to the intasome footprint.Sequence conservation and relative base frequencies in this region were evaluated using sequence logos.Figure 3A shows the local,palindromic integration site sequence logo for WT HIV-1NL4-3(IN S119-T122-R231).Most notable is the bias against thymine at the site of strand transfer (position 0)and a corresponding symmetrical bias against adenine at po-sition 4(the point of strand transfer on the complementary strand).The intasome structure suggests that a thymine C5-methyl is incompatible with the transesterification reaction (Maertens et al.,2010).Further away from the palindromedyadFigure 2.Replication of tDNA Contact Variant Viruses(A)Transduction efficiency as measured by fLuc reporter activity (relative light units,RLUs/m g protein)3days postinfection of SupT1cells for the indicated HIV-1NL4-3-fLuc carrying IN mutants.Activity is reported as percentage of HIV-1NL4-3-fLuc carrying WT IN.Data represent averages and SDs from triplicate mea-surements.Significant deviations from WT are indicated (*p <0.01,t test).(B and C)Viral breakthrough experiments of HIV-1NL4-3WT and IN S119A/P/T viruses (colored as in A)in MT-4cells (B)and PBMCs (C).See also Figure S2.Cell Host &MicrobeHIV-1Integrase Variants Retarget Integrationaxis,at positions À2,À3on the plus and 6,7on the minus strand,are the bases contacting IN 119(Figures 3A and S3A).WT HIV-1NL4-3IN codes for Ser at this position,inducing a small predilec-tion for thymine at position À3through a weak hydrogen bonding interaction between the S119hydroxyl and the complementary adenine N3at position 7(Figure S3A).G:C bp are disfavored at position À3,as the presence of a guanine C2amino group in the minor groove would sterically hinder the Ser sidechain.Figure 3.Integration Site Sequence Logos of HIV-1NL4-3IN VariantsSequence conservation is indicated as the total height of each stack (measured in bits),while the relative height of bases in a stack reflects base frequencies at that position.Arrows on the HIV NL4-3WT logo (A)indicate the sites of strand transfer (position 0)on the plus (black)and minus (gray)target DNA strands.On top is a B-DNA cartoon of the tDNA and aa-base contacts where positions directly correspond to the sequence logo below.(B–I)Integration site sequence logos for the indicated HIV-1NL4-3variants.Positions showing an altered base distribution compared to WT HIV-1NL4-3are colored (*p <10À4,c 2test).(J)Integration site sequence logo for EIAV.See also Figures S3and S4.Cell Host &MicrobeHIV-1Integrase Variants Retarget IntegrationIN S119A substitution in HIV-1NL4-3,as observed in SIV and HIV-2IN,resulted in stronger nucleotide biases(Figure3B).c2ana-lyses comparing base distributions at each position to those observed for WT HIV-1NL4-3indicate significantly different prefer-ences at positionsÀ4,À3,À2,and symmetrically6and7(p< 10À4).The smaller Ala allows the CCD a2helix to approach the tDNA bases more closely.Van der Waals interactions with the methyl side chain are established,preferably involving a guanine base at positionÀ3and a complementary cytosine at7(Fig-ure S3B).Modeling also suggests a polar contact between the Ala backbone NH and the neighboringÀ2thymine C2carbonyl, or adenine N3.This interaction is not possible with a G:C bp due to its bulky guanine C2amino group in the minor groove,explain-ing the bias for A:T bp at this position.IN S119P substitution gives rise to small additional sequence biases at positionsÀ4and complementary8,suggesting that contacts may be established with these bases as well(Figures 3C and S3C).Preferences distinct from those of WT HIV-1NL4-3 are also observed at positionsÀ2,À3and6,7.Pro residues are frequently observed to make extensive ring-stacking interac-tions with adenine,and to a lesser extent,thymine(Luscombe et al.,2001),rationalizing the novel predilections.Due to the more optimal van der Waals interactions,IN S119P virus generally prefersÀ3R:Y7bp toÀ3Y:R7bp(International Union of Biochemistry and Molecular Biology base notation). Increasing the volume of the IN S119side chain by substitution with Thr,as present in EIAV,results in a strong preference for A:T bp at positionsÀ3and7,while leaving the remainder of the logo unaffected(Figure3D).Our model suggests that the IN S119T side chain can engage either a thymine C2carbonyl or adenine N3in a hydrogen bonding interaction(Figure S3D),whereas the addi-tional minor groove bulk associated with a G:C bp is sterically disfavored.Sequence logo analysis of HIV-1NL4-3IN T122K integration sites shows a decreased bias at positionÀ3(and7)compared to WT (Figure3E),suggesting that additional tDNA contacts can be introduced at IN122.While we cannot exclude the possibility that the IN T122K substitution causes a slight shift of the CCD a2 helix,altering interactions with IN S119,the observed loss of bias likely reflects the broad hydrogen bonding capacities of Lys with minor groove bases(Figure S3E)(Luscombe et al.,2001). As expected,the conservative IN R231K substitution,occurring in several lentiviral INs,does not alter the local integration site preferences(Figure3F).Both the WT Arg(Figure S3F)and the variant Lys side chain can contact tDNA bases at positionsÀ1, 0and4,5to balance the energetic penalty accompanying the strong central tDNA bending(Figure S3G).Both aa are relatively promiscuous in establishing hydrogen bonding interactions with major groove bp and do not impose overly specific sequence re-quirements(Luscombe et al.,2001;Maertens et al.,2010). Analysis of IN R231G virus integration sites reveals that sequence biases at positionsÀ1,0and complementary4,5 are reduced(Figure3G),with significant decreases(>five SDs) in the proportion of guanine at the former two positions and of cytosine at the latter.Both the WT IN R231and the variant IN R231K side chains will optimally engage a position0guanine base in a bidentate hydrogen bonding interaction(Figures S3F and S3G)(Luscombe et al.,2001).The IN R231G virus,however, is stripped of this interaction(Figure S3H).Simultaneously,increased preferences for thymine and adenine are observed at positions1and3,respectively,hinting at an increased fre-quency offlexible TAA,AAA,AAT trinucleotides at the integration site center(positions1–3;Figure3G)(Gabrielian and Pongor, 1996;Satchwell et al.,1986).To balance the energetic penalty for tDNA bending,integration into moreflexible sequences may be required in the absence of a central Arg or Lys tether. The IN R231E virus integration site sequence logo showed an increased bias toward cytosine at the site of strand transfer(Fig-ure3H).In this case a novel hydrogen bonding interaction can be established between the Glu side chain and a cytosine C4-amino group(Figure S3I)(Luscombe et al.,2001).An increased bias for cytosine was observed for PFV IN R329E-mediated integration in vitro(Maertens et al.,2010).However,in this context,the bias appeared at the base just50to the site of strand transfer. In conclusion,substitutions at positions IN119,IN122,and IN231 result in local retargeting of HIV-1integration.HIV-1NL4-3IN S119T-R231G Mimics EIAV Local Integration Site PreferencesDifferent retroviruses exhibit distinct nucleotide preferences at their local integration site.Introduction of single aa substitutions in HIV-1NL4-3IN allows mimicry of local preferences of the respective paring positionsÀ4toÀ2in HIV-1NL4-3 IN S119P and FIV(Figures3C and S4B)reveals preferences for the same nucleotides(W,R,and T atÀ4,À3,andÀ2,respec-tively).Likewise,biases toward R and W at respective positions À3andÀ2for HIV-1NL4-3IN S119A are also observed for HIV-2and SIV(Figures3B,S4C,and S4D).To assess whether the same molecular interaction patterns govern local integration site selec-tion for lentiviruses,and by extension,retroviruses,we attemp-ted to shift the HIV-1local integration site preference toward that of EIAV(Figure3J).EIAV IN contains T119,V122,and G229,matching HIV-1IN positions119,122,and231.Intro-ducing either single aa substitution IN S119T or IN R231G altered the integration site nucleotide preferences of HIV-1NL4-3.As IN V122is unlikely to interact directly with tDNA,we created an HIV-1NL4-3IN S119T-R231G double mutant.Sequence logo analysis of viral integration sites in SupT1cells reflects a merger between the effects observed for the single IN substitutions:a strong bias toward A:T bp at positionsÀ3and7and increases in their pres-ence around the palindrome dyad axis(Figure3I).Of note,the central base itself(position2)also exhibits a significantly height-ened bias toward A:T bp,which was not observed for the IN S119T or IN R231G viruses(Figures3D and3G).Overall,integration site base preferences of the HIV-1NL4-3IN S119T-R231G virus are similar to those of EIAV,indicating close mimicry of the tDNA base inter-actions in the EIAV intasome.Patient-Derived HIV Sequences Reveal Further Polymorphisms at IN119Complementary to our breadth-first approach evaluating IN se-quences of different lenti-and retroviruses,we exploited the large number of patient-derived HIV IN sequences contained in the Los Alamos HIV Sequence Database to obtain an in-depth view on the variability at tDNA base contact positions.Figure4A summarizes the sequence conservation and aa frequencies at positions IN119,IN122,and IN231for2,276 aligned HIV-1sequences.Interestingly,all aa observed in otherCell Host&MicrobeHIV-1Integrase Variants Retarget Integrationlentiviruses occur as natural polymorphisms in HIV-1.Additional substitutions to Gly or Arg and less frequently to Lys or Ile are found at IN 119.HIV-1IN 122allows less diversity,with Thr occur-ring most frequently,followed by Ile,Val,and Ala.The strongest conservation is found at position IN 231,where the entropy ap-proaches four bits;Arg is preferred,followed by Lys,and occa-sionally Gly.Of note,we also split up the results with respect to the different HIV-1groups and subtypes.Distinct aa frequencies exist at IN 119when comparing viral groups,subtypes,and circulating recom-binant forms (Figure S5).Regardless of the higher conservation,similar observations could be made for IN positions 122and 231.In particular,IN R231K was found among subtype B only,while IN R231G substitution was unique for subtype C,although the limited number of sequences may preclude finding of these var-iants among other subtypes.Analysis of 72HIV-2sequences is shown in Figure 4B.Aside from the highly frequent Ala at position IN 119,Pro,Ser,and Val are also encountered.At position IN 122,Thr and Ala are found with nearly equal probabilities.Arg is the only aa found at HIV-2IN 231.Indubitably,the smaller sample size plays a role in this finding.Nevertheless,it further underscores the contribution of IN R231to tDNA binding.Based on these results,we generated and characterized additional single-round HIV-1NL4-3-fLuc and multiple-round HIV-1NL4-3IN S119G/R/K/V/I variants.Transduction efficiency in SupT1and HeLa P4cells revealed that IN S119G/R/K viruses main-tained WT transduction efficiency (Figures 4C and S2D,respec-tively).Substitution by the more inflexible,bulky Val and Ile at this position caused a 2-and 30-fold reduction in fLuc signal,respec-tively (p <0.01,t test).Clinically relevant IN S119G/R variantsexhibited growth kinetics similar to WT virus in a multiple-round infection assay in MT-4cells and PBMCs (Figures 4D and 4E).IN S119K virus also replicated similarly to WT,while the IN S119I variant did not break through,and IN S119V virus exhibited an in-termediate phenotype (Figures S2E and S2F).Naturally Occurring HIV-1IN 119Variants Exhibit Distinct Local Integration PreferencesIf a close approach between the CCD a 2helix and tDNA allows the formation of additional polar contacts as suggested for HIV-1NL4-3IN S119A ,then the IN S119G variant should demonstrate this more clearly.Indeed,integration site analysis shows an increased preference for thymine at position À2(Figure 5B).Additionally,a second weak electrostatic interaction is estab-lished in this case,between the polar Gly C a -H and the C2carbonyl group of a pyrimidine base (Figure S3J).Together with increased van der Waals contacts with a À3G:C 7bp,this likely results in the observed preferences at nucleotide À3.The close approach allows contacts to be established at posi-tions À4and 8as well,giving rise to clear nucleotide biases.Base distributions at the sites of strand transfer are distinct from WT.Biases against thymine at position 0and the comple-mentary adenine at 4are alleviated (>five SDs).Slight changes in the overall tDNA binding mode permitted by the close fit be-tween the IN S119G CCD a 2helix and the tDNA minor groove may be enough to reduce clashes with the thymine C5-methyl during transesterification.A trend toward this effect was also observed for the IN S119A virus (p values of 0.0017and 0.024for positions 0and 4,respectively).Comparing sequence logos for the IN S119V (Figure 5C)and IN S119T viruses underscores the value of the hydrogenbondFigure 4.HIV IN tDNA Contacts Variants and Their Replication(A and B)Sequence conservation (indicated in bits)and aa frequencies of (A)2,276aligned HIV-1and (B)72HIV-2sequences at positions IN 119,IN 122,and IN 231.(C)Transduction efficiency as measured by fLuc reporter activity (relative light units,RLUs/m g protein)3days postinfection of SupT1cells for the indicated HIV-1NL4-3-fLuc single-round viruses.fLuc reporter activity is reported as percentage of HIV-1NL4-3-fLuc carrying WT IN.Data represent averages and SDs from triplicate measurements.Significant deviations from WT are indicated (*p <0.01,t test).(D and E)Viral breakthrough experiments of HIV-1NL4-3WT and IN S119G/R viruses,colored as in (C),on MT-4cells (D)and PBMCs (E).See also Figures S2and S5.Cell Host &MicrobeHIV-1Integrase Variants Retarget Integrationwith Thr as well as the limitations for steric bulk at this site.HIV-1NL4-3-fLuc IN S119V virus indeed showed a 2-fold reduction in infectivity.With the loss of the hydrogen bonding potential,the selectivity for A:T bp at positions À3and 7is weakened.Howev-er,the slightly larger Val side chain also establishes contacts around this position (Figure S3K).Increased preferences for the smaller A:T bp are apparent at positions À4to À2(and 6–8)at the cost of the more bulky G:C.Further increase in the aa bulk in the IN S119I mutant produced a considerably crippled virus.As a result,we retrieved insufficient canonical integration sites (n =114)to draw solid conclusions on its nucleotide biases (Figure 5D).The IN S119K virus biases against G:C bp at positions À2,À3(and 6,7)of the viral integration site (Figure 5E),highlighting the space factor,but also suggesting the establishment of hydrogen bonding between the Lys ε-amino group and a thymine C2carbonyl or adenine N3(Figure S3L).The IN S119R variant also exhibits a bias against G:C bp at the same two posi-tions (Figure 5F).Additionally,preferences for thymine at position À2and complementary adenine at 6are apparent.Interactions are also established at positions À4or 8,as a significant prefer-ence for A:T bp appears (Figure S3M).Base distributions at the sites of strand transfer are also affected.It is possible that the size or the interactions of the Arg side chain induce a subtle shift in the conformation of the tDNA,which is propagated to the active sites.IN Amino Acid—tDNA Base Contacts Affect tDNA Flexibility RequirementsIn the intasome,the tDNA major groove is pried open at the center of the integration site while the minor groove is com-pressed,resulting in a partial destacking of the central bp.In the case of HIV-1,which catalyzes strand transfer with a 5bp instead of a 4bp stagger as in PFV,this severe bending can extend over a central trinucleotide instead of a dinucleotide.In order to investigate the effect of tDNA flexibility on our HIV-1NL4-3variants,we grouped the 64possible trinucleotides (32considering base complementarity)into three classes (Gabrie-lian and Pongor,1996;Satchwell et al.,1986):(i)those that pre-fer minor groove compression,(ii)those that are stiff/show no preference,and (iii)those inclined toward major groove compression.Next,the frequencies of these classes were determined and compared to those observed for WT HIV-1NL4-3(Figure 6A).The IN S119I virus was excluded due to its limited data set.As expected,the IN R231G variant shows an increased ten-dency to integrate into minor groove-compressible sequences when compared to WT.This is accompanied by biases against stiff and major groove-compressible trinucleotides.Both the IN R231K and IN R231E viruses did not show altered preferences regarding central tDNA flexibility.The novel interactions of these viruses with tDNA bases may balance the energetic penalty associated with bending.A remarkable correlation emerges between the bias toward minor groove-compressible trinucleotides and steric bulk at po-sition IN 119.While the IN S119G variant shows a preference for more rigid sequences,the IN S119A/P viruses behave more like WT,and the bulkier HIV-1NL4-3IN S119T/V show the strongest bias toward minor groove-compressible trinucleotides.The IN S119K/R viruses,due to their flexible side chains,do not alter the tDNA flexibility requirements for integration.The EIAV mimic,HIV-1NL4-3IN S119T-R231G ,shows the combined effect oftheFigure 5.Integration Site Sequence Logos of HIV-1NL4-3IN Variants(A–F)Local integration sites obtained for (A)HIV-1NL4-3WT and variants (B)IN S119G ,(C)IN S119V ,(D)IN S119I ,(E)IN S119K ,and (F)IN S119R .Positions showing an altered base distribution compared to WT HIV-1NL4-3(A)are colored (*p <10À4,c 2test).See also Figure S3.Cell Host &MicrobeHIV-1Integrase Variants Retarget Integration。

Package‘BRINDA’October16,2022Type PackageTitle Computation of BRINDA Adjusted Micronutrient Biomarkers forInflammationVersion0.1.5Maintainer Hanqi Luo<******************>Description Inflammation can affect many micronutrient biomarkers and can thus lead to incorrect di-agnosis of individuals and to over-or under-estimate the prevalence of deficiency in a popula-tion.Biomarkers Reflecting Inflammation and Nutritional Determinants of Ane-mia(BRINDA)is a multi-agency and multi-country partnership designed to improve the inter-pretation of nutrient biomarkers in settings of inflammation and to generate context-specific esti-mates of risk factors for ane-mia(Suchdev(2016)<doi:10.3945/an.115.010215>).In the past few years,BRINDA pub-lished a series of papers to provide guidance on how to adjust micronutrient biomark-ers,retinol binding protein,serum retinol,serum ferritin by Namaste(2020),soluble transferrin re-ceptor(sTfR),serum zinc,serum and Red Blood Cell(RBC)folate,and serum B-12,using in-flammation markers,alpha-1-acid glycoprotein(AGP)and/or C-Reactive Protein(CRP)by Na-maste(2020)<doi:10.1093/ajcn/nqaa141>,Rohner(2017)<doi:10.3945/ajcn.116.142232>,Mc-Don-ald(2020)<doi:10.1093/ajcn/nqz304>,and Young(2020)<doi:10.1093/ajcn/nqz303>.The BRINDA in-flammation adjustment method mainly focuses on Women of Reproduc-tive Age(WRA)and Preschool-age Children(PSC);however,the general princi-ple of the BRINDA method might apply to other population groups.The BRINDA R pack-age is a user-friendly all-in-one R package that uses a series of functions to imple-ment BRINDA adjustment method,as described above.The BRINDA R pack-age willfirst carry out rigorous checks and provides users guidance to correct data or input er-rors(if they occur)prior to inflammation adjustments.After no errors are detected,the pack-age implements the BRINDA inflammation adjustment for up tofive micronutrient biomark-ers,namely retinol-binding-protein,serum retinol,serum ferritin,sTfR,and serum zinc(when ap-propriate),using inflammation indicators of AGP and/or CRP for various popula-tion groups.Of note,adjustment for serum and RBC folate and serum B-12is not in-cluded in the R package,since evidence shows that no adjustment is needed for these micronutri-ent biomarkers in either WRA or PSC groups(Young(2020)<doi:10.1093/ajcn/nqz303>). BugReports https:///hanqiluo/BRINDA/issuesLicense CC BY4.01URL https:///hanqiluo/BRINDADepends R(>=4.1.0)Imports berryFunctions,data.table,dplyr,Hmisc,rlangSuggests testthat(>=3.0.0)Config/testthat/edition3Encoding UTF-8Language en-USLazyData trueRoxygenNote7.2.1NeedsCompilation noAuthor Hanqi Luo[cre,aut](<https:///0000-0001-6253-5818>),O Yaw Addo[aut](<https:///0000-0003-1269-759X>),Jiaxi Geng[ctb]Repository CRANDate/Publication2022-10-1618:55:20UTCR topics documented:BRINDA (2)sample_data (6)Index8 BRINDA Computation of BRINDA Adjusted Micronutrient Biomarkers for in-flammationDescriptionInflammation can affect many micronutrient biomarkers and can thus lead to incorrect diagnosis of individuals and to over-or under-estimate the prevalence of deficiency in a population.Biomark-ers Reflecting Inflammation and Nutritional Determinants of Anemia(BRINDA)is a multi-agency and multi-country partnership designed to improve the interpretation of nutrient biomarkers in set-tings of inflammation and to generate context-specific estimates of risk factors for anemia(Suchdev (2016)<doi:10.3945/an.115.010215>).In the past few years,BRINDA published a series of pa-pers to provide guidance on how to adjust micronutrient biomarkers,retinol binding protein,serum retinol,serum ferritin by Namaste(2020),soluble transferrin receptor(sTfR),serum zinc,serum and Red Blood Cell(RBC)folate,and serum B-12,using inflammation markers,alpha-1-acid glyco-protein(AGP)and/or C-Reactive Protein(CRP)by Namaste(2020)<doi:10.1093/ajcn/nqaa141>, Rohner(2017)<doi:10.3945/ajcn.116.142232>,McDonald(2020)<doi:10.1093/ajcn/nqz304>,and Young(2020)<doi:10.1093/ajcn/nqz303>.The BRINDA inflammation adjustment method mainly focuses on Women of Reproductive Age(WRA)and Preschool-age Children(PSC);however,the general principle of the BRINDA method might apply to other population groups.The BRINDAR package is a user-friendly all-in-one R package that uses a series of functions to implement BRINDA adjustment method,as described above.The BRINDA R package willfirst carry out rigorous checks and provides users guidance to correct data or input errors(if they occur)prior to inflammation adjustments.After no errors are detected,the package implements the BRINDA inflammation adjustment for up tofive micronutrient biomarkers,namely retinol-binding-protein, serum retinol,serum ferritin,sTfR,and serum zinc(when appropriate),using inflammation in-dicators of AGP and/or CRP for various population groups.Of note,adjustment for serum and RBC folate and serum B-12is not included in the R package,since evidence shows that no adjust-ment is needed for these micronutrient biomarkers in either WRA or PSC groups(Young(2020) <doi:10.1093/ajcn/nqz303>).UsageBRINDA(dataset,retinol_binding_protein_varname,retinol_varname,ferritin_varname,soluble_transferrin_receptor_varname,zinc_varname,crp_varname,agp_varname,population_group,crp_ref_value_manual=NULL,agp_ref_value_manual=NULL,output_format)Argumentsdataset Enter the name of the dataset(should be already loaded in the R environment;micronutrient biomarkers should NOT be log-transformed).retinol_binding_protein_varnameEnter the variable name of retinol binding protein(if available)in your dataset.The variable can be in either international or conventional units.The adjustedvalues in the output dataset will be in the same unit as retinol binding proteinvariable in the input dataset.retinol_varnameEnter the variable name of serum/plasma retinol(if available)in your dataset.The variable can be in either international or conventional units.The adjustedvalues in the output dataset will be in the same unit as retinol variable in theinput dataset.ferritin_varnameEnter the variable name of serum/plasma ferritin(if available)in your dataset.The variable can be in either international or conventional units.The adjustedvalues in the output dataset will be in the same unit as serum ferritin in the inputdataset.soluble_transferrin_receptor_varnameEnter the variable name of serum/plasma soluble transferrin receptor(if avail-able)in your dataset.The variable can be in either international or conventionalunits.The adjusted values in the output dataset will be in the same unit as solu-ble transferrin receptor in the input dataset.zinc_varname Enter the variable name of serum/plasma zinc(if available)in your dataset.The variable can be in either international or conventional units.The adjusted valuesin the output dataset will be in the same unit as serum zinc in the input dataset.crp_varname Enter the variable name of CRP(if available)in your dataset.Unit must be mg/L agp_varname Enter the variable name of AGP(if available)in your dataset(unit must be g/L) population_groupPlease write WRA,PSC,Other,or Manual.The BRINDA R package can onlyanalyze one population group at one time.If users select WRA or PSC,externalCRP/AGP reference values will be used If users select Other,the lowest decileof the CRP and AGP will be calculated and used as CRP and AGP referencevalues If users select Manual as the population group,users can define their ownAGP and CRP reference values for the BRINDA adjustment.crp_ref_value_manualLeave it empty if users select population_group as WRA,PSC,or Other.If usersselect population_group as Manual,and there is a CRP variable in the dataset,enter a user-specified CRP reference valueagp_ref_value_manualLeave it empty if users select population_group as WRA,PSC,or Other If usersselect population_group as Manual,and there is an AGP variable in the dataset,enter a user specified AGP reference valueoutput_format Please write FULL or SIMPLE(SIMPLE by default if users leave it empty).The SIMPLE output only provides users adjusted micronutrient biomarker val-ues.The FULL output provides users all the intermediate parameters for theBRINDA adjustment,such as coefficients of log(AGP)and log(CRP)andassociated standard errors and P values,in addition to adjusted micronutrientbiomarker values.Value‘brinda()‘returns a data frame object that contains additional variables of adjusted micronutrient biomarkers(by default).If users specify output format=full,the output dataset will also include additional variables such as coefficients of regressions of micronutrient biomarkers on AGP and CRP,natural logs of AGP/CRP reference values.Author(s)Hanqi Luo,O.Yaw AddoExamplesdata(sample_data)#Example1#Calculate BRINDA inflammation adjustment values for preschool-age children#(Assuming the data set contains information of preschool-age children) sample_data_adj<-BRINDA(dataset=sample_data,retinol_binding_protein_varname=rbp,retinol_varname=sr,ferritin_varname=sf,soluble_transferrin_receptor_varname=stfr,zinc_varname=zinc,crp_varname=crp,agp_varname=agp,population=Psc,crp_ref_value_manual=,agp_ref_value_manual=,output_format=)#Example2#Calculate BRINDA inflammation adjustment values for non-pregnant women of#reproductive age assuming the sample data set contains information of women #of reproductive age).sample_data_adj2<-BRINDA(dataset=sample_data,retinol_binding_protein_varname=rbp,retinol_varname=sr,ferritin_varname=sf,soluble_transferrin_receptor_varname=stfr,zinc_varname=zinc,crp_varname=,agp_varname=agp,population=WRA,crp_ref_value_manual=,agp_ref_value_manual=,output_format=)#Example3#Calculate BRINDA inflammation adjustment values for other population assuming #the study population is neither women of reproductive age nor preschool-age #childrensample_data_adj3<-BRINDA(dataset=sample_data,retinol_binding_protein_varname=rbp,retinol_varname=sr,ferritin_varname=sf,soluble_transferrin_receptor_varname=stfr,zinc_varname=zinc,crp_varname=crp,agp_varname=,population=OTHER,crp_ref_value_manual=,agp_ref_value_manual=,output_format=FULL)#Example4#Calculate BRINDA inflammation adjustment values for a population when users#would like to apply user-defined CRP and AGP reference valuessample_data_adj4<-BRINDA(dataset=sample_data,retinol_binding_protein_varname=rbp,retinol_varname=sr,ferritin_varname=sf,soluble_transferrin_receptor_varname=stfr,zinc_varname=zinc,crp_varname=crp,agp_varname=agp,population=MANUAL,crp_ref_value_manual=0.2,agp_ref_value_manual=1.4,output_format=FULL)sample_data Micronutrient biomarker datasetDescriptionA biomarker data set that was subset from a cross-sectional survey in Malawi.It provides de-identified information for serum ferritin,soluble transferrin receptor,retinol binding protein,retinol, zinc,C-reactive protein(CRP),Alpha1-acid glycoprotein(AGP)to illustrate the use of the package. Usagedata(sample_data)FormatAn object of class"data.frame"id Unique identification numberssf Serum ferritin,µg/lstfr Soluble transferrin receptor,mg/Lrbp Retinol binding protein,µmol/Lsr Serum retinol,µmol/Lzinc Zinc,µg/Lcrp C-reactive Protein,mg/Lagp Alpha1-acid glycoprotein,g/LReferencesNational Statistical Office(NSO),Community Health Sciences Unit(CHSU)[Malawi],Centers for Disease Control and Prevention(CDC),and Emory University.2017.Malawi Micronutrient Survey 2015-16.Atlanta,GA,USA:NSO,CHSU,CDC,and Emory UniversityIndex∗datasetssample_data,6BRINDA,2sample_data,68。

VLDB201440th International Conference on Very Large Data Bases, Hangzhou, ChinaProceedings of the Array VLDB EndowmentVolume 7, No. 11 – July 2014Proceedings of the 40th International Conference on Very Large Data Bases, Hangzhou, ChinaProgram Chairs and Editors-in-Chief:H. V. Jagadish, Aoying ZhouAssociate Editors – Research and Innovative Systems Tracks:Shivnath Babu, Lei Chen, Graham Cormode, Bin Cui, Wynne Hsu, Martin Kersten,Donald Kossman, Elke Rundensteiner, Kyuseok Shim, Wang-Chiew Tan, Letizia Tanca, Jeffrey YuAssociate Editors – Experiments and Analysis Track:Gao Cong, Jens DittrichAssociate Editors – Vision Track:Zachary IvesProceedings Chairs:Li Xiong, Cong YuPVLDB – Proceedings of the VLDB EndowmentVolume 7, No. 11, July 2014.The 40th International Conference on Very Large Data Bases, Hangzhou, China.Copyright 2014 VLDB EndowmentThis work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visit /licenses/by-nc-nd/3.0/. Obtain permission prior to any use beyond those covered by the license. Contact copyright holder by emailing *************.Volume 7, Number 11, July 2014: VLDB 2014Pages ii - ix and 931 - 1022ISSN 2150-8097Additional copies only online at: , /corr, and TABLE OF CONTENTSFront MatterCopyright Notice (ii)Table of Contents (iii)VLDB 2014 Organization and Review Board (iv)LettersFirst VLDB in Mainland China .................................................................................. L idan Shou ix Research PapersTrekking Through Siberia: Managing Cold Data in a Memory-Optimized Database (931)................................................................... Ahmed Eldawy, Justin Levandoski, Per-Åke Larson The Case for Personal Data-Driven Decision Making .. (943)....................................................................................................................... Jennie Duggan ConfluxDB: Multi-Master Replication for Partitioned Snapshot Isolation Databases . (947)......................................................... Prima Chairunnanda, Khuzaima Daudjee, M. Tamer Ozsu γ-DB: Managing scientific hypotheses as uncertain data .. (959)............................................................................................. Bernardo Goncalves, Fabio PortoIbex — An Intelligent Storage Engine with Support for Advanced SQL Off-loading (963).............................................................................. Louis Woods, Zsolt Istvan, Gustavo AlonsoNOMAD: Nonlocking, stOchastic Multi-machine algorithm for (975)Asynchronous and Decentralized matrix completion .......................................................................................... H yokun Yun, Hsiang-Fu Yu, Cho-Jui Hsieh, S V N Vishwanathan, Inderjit Dhillon Repairing Vertex Labels under Neighborhood Constraints (987)................................................................. Shaoxu Song, Hong Cheng, Jeffrey Xu Yu, Lei ChenProgressive Approach to Relational Entity Resolution (999)........................................................... Y asser Altowim, Dmitri V. Kalashnikov, Sharad MehrotraConcurrent Analytical Query Processing with GPUs (1011)...... K aibo Wang, Kai Zhang, Yuan Yuan, Siyuan Ma, Rubao Lee, Xiaoning Ding, Xiaodong ZhangVLDB 2014 ORGANIZATION AND REVIEW BOARDHonorary ChairYunhe Pan, Chinese Academy of EngineeringGeneral ChairsChun Chen, Zhejiang UniversitySharad Mehrotra, University of California, IrvineProgram Chairs and Editors-in-Chief of PVLDB 7H. V. Jagadish, University of MichiganAoying Zhou, East Normal University, ChinaResearch and Innovative Systems Tracks Associate EditorsShivnath Babu, Duke UniversityLei Chen, Hong Kong University of Science and TechnologyGraham Cormode, University of WarwickBin Cui, Peking University, ChinaWynne Hsu, NUSMartin Kersten, CWIDonald Kossman, ETHElke Rundensteiner, WPIKyuseok Shim, Seoul National UniversityWang-Chiew Tan, University of California, Santa CruzLetizia Tanca, Poli MilanoJeffrey Yu, Chinese University of Hong KongExperiments and Analysis Track Associate EditorsGao Cong, Nanyang Technology UniversityJens Dittrich, SaarlandVisions Track Associate EditorZachary Ives, University of PennsylvaniaIndustrial and Applications Track Associate EditorsUmeshwar Dayal, HPC. Mohan, IBMGe Yu, Northeastern University, ChinaDemonstration ChairsMong-Li Lee, NUSFeifei Li, University of UtahSunil Prabhakar, PurdueTutorial ChairsXiaoyong Du, Renmin UniversityMurat Kantarcioglu, University of Texas, Dallas Divesh Srivastava, AT&T Labs Workshop ChairsAnastasia Ailamaki, EPFLKaushik Chakrabarti, MicrosoftPanel ChairsHakan Hacigumus, NEC Labs Jignesh Patel, University of Wisconsin Xiaoyang Sean Wang, Fudan UniversityResearch Track Review BoardSibel Adali, Rensselear Polytechnic InstituteFoto Afrati, NTU AthensYanif Ahmad, JHUJose Luis Ambite, ISI - USCWalid Aref, Purdue UniversityClaudia Bauzer Medeiros, University of Campinas Srikanta Bedathur, IIIT DelhiMichael Benedikt, Oxford UniversitySonia Bergamaschi, Universita ModenaLaure Berti-Equille, IRD, FranceLeopoldo Bertossi, Carleton University, Ottawa Subhash Bhalla, University of Aizu, JapanPeter Boncz, CWIAngela Bonifati, University of Lille 1Rajesh Bordawekar, IBM Watson Research Center Omar Boucelma, Aix-Marseille UniversityNico Bruno, Microsoft ResearchAndrea Cali, University of London, Birkbeck College Malu Castellanos, HP LabsBadrish Chandramouli, Microsoft Research Adriane Chapman, MitreGang Chen, Zhejiang UniversityYi Chen, New Jersey Institute of Technology James Cheng, CUHKReynold Cheng, University of Hong Kong Brian Cooper, Google, USAPhilippe Cudré-Mauroux, University of Fribourg Carlo Curino, MITGautam Das, UT Arlington and QCRISudipto Das, Microsoft ResearchAnish Das SarmaAtish Das Sarma, eBay Research LabsKhuzaima Daudjee, University of Waterloo Antonios Deligiannakis, Technical University of Crete Daniel Deutch, Ben Gurion UniversityYanlei Diao, University of Massachusetts Amherst Xin (Luna) Dong, Google, USASameh Elnikety, Microsoft ResearchMohamed Eltabakh, Worcester Polytechnic Institute Ihab F. Ilyas, QCRIHakan Ferhatosmanoglu, Bilkent UniversityAda Wai-Chee Fu, Chinese University of Hong Kong Minos Garofalakis, Technical University of Crete Wolfgang Gatterbauer, Carnegie Mellon University Tingjian Ge, University of Massachussets, LowellBuğra Gedik, Bilkent UniversityRainer Gemulla, Max-Plack-Institut Saarbr點ken Gabriel Ghinita, University of Massachusetts Boston Parke Godfrey, York UniversityLukasz Golab, University of WaterlooSergio Greco, University of CalabriaLe Gruenwald, University of OklahomaGiovanna Guerrini, Universita GenovaKrishna Gummadi, MPI-SWSRahul Gupta, Google ResearchRajeev Gupta, IBM ResearchShyam Gupta, IIT DelhiMarios Hadjielefhteriou, AT&T labsWook-Shin Han, KNU, KoreaKuno Harumi, HP LabsBingsheng He, NTU SingaporeSven Helmer, Free University of Bozen-BolzanoJan Hidders, TUDelftWei Hong, Cisco System Inc.Katja Hose, Aalborg UniversityZi Huang, University of QueenslandJeong-Hyon Hwang, SUNY - AlbanySeung-won Hwang, POSTECH, KoreaStratos Idreos, CWIYoshiharu Ishikawa, Nagoya UniversityZachary Ives, University of PennsylvaniaRicardo Jimenez-Peris, Technical University of Madrid Cheqing Jin, East China Normal University Ruoming Jin, Kent State UniversityAlekh Jindal, Saarland University/MITRyan Johnson, University of TorontoDmitri V Kalashnikov, UC IrvinePanos Kalnis, KAUST, Saudi ArabiaBen Kao, Hong Kong UniversityPanagiotis Karras, Rutgers UniversityYiping Ke, Institute of High Performance Computing Bettina Kemme, McGill UniversityDaniel Kifer, PSUBenny Kimelfeld, IBMHideaki Kimura, Microsoft Jim Gray Systems Lab George Kollios, Boston UniversityChristian König, Microsoft ResearchTim Kraska, Brown University Laks V. S. Lakshmanan, University of British Columbia Mounia Lalmas, Yahoo Inc.Mong-Li Lee, National University of Singapore Wolfgang Lehner, Technische University Dresden Justin Levandoski, Microsoft ResearchChengkai Li, The University of Texas at Arlington Cuiping Li, Renmin University of ChinaFeifei Li, University of UtahGuoliang Li, Tsinghua UniversityJianzhong Li, Harbin Institute of Technology Yunyao Li, IBM AlmadenZhanhuai Li, Northwestern Polytechnical University Dan Lin, Missouri S&T, USAXuemin Lin, University of New South WalesBin Liu, NEC Labs AmericaZiyang Liu, NEC Labs AmericaEric Lo, The Hong Kong Polytechnic University Qiong Luo, HKUSTShuai Ma, Beihang UniversityAshwin Machanavajjhala, Duke UniversityBrad Malin, Duke UniversityNikos Mamoulis, University of Hong KongStefan Manegold, CWIMurali Mani, University of MichiganIoana Manolescu, INRIA, FranceAmélie Marian, Rutgers UniversityVolker Markl, TU BerlinMarta Mattoso, Federal University of Rio de Janeiro Frank McSherry, MicrosoftAlexandra Meliou, Umass AmherstMarco Mesiti, University of MilanoDan Miranker, The University of Texas at Austin Mohamed Mokbel, University of MinnesotaBongki Moon, Seoul National UniversityYasuhiko Morimoto, Hiroshima UniversityMirella Moro, Universidade Federal de Minas Gerais Kyriakos Mouratidis, SMU, SingaporeKarin Murthy, IBM IndiaArnab Nandi, Ohio State UniversityWolfgang Nejdl, University of HannoverThomas Neumann, Technology University Munchen Boris Novikov, St Petersburg UniversityDan Olteanu, Oxford UniversityGultekin Ozsoyoglu, Case Western Reserve University Tamer Ozsu, University of WaterlooEsther Pacitti, University of MontpellierThemis Palpanas, University of TrentoIppokratis Pandis, IBM AlmadenStelios Paparizos, Microsoft ResearchAditya Parameswaran, Stanford University Srinivasan Parthasarathy, The Ohio State University Jignesh Patel, University of WisconsinAndrew Pavlo, Brown UniversityPeter Pietzuch, Imperial College LondonNeoklis Polyzotis, University of California - Santa Cruz Cecilia M. Procopiuc, AT&T LabsLi Qian, University of MichiganJorge Quiané-Ruiz, QCRIElisa Quintarelli, Politecnico di MilanoMaya Ramanath, IIT DelhiLouiqa Raschid, University of MarylandVibhar Rastogi, YahooMatthias Renz, University of MunichKenneth Ross, Columbia UniversitySourav S Bhowmick, NTU, SingaporeDimitris Sacharidis, IMIS Athena, GreeceKenneth Salem, Univesity of WaterlooMaria Sapino, University of TorinoKai-Uwe Sattler, TU IlmenauMonica Scannapieco, ISTATBernhard Seeger, University of MarburgLidan Shou, Zhejiang UniversityAdam Silberstein, TrifactaLisa Singh, Georgetown UniversityRadu Sion, Stony Brook University Yufei Tao, Chinese University of Hong Kong Nesime Tatbul, ETH ZurichArash Termehchy, Oregon State University Evimaria Terzi, University of BostonMartin Theobald, Max Planck Institute, Germany Srikanta Tirthapura, Iowa State University Riccardo Torlone, Roma Tre UniversityAnthony Tung, National University of Singapore Kostas Tzoumas, Technical University of Berlin Sergei Vassilvitskii, Google ResearchMarcos Vaz Salles, University of Copenhagen (DIKU) Stratis Viglas, University of EdinburghHoang Tam Vo, National University of Singapore Daisy Zhe Wang, University of FloridaHaixun Wang, Microsoft Research AsiaKe Wang, Simon Fraser UniversityWei Wang, University of New South Wales Xiaoling Wang, East China Normal University Ingmar Weber, YahooRaymond Chi Wing Wong, HKUSTSai Wu, Zhejiang UniversityYuqing Wu, Indiana UniversityXiaokui Xiao, NTUDong Xin, GoogleJianliang Xu, Hong Kong Baptist UniversityJun (Jim) Xu, Georgia Institute of Technology Xifeng Yan, University of Santa BarbaraXiaoyan Yang, Advanced Digital Science CenterKe Yi, HKUSTGe Yu, Northeastern University, ChinaHwanjo Yu, POSTECH, KoreaMeihui Zhang, National University of Singapore Wenjie Zhang, The University of New South Wales Ying Zhang, The University of New South Wales Zhenjie Zhang, Advanced Digital Science Center Wenzhao Zhou, Georgetown UniversityXiaofang Zhou, University of QueenslandPhD Workshop ChairsErich Neuhold, University of Vienna Yunyao Li, IBM Proceedings ChairsLi Xiong, Emory University Cong Yu, Google ResearchSponsorship ChairsMike Carey, University of California, Irvine Lizhu Zhou, Tsinghua UniversityLocal Organization ChairLidan Shou, Zhejiang UniversityWeb Management ChairSai Wu, Zhejiang UniversityConference and Registration Chairs Ke Chen, Zhejiang UniversityCuiping Li, Renmin UniversityPublicity ChairsVasilis Vassalos, AUEB, GreeceDunlu Peng, USST, China Treasury ChairLi (Eric) Qian, University of MichiganVLDB Endowment LiaisonKyu-Young Whang, KAISTPVLDB Managing EditorDivesh Srivastava, AT&T LabsPVLDB Information DirectorGerald Weber, University of AucklandPVLDB Advisory CommitteePhilip Bernstein, Michael Böhlen, Peter Buneman, Susan Davidson, Z. Meral Ozsoyoglu, S. Sudarshan, Gerhard WeikumLogo DesignGuanmin GuoFIRST VLDB IN MAINLAND CHINAAs the Local Organization Chair of this year's VLDB, I would like to take this opportunity to introduce toyou the wonderful city of Hangzhou, which is to host the upcoming VLDB 2014. Located at the southern tip of the Yangtze River Delta, Hangzhou is the capital of the highly developed Zhejiang province ineastern China. Having a culture-rich history dating back to two centuries B.C., Hangzhou boasts of its influential position in ancient poetry, textile design, calligraphy, and other numerous forms of traditional Chinese arts. Besides all these man-made wonders, the city is most famous for the natural scenes around the mythical West Lake, a major tourist area surrounded by tens of attractions. While boating on the lake is a must-go tour for most, you may be more excited about a detour into the serenity of the HuPao Ancient Temple, a light hiking at dawn towards the Baochu Pagoda, or cycling at dusk on the Bai Causeway.While maintaining its historic, romantic name of "earthly paradise", Hangzhou is striving to earn the title of "IT paradise" of China today. The city is home to several major players in the Chinese ICT industry. It has also aggregated tens of thousands of young technological entrepreneurs venturing out of its High-Tech District and University Incubators.This year's VLDB is not only a fiesta for the world's database academics and industry, but also a landmark event for the Chinese database community. On behalf of the local organizing team at Zhejiang University, I would like to deliver my heartfelt thanks to all those who have helped and sponsored this event. Please join us in the picturesque city of Hangzhou!Lidan Shou, Zhejiang University, Hangzhou, Zhejiang, ChinaLocal Organization Chair, VLDB 2014。

IMDG CODE UN PACKING CODEThe Un ited Nati ons Committee of Experts on the Tran sportati on of Dan gerous Goods has established a uni form intern ati onal system for ide ntify ing and packag ing Class 3, 4, 5, 6.1, 8 and 9 dan gerous goods for tran sport. These UN requireme nts are found in sta ndard CAN/CGSB 43.150- 97 “ Performa nee Packag ing for the Tran sportation of Dan gerous GoodsHere?s a summary:How the UN Paekagi ng System WorksThe UN Committee has assig ned all dan gerous goods to one of three Pack ing Groups:Pack ing Group I (high dan ger), II (medium dan ger) and III (low dan ger). The list of dan gerous goods and the Pack ing Group for each can be found in the ICAO Tech ni cal In structi ons, the IMDG Code or the TDG Regulati ons. The Committee has also developed what is referred to as ,UN packagi ng?. UN packagi ngs have bee n performa nee tested for their resista nee to drop, stacking and internal pressure, the severity of the test varying with the Packing Group. Each packag ing is marked with a code that in dicates the type of packag ing, Pack ing Group, form (liquid or solid), relative den sity, inner packag in gs, etc. for which the packagi ng was tested by the man ufacturer and can, therefore, be used.It is the shipper?s resp on sibility to select the appropriate packagi ng for dange rous goods. Shippers should become familiar with the code used in the UN package mark. To select a UN packag ing that is suitable for their product, they n eed the follow ing in formati on:* the Pack ing Group for their product,* the compatability of their product with the packag ing material,* the vapour pressure at 55o C or 50 °and the relative density (liquids),* the net mass (solids).The UN Package MarkHere?s a typical UN package mark,1. UN Symbol2. Packagi ng Code:3. Pack ing Group:X -acceptable for Packing Groups I, II and III substancesY -acceptable for Packing Groups II and III substances onlyZ -acceptable for Pack ing Group III substa nces only4. The relative density (liquids. If the relative density isn?t marked, it is considered to be 1.2) or the gross mass in kg (solids) for which the packag ing was tested.5. The hydrostatic test pressure in kPa (liquids) or the letter ,S? meaning the package was tested for solids or inner packagi ngs.6. Year of man ufacture.7. Country code for the country authorizing the allocation of the mark.8. Name or registered symbol of the man ufacturer.9. Tran sport Can ada desig n registrati on n umber.10. Nominal thick ness of the material of con structi on (metal drums onl y); top head/body/bottom headSelecting the Right UN Packaging for the Product1. Single Packagings . Single packagings are constructed of a single component (e.g., steel drums).When order ing packagi ng from a supplier, specify a UN sin gle packagi ng« permitted for the product by Part II of stan dard CAN/CGSB 43.150-97«with a marked Pack ing Group at or above that for the product;・in which the material in con tact with the product is compatible with and impermeable to the product;・with a marked relative den sity at or above that for the product (liquids);・with a marked te st pressure suitable for the product (liquids):* with a marked gross mass that is adequate for the product (solids);* with a nominal thick ness 1.1/0.8/1.1 (top head/body/bottom head) or greater if it?s asteel drum over 150 litres capacity and to be reused in Can ada for dan gerous goods. There are also requireme nts for recon diti oning some steel drums reused for liquidtran sport, see section 18 of sta ndard CAN/CGSB -43.150-97.Packagi ngs may be usedfor products hav ing a form or Pack ing Group differe nt from that in the marking, with in thefollowing limits:-PG I packag ings for liquids may be used for PG II liquids with a relative den sity not exceed ing the greater of 1.8 or [1.5 X the marked relative den sity]*;-PG I packag ings for liquids may be used for PG III liquids with a relative den not exceed ing the greater of 2.7 or [2.25 X the marked relative den sity]*;sity-PG II packagi ngs for liquids may be used for PG III liquids with a relative den sitynot exceed ing the greater of 1.8 or [1.5 X the marked relative den sity]*;-A packagi ng for liquids may be used for solid products if the gross mass (kg)does n?t exceed the packag in g?s capacity (litres) X the marked relative den sity;-PG I packag ings for liquids may be used for PG II solid products if the grossmass (kg) does n?t exceed the packag in g?s capacity (litres) X 1.5 X th e marked relative den sity*;-PG I packag ings for liquids may be used for PG III solid products if the grossmass (kg) does n?t exceed the packag in g?s capacity (litres) X 2.25 X the marked relative den sity*;-PG II packagi ngs for liquids may be used for PG III solid products if the grossmass (kg) does n?t exceed the packag in g?s capacity (litres) X 1.5 X the marked relative den sity*;*packag ings should be capable of withsta nding a 3m high stack ing load at the higher relativeden sity2. Combin ati on Packagings. __ Containers hav ing inner packag ings are called,combination packagings? (e.g., fibreboard box containing bottles):When orderi ng packag ing from a supplier, specify a UN comb in ati on packag ing*o permitted for the product by Part II of stan dard CAN/CGSB 43.15 0-97o with a marked Pack ing Group at or above that for the product;o in which the material in con tact with the product is compatible with andimpermeable to the product;o with a marked gross mass that is adequate for the product and packag ing;o that has been te sted with the inner packagings to be used for the product;A packaging may be used to contain inner packagings that differ from those that were used in thetests, within the following limits:- A lesser number of inner packagings may be shipped in the box if voids are filled and cushioning is maintained.- If a box was tested with several different types of inner packagings, inners from each design may be shipped together in the box.- Inner packagings that are similar in design to the inners that wer e tested with the box (shape, same or smaller openings, similar type of closure, equivalent or smaller size, equivalent or better materials) may be shipped in the box.。