Tmarus Simon, 1875 is a relatively large spider genus, currently includes 227 species distributed worldwide. Fanjing Mountain Nature Reserve is one of China’s most biodiverse regions. However, Tmarus can be regarded as being poorly represented in Fanjing Mountain, with only one species having been recorded so far: T.fanjing Yang & Yu, 2022.Tmarus species were brought to light by those expeditions: T.fanjing Yang & Yu, 2022 and T.circinalis Song & Chai, 1990. T.fanjing is redescribed, based on new material and the female is described and illustrated for the first time. The supplementary micrographs of T.circinalis are given for the first time. The DNA barcodes and a distribution map of both species are provided for future use.Recently, various expeditions to Fanjing Mountain Nature Reserve were carried out by the authors. In this paper, two  Tmarus Simon, 1875 is the second most speciose genus of Thomisidae Sundevall, 1833, with 227 valid species distributed worldwide so far, after Xysticus C. L. Koch, 1835 (293 species), 27 species of which are recorded from China  ; for manThomisidae represents a substantial fraction of southwest China foliage-dwelling spiders , Lysitelesfanjingensis Wang, Gan & Mi, 2020, L.inflatus Song & Chai, 1990, Phartatangi Wang, Mi & Peng, 2016, Phrynarachnemammillata Song, 1990, Strigoplusguizhouensis Song, 1990, Tmarusfanjing Yang & Yu, 2022, Thomisuslabefactus Karsch, 1881 and Xysticuskurilensis Strand, 1907  to redescribe the male and report the female of T.fanjing for the first time; 2) to re-illustrate T.circinalis, based on new material from Mt. Fanjing and give supplementary micrographs; 3) to provide the DNA barcodes and a distribution map of T.fanjing and T.circinalis for future use.Recently various short, but intensive field collections in Fanjing Mountain have been conducted by staff of the Guizhou Normal University and Guizhou Education University. This paper reports our findings on the study of recently-available samples from the area, which revealed a new record species of Fanjing Mountain, Specimens in this study were collected by beating vegetation. Spiders were fixed and preserved in 95% ethanol. Specimens were examined with an Olympus SZX7 stereomicroscope; details were studied with an Olympus CX41 compound microscope. Female epigynes and male palps were examined and illustrated after being dissected. Epigynes were removed and cleared in warm lactic acid before illustration. The vulva was also imaged after being embedded in Arabic gum. Photos were made with a Cannon EOS70D digital camera mounted on an Olympus CX41 compound microscope. The digital images were taken and assembled using Helifocus 3.10.3. software package .T.circinalis in Hubei Province and Chongqing City were originated from ArcGIS . Due to lack of locality coordinates in previous publications, locality coordinates for cGIS see .Tmarusfanjing: YHTHO014, ♂, GenBank ON796486; YHTHO013, ♀, GenBank ON796487. Tmaruscircinalis: YHTHO015, ♂, GenBank OR075896; YHTHO016, ♀, GenBank OR075897.A DNA barcode was also obtained for the species matching. A partial fragment of the mitochondrial cytochrome oxidase subunit I (CO1) gene was amplified and sequenced for one male and one female specimen, respectively, using the primers LCOI1490 (5’-GGTCAACAAATCATAAAGATATTG-3’) and HCOI2198 (5’-TAAACTTCAGGGTGACCAAAAAAT-3’) . For addAll measurements were obtained using an Olympus SZX7 stereomicroscope and given in millimetres. Eye diameters are taken at the widest point. The total body length does not include chelicerae or spinnerets length. Leg lengths are given as total length . Most of the terminologies used in text and figure legends follows All specimens are deposited Museum of Guizhou Normal University, Guiyang, Guizhou, China.Yang & Yu, 202242208F9D-B77B-5D32-BABA-3628DD1444C6Type status:Holotype. Occurrence: recordedBy: Da Wang; Jaiyuan Xin; individualID: YHTHO001; individualCount: 1; sex: 1 male; lifeStage: 1 adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: ON392063GenBank: ; occurrenceID: 7FC42291-EC27-5A7D-9D64-6E9F98746510; Taxon: order: Araneae; family: Thomisidae; genus: Tmarus; specificEpithet: fanjing; taxonRank: species; scientificNameAuthorship: Yang & Yu; taxonomicStatus: accepted; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Jiangkou; locality: Fanjingshan Nature Reserve; verbatimElevation: 755 m; decimalLatitude: 27.87; decimalLongitude: 108.80; Identification: identifiedBy: Jianshuang Zhang; dateIdentified: 12-12-2022; identificationReferences: Yang et al. 2022; Event: samplingProtocol: Beating; samplingEffort: 10 km by foot; year: 2021; month: 4; day: 18; Record Level: basisOfRecord: PreservedSpecimenType status:Other material. Occurrence: recordedBy: Haonan Zhang; individualCount: 2; sex: 1 male, 1 female; lifeStage: 2 adults; behavior: foraging; preparations: whole animal (ETOH); occurrenceID: A38DE598-7685-5DA5-AB10-C63D1031394B; Taxon: order: Araneae; family: Thomisidae; genus: Tmarus; specificEpithet: fanjing; taxonRank: species; scientificNameAuthorship: Yang & Yu; taxonomicStatus: accepted; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Jiangkou; locality: Fanjingshan Nature Reserve; verbatimElevation: 1060 m; decimalLatitude: 27.90; decimalLongitude: 108.58; Identification: identifiedBy: Hao Yu; dateIdentified: 12-12-2022; identificationReferences: Yang et al. 2022; Event: samplingProtocol: Beating; samplingEffort: 10 km by foot; year: 2022; month: 7; day: 19; Record Level: basisOfRecord: PreservedSpecimenType status:Other material. Occurrence: recordedBy: Cheng Wang; Xiaoqi Mi; individualID: YHTHO013, YHTHO014; individualCount: 4; sex: 2 males, 2 females; lifeStage: 4 adults; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: ON796487; ON796486; occurrenceID: EE48AA39-2206-5DA9-8F4B-82BB2DA9B1D3; Taxon: order: Araneae; family: Thomisidae; genus: Tmarus; specificEpithet: fanjing; taxonRank: species; scientificNameAuthorship: Yang & Yu; taxonomicStatus: accepted; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Shiqian; locality: Fodingshan Nature Reserve; verbatimElevation: 858 m; decimalLatitude: 27.36; decimalLongitude: 108.0; Identification: identifiedBy: Cheng Wang; dateIdentified: 12-05-2022; identificationReferences: Yang et al. 2022; Event: samplingProtocol: Beating; samplingEffort: 10 km by foot; year: 2017; month: 4; day: 28; Record Level: basisOfRecord: PreservedSpecimenFemale . Overall body colour is dull brown in ethanol. Total length 6.34; carapace 2.33 long, 2.25 wide; abdomen 4.01 long, 2.23 wide.Chelicerae coloured as ocular area, both margins without teeth. Labium and endites uniformly light brown, endites depressed posteriorly, slightly convergent anteriorly, with dense scopulae on anterior margin; labium nearly diamond-shaped, anterior margin with sparse setae. Sternum yellowish-white, more or less cordiform or shield-shaped, 1.20 long, 0.93 wide.Carapace Fig. A dull brAbdomen Fig. A–C elongLegs uniformly yellowish-white Fig. A and B. Epigyne Fig. C–F. EpigMale .5'TATTTGGAGCGTGATCGGCTATAGTAGGAACTGCTATAAGAGTATTGATTCGAATAGAATTAGGTAATTCAGGAAGACTTTTTGGAAATGATCATTTATATAATGTAATTGTGACTGCTCATGCTTTTGTGATAATTTTTTTTATAGTTATACCTATTTTAATTGGAGGATTTGGTAATTGATTAGTACCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTATTTTTATTATTTATATCTTCTATAGTAGAAATAGGAGTAGGAGCTGGATGAACTGTATATCCACCTTTGGCTTCTAGTTTAGGTCATATAGGGAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCTTCAATTATAGGGGCTGTAAATTTTATTTCTACTATTATTAATATACGAAGAGTAGGAATGACTATAGAAAAGGTGCCTTTATTTGTCTGATCGGTGTTAATTACTGCTATTTTACTTTTATTATCATTACCTGTTTTAGCAGGAGCTATTACTATATTATTAACAGATCGAAATTTTAATACTTCGTTTTTTGACCCTGCTGGTGGAGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTT3' .5'TATTTGGAGCGTGATCGGCTATAGTAGGAACTGCTATAAGAGTATTGATTCGAATAGAATTAGGTAATTCAGGAAGACTTTTTGGAAATGATCATTTATATAATGTAATTGTGACTGCTCATGCTTTTGTGATAATTTTTTTTATAGTTATACCTATTTTAATTGGAGGATTTGGTAATTGATTAGTACCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTATTTTTATTATTTATATCTTCTATAGTAGAAATAGGAGTAGGAGCTGGATGAACTGTATATCCACCTTTGGCTTCTAGTTTAGGTCATATAGGGAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCTTCAATTATAGGGGCTGTAAATTTTATTTCTACTATTATTAATATACGAAGAGTAGGAATGACTATAGAAAAGGTGCCTTTATTTGTCTGATCGGTGTTAATTACTGCTATTTTACTTTTATTATCATTACCTGTTTTAGCAGGAGCTATTACTATATTATTAACAGATCGAAATTTTAATACTTCGTTTTTTGACCCTGCTGGTGGAGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTT3'  . However, T.fanjing can be distinguished from T.piger by the following characters: for the males, embolus apex blunt in T.fanjing ; associatedSequences: GenBank: prepare to upload; occurrenceID: 9E9A0A7F-27B9-5732-81EE-36AD91E7A4E2; Taxon: order: Araneae; family: Thomisidae; genus: Tmarus; specificEpithet: Tmaruscircinalis; scientificNameAuthorship: Song & Chai; taxonomicStatus: accepted; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; municipality: Jiangkou; locality: Fanjingshan Nature Reserve; verbatimElevation: 1025 m; decimalLatitude: 27.98; decimalLongitude: 108.69; Identification: identifiedBy: Hao Yu; dateIdentified: 15-01-2023; identificationReferences: Song and Zhu 1997; Event: samplingProtocol: Beating; samplingEffort: 10 km by foot; year: 2021; month: 7; day: 20; Record Level: basisOfRecord: PreservedSpecimenSee OR075896).5'TATTTGGGGCGTGGTCAGCTATAGTAGGAACTGCTATAAGAGTATTAATTCGAATAGAATTGGGTAATTCAGGAAGACTTCTTGGTAATGATCATTTATATAATGTAATTGTGACTGCTCATGCTTTTGTAATAATTTTTTTTATAGTTATGCCTATTTTAATTGGAGGTTTTGGTAATTGATTAGTACCTTTGATATTAGGAGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTATTTTTATTATTTATATCTTCTATAGTGGAGATAGGAGTAGGGGCTGGGTGAACTGTATATCCACCTTTAGCTTCTAGTTTGGGTCATATAGGAAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCTTCAATTATAGGGGCTGTAAATTTTATTACTACTATTATTAATATACGTAGAGTAGGAATAACTATAGAAAAAGTGCCTTTATTTGTTTGATCAGTGTTAATTACTGCTATTTTACTTTTACTATCATTACCTGTTTTAGCAGGAGCTATTACTATATTATTAACAGATCGAAATTTTAATACATCGTTTTTTGACCCTGCTGGAGGGGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTT3' .5'TATTTGGGGCGTGGTCAGCTATAGTAGGAACTGCTATAAGAGTATTAATTCGAATAGAATTGGGTAATTCAGGAAGACTTCTTGGTAATGATCATTTATATAATGTAATTGTGACTGCTCATGCTTTTGTAATAATTTTTTTTATAGTTATGCCTATTTTAATTGGAGGTTTTGGTAATTGATTAGTACCTTTGATATTGGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTATTTTTATTATTTATATCTTCTATAGTGGAGATAGGAGTAGGGGCTGGGTGAACTGTGTATCCACCTTTAGCTTCTAGTTTGGGTCATATAGGGAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTGGCTGGGGCTTCTTCAATTATAGGGGCTGTAAATTTTATTACTACTATTATTAATATACGTAGAGTAGGAATAACTATAGAAAAAGTGCCTTTATTTGTTTGATCAGTGTTAATTACTGCTATTTTACTTTTACTATCATTACCTGTTTTAGCAGGAGCTATTACTATATTATTGACAGATCGAAATTTTAATACATCGTTTTTTGACCCTGCTGGAGGGGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTT3' , Chongqing City (Xiushan County) and Guizhou Province (Mt. Fanjing), China Fig. .


In contrast, mutation of theC. elegansBRCA1 ortholog,brc-1,or itsbinding partner,brd-1, lead to only mild embryonic lethality. We show that inC. elegans,brc-1andbrd-1embryonic lethality is enhanced when53bp1 ortholog,hsr-9,is also mutated. This is not a consequence of activatingpolq-1-dependent microhomology-mediated end joining, aspolq-1mutation does not suppress embryonic lethality ofhsr-9;brc-1mutants. Together, these results suggest thatBRC-1-BRD-1andHSR-9function in parallel pathways and do not act antagonistically as in mammals.In mice, mutation of In contrast to what has been reported in mice, we observed elevated embryonic lethality inbrc-1andbrd-1null alleles . We found that thehsr-9;brc-1polq-1triple mutant had levels of embryonic lethality similar tobrc-1polq-1but higher thanhsr-9;brc-1, suggesting that the elevated embryonic lethality ofhsr-9;brc-1is not a consequence of activation of MMEJ . On thethality . To detof MMEJ . TherefCRISPR-mediated genome editing:hsr-9(xoe17)andpolq-1(xoe51)alleles were engineered by incorporating the stop-in cassettehsr-9repair template (gattttgcctcttaaataaaatttcagCAAAAAACCGAGGGGAGACTTGCAATAGGGAAGTTTGTCCAGAGCAGAGGTGACTAAGTGATAAGCTAGCTCTCGGATCATCTTGCAAACATGCTTATTGCTGgtaggtattgcaacc) and guide RNA (AGGGGAGACTTGCAATATCT) were injection intoN2and the resulting progeny were analyzed by PCR using TGAAATTAAGGTGGTCACTCGAAG and GTTGTTGTGGGGAGGCTGAA. Thepolq-1repair template (AGAGAATTCTCTGAAGATCCATTAATATTGCTTACCGAAGGGGAAGTTTGTCCAGAGCAGAGGTGACTAAGTGATAAGCTAGCAGAGTTTTCGCCGCAATTCTCAGACTTTGGTAATGATTTC) and guide RNA (ATTGCGGCGAAAACTCTCTT) were injected intoN2and the resulting progeny were analyzed by PCR using ATAGGCAAATGGCTGGACGG and TCAAAGCAGTCTTCTCGGCA. Worms were outcrossed a minimum of three times.Embryonic lethality:L4 hermaphrodites of indicated genotypes were picked onto individual plates and transferred to new plates every 24hr for 3 days. Embryonic lethality was determined by counting eggs and hatched larvae 24hr after removing the hermaphrodite and calculating percent as eggs/(eggs + larvae).Strains:


Ribosome footprint profiling has demonstrated that ribosomes can be slowed or stalled on select mRNAs, often due to the presence of rare codons, stalling motifs, or via a ribosome-binding protein . Stalled ribosomes can act as physical roadblocks for trailing ribosomes and ultimately can cause ribosome collisions that stimulate no-go mRNA decay. Detecting stalled or slowed ribosomes in cells by ribosome footprint profiling or classic polysome profiling is laborious, technically challenging, and low throughput. Here, we present a protocol to assay for stalled ribosomes on in vitro–transcribed reporter mRNAs using a robust, commercially available mammalian in vitro translation lysate and an optimized low-speed sucrose cushion. In short, we take advantage of the ability of puromycin to incorporate into the nascent polypeptide and cause the ribosome to dissociate from the mRNA during active elongation, as well as the ability to selectively pellet ribosomes through a low-speed sucrose cushion due to their large molecular weight. Stalled ribosomes are not actively elongating and do not incorporate puromycin, allowing the ribosome-bound mRNA to pellet in the low-speed sucrose cushion. RT-qPCR is used to quantify the amount of ribosome-bound reporter mRNA in the pellet. This workflow allows for direct assessment of stalled ribosomes and is fully amendable to insertion of putative stalling motifs in the target mRNA, as well as supplementation with recombinant proteins or small molecule inhibitors that target translation elongation.Key featuresThis protocol is optimized for cap-dependent in vitro translation in the dynamic linear range.Details for generating capped reporter mRNA in one day are provided.Requires as little as one day to complete if starting with in vitro–transcribed mRNA.This protocol requires access to an ultracentrifuge and a real-time PCR system. Graphical overviewCaenorhabditis elegans) (Caenorhabditis elegans)  . Elongatelegans) , which uelegans) . In yeaselegans) . Subsequelegans) . Upon laelegans) .Ribosome footprint profiling and classic polysome profiling can be used to detect stalled ribosomes; however, these approaches can be technically challenging, laborious, and rather low throughput. Additionally, ribosome profiling is not cost-effective when testing multiple specific mutations within reporter mRNAs or effector proteins. Here, we present a validated protocol that can be used to assess ribosome stalling in vitro that is medium-to-high throughput and can be performed in as little as one day if starting with in vitro–transcribed mRNA.We take advantage of the selective nature of puromycin, an amino-acyl transfer RNA analog to incorporate into nascent polypeptides of only actively elongating ribosomes . PuromycReagents2-Propanol (isopropanol) 3′-O-Me-m7G(5′)ppp(5′)G RNA Cap Structure Analog  Agarose LE, quick dissolve Bromophenol blue Chloroform, ethanol stabilized 10× CutSmart buffer Cycloheximide (CHX) Dimethyl sulfoxide (DMSO) Dithiothreitol (DTT) DNA Clean & Concentrator-25 kit DNase I (Rnase-free) E. coli Poly(A) polymerase 500 mM EDTA, pH 8.0 ULTROL grade Ethanol, 200 proof 10 mg/mL ethidium bromide Flexi Rabbit Reticulocyte Lysate System 37% (w/v) formaldehyde Glycerol, biotechnology grade Glycogen, molecular biology grade Hi-Di Formamide HiScribe T7 High Yield RNA Synthesis kit iScript Reverse Transcription Supermix iTaq Universal SYBR Green Supermix, 5 mL 2 M KCl, pH 7.4 2 1 M MgClMillennium RNA size marker 10× MOPS buffer Nuclease-free water Puromycin dihydrochloride 6× purple gel loading dye Quick-Load Purple 1 kb Plus DNA ladder RNA Clean & Concentrator-25 kit Rnase inhibitor, murine Sucrose, Ultra-pure, Rnase- & Dnase-free 10× TBE buffer 1 M Tris-HCl, pH 7.4 TRIzol reagent Xylene cyanol FF pcDNA3.1(+)/nLuc-3XFLAG Dual promoter plasmid pCR II pGL4.13 Solutions100 mg/mL cycloheximide (CHX) (see Recipes)10 mg/mL CHX (see Recipes)5 mg/mL CHX (see Recipes)1 M DTT, Cleland’s Reagent (see Recipes)70% (v/v) ethanol (see Recipes)1 mg/mL ethidium bromide (see Recipes)10 mM GTP (see Recipes)1× MOPS buffer (see Recipes)10 mg/mL (~18 mM) puromycin (see Recipes)0.6 mM puromycin (see Recipes)2× ribosome dilution buffer (see Recipes)RNA loading dye (see Recipes)RNA sample buffer (see Recipes)60% (w/v) sucrose (see Recipes)35% (w/v) sucrose, buffered (see Recipes)1× TBE buffer (see Recipes)Recipes100 mg/mL cycloheximide (CHX) (store at -20 °C)10 mg/mL CHX 5 mg/mL CHX 1 M DTT, Cleland’s Reagent (store at -20 °C)70% (v/v) ethanol1 mg/mL ethidium bromide10 mM GTP (store at -20 °C)1× MOPS buffer10 mg/mL (~18 mM) puromycin (store at -20 °C)0.6 mM puromycin (store at -20 °C)2× ribosome dilution buffer (store at 4 °C)RNA loading dyeRNA sample buffer (make fresh day of use)60% (w/v) sucrose35% (w/v) sucrose, buffered (store at 4 °C)1× TBE bufferLaboratory supplies0.2 mL, open-top thick wall polycarbonate tube, 7 mm × 20 mm 1.7 mL microcentrifuge tube, clear 500 mL Erlenmeyer flask8-strip PCR tubes Hard-shell PCR plates, 96-well, thin-well IceIce bucketsLight-dry tissue wipes Microseal ‘B’ seals Nitrile glovesP10, P20, P200, and P1000 calibrated pipettesP10, P20, P200, and P1000 pipette tips ParafilmPlastic wrapTube racks-80 °C freezer-20 °C freezer4 °C refrigeratorAspiratorCFX Connect Real-Time System Eppendorf centrifuge 5430 Fume hoodGelDoc Go Gel imaging system with Image Lab Touch software MicrowaveMilliporeSigma Synergy ultrapure water purification system NanoDrop One Microvolume UV-Vis spectrophotometer OWL EasyCast B1 Mini Gel electrophoresis system OWL EasyCast B1A Mini Gel electrophoresis system Paper towelsPlate centrifuge, PerfectSpin P Pointed tweezersS100-AT3 fixed angle rotor Sorvall Discovery M120 SE micro-ultracentrifuge (Hitachi)T100 thermal cycler Vortex mixer Bio-Rad CFX MaestroMicrosoft Excelhttps://www.graphpad.com/quickcalcs/molarityform/)Prism GraphPad Molarity Calculator Oligo Calc: Oligonucleotide Properties Calculator Transcription and Translation Tool  subcloned from pGL4.13 into pCR II, which is linearized with HindIII. For the experimental reporter, we typically use nanoLuciferase (nLuc) subcloned from pNL1.1 into pcDNA3.1(+), which is linearized with either XbaI or PspOMI. Both plasmids are available upon request from the corresponding author; pcDNA3.1(+)/nLuc-3XFLAG is also available from Addgene.ii. Choose a restriction endonuclease that cleaves downstream of the reporter coding sequence and either produces blunt ends or 5 overhangs. Restriction endonucleases that produce 3′ overhangs should be avoided as they can elicit aberrant and antisense transcription . Choose In a microcentrifuge tube, combine 100 μL of plasmid DNA (300 ng/μL), 20 μL of 10× CutSmart buffer, 20 μL of restriction enzyme , and 60 μL of Milli-Q water. Gently mix by inversion 20 times and collect contents by a short centrifugation spin .Pause point: Store completed digest at -20 °C.Digest the plasmid for 4 h at 37 °C and then hold at 4 °C. Note: Despite the excess of restriction enzyme in the reaction, we suggest a 4 h incubation for complete linearization due to the relatively large massof plasmid DNA.During the restriction digest, cast a 0.8% (w/v) agarose gel in an OWL EasyCast B1A Mini Gel electrophoresis system with a 1.5 mm 10-well comb. Add 100 mL of 1× TBE buffer with 0.8 g of agarose in a 500 mL Erlenmeyer flask and then loosely plug the flask with a folded-up paper towel. Heat in the microwave for 1.5 min or until the agarose is completely dissolved. Allow to cool for 10 min on the benchtop and then add 5 μL of 10 mg/mL ethidium bromide. Gently swirl to mix and let the flask cool on the benchtop until it can be held comfortably for 10 s (or place in a 65 °C water or bead bath for 30 min). Pour ~70 mL into the casting tray and let the gel solidify at room temperature for ~1 h. Remove combs by gently pulling them up vertically. Remove and rotate the casting tray so that the wells are near the cathode. Fill the gel tank and completely cover the agarose gel with 1× TBE buffer .Purify linearized plasmids using the DNA Clean and Concentrator-25 kit and the supplied solutions.g for 30 s, unless specified. To the 200 μL digest, add 800 μL of DNA binding buffer and mix by gentle inversion 20 times. Transfer mixture to the Zymo-spin column in a collection tube. Centrifuge and discard flowthrough. Wash the column with 200 μL of DNA wash buffer by centrifugation. Discard flowthrough and repeat wash step. Transfer column into a fresh and pre-labeled 1.7 mL microcentrifuge tube. Add 30 μL of nuclease-free water directly to the column (the white resin at the bottom of the column) and incubate at room temperature for 1 min. Elute by centrifugation . Store on ice for immediate use or at -20 °C for long-term storage.Perform all steps at room temperature and centrifugation at 16,000× Determine DNA concentration and purity by UV spectrophotometry .Note: The Zymo-spin IICR column has a reported capacity of 25 μg of DNA. This protocol calls for 30 μg of DNA, which allows us to max out the column, resulting in most reporter plasmids eluting at ~1,000 ng/μL.Confirm linear plasmid integrity by 0.8% (w/v) agarose gel electrophoresis. Add 5 μL of Quick-Load Purple 1 kb Plus DNA ladder to the first well. On a piece of parafilm or in a microcentrifuge tube, gently mix 1 μL of purified linear plasmid DNA (250–500 ng/μL), 1 μL of purple gel loading dye (6×), and 4 μL of Milli-Q water, and then load the entire sample into a single well. Run the agarose gel at 120 V (constant) for 30–45 min or until the desired resolution. Image gel via UV transillumination to confirm a single band that is running at the expected molecular weight.Note: If using two combs per casting tray, use the bottom half of the agarose gel before using the top half, as ethidium bromide in the gel runs toward the cathode. Unused parts of the gel may be stored for up to one week in plastic wrap at 4 °C.Synthesize in vitro–transcribed reporter mRNA using the HiScribe T7 High Yield RNA Synthesis kit.To resuspend the ARCA cap analog to 40 mM, first quickly collect the contents (1 μmol) by a short centrifugation and then add 25 μL of nuclease-free water to the pellet. Gently mix by flicking the tube and collect the contents by brief centrifugation. Repeat the gently mixing procedure for a total of five times.It is critical to use 1 μL of 10 mM GTP and not 1 μL of 100 mM GTP. The 8:1 ARCA cap analog to GTP ratio ensures 90% co-transcriptional capping efficiency , 0.5 μL of RNase inhibitor, 1 μL of 10× T7 reaction buffer, 1 μL of 100 mM ATP, 1 μL of 100 mM UTP, 1 μL of 100 mM CTP, 1 μL of 10 mM GTP, 2 μL of 40 mM ARCA cap analog, 1 μL of T7 RNA polymerase mix, and 0.5 μL of nuclease-free water in a PCR tube or microcentrifuge tube. ficiency .Notes:i. If transcribing multiple mRNAs at one time, assemble a master mix of all components except linear plasmid templates. Use 9 μL of master mix with 1 μL of linear plasmid template (~500 ng/μL). Reactions can also be scaled up linearly to 20 μL but beware of exceeding the binding capacity of the RNA clean up columns (see below).ii. Most T7 RNA polymerase–mediated mRNA synthesis kits with a cap analog pre-mixed with the NTPs use a 4:1 ARCA cap to GTP ratio, which only provides ~80% co-transcriptional capping efficiency. These cap analog pre-mixed kits also do not provide the ability to generate A-capped or non-methylated G-capped mRNAs to test cap-dependency.Perform in vitro transcription for 2 h at 30 °C using a thermal cycler (for PCR tube) or heat block (for microcentrifuge tube).Note: In our hands, transcription at 30 °C yielded purer mRNA than transcription at 37 °C.To remove the DNA template, add 1 μL of DNase I (RNase-free) to each 10 μL reaction and gently mix by inversion. Incubate at 37 °C for 15 min.E. coli Poly(A) polymerase, and 28 μL of nuclease-free water, followed by incubation at 37 °C for 1 h.If polyadenylation of the RNA is desired, combine and gently mix on ice the 11 μL DNase-treated capped and transcribed mRNA reaction with 5 μL of 10× Poly(A) polymerase buffer, 5 μL of 10 mM ATP, 1 μL of Note: This protocol uses thenuclease-treatedFlexi rabbit reticulocyte lysate (RRL) system from Promega. It is not entirely necessary to polyadenylate in vitro–transcribed mRNA for efficient translation usingnuclease-treatedRRL. Polyadenylation provides a less than two-fold enhancement of reporter mRNA translation innuclease-treatedRRL Soto . If polyIf using a PCR tube, transfer the entire contents to a microcentrifuge tube.Purify mRNA using the RNA Clean and Concentrator-25 kit and the supplied solutions.g for 1 min, unless specified. To each 50 μL of mRNA sample, add 100 μL of RNA binding buffer and gently mix by inversion 20 times. Add 150 μL of 100% ethanol to each reaction and gently mix by inversion 20 times. Transfer the entire sample to the Zymo-Spin IICR Column in a collection tube and centrifuge. Discard the flowthrough. Add 400 μL of RNA prep buffer to the column, centrifuge, discard flowthrough, and place column back into the collection tube. Add 700 μL of RNA wash buffer to the column, centrifuge, discard flowthrough, and place column back into the collection tube. Add 400 μL of RNA wash buffer to the column, centrifuge for 4 min, discard flowthrough, and place column into a new RNase-free 1.7 mL microcentrifuge tube. Add 75 μL of nuclease-free water directly to the column matrix and allow to incubate at room temperature for 1 min. Elute by centrifugation. Store mRNA on ice moving forward.Perform all steps at room temperature and centrifugation at 16,000× Determine RNA concentration and purity by UV spectrophotometry .Pause point.Aliquot mRNA in 3 μL volumes in PCR strips and store at -80 °C. Confirm in vitro–transcribed mRNA quality and purity by denaturing agarose gel electrophoresis.Cast a denaturing 0.8%–1% (w/v) agarose formaldehyde gel in OWL EasyCast B1 Mini Gel electrophoresis system with a 1.5 mm 10-well comb placed in the top comb slot. Add 0.8–1 g of agarose to 80 mL of Milli-Q water in a 500 mL Erlenmeyer flask loosely plugged with a folded-up paper towel. Microwave for 1.5 min or until the agarose is dissolved and gently swirl to mix. Cool on countertop for 2 min.In a fume hood, add 10 mL of 10× MOPS buffer and 10 mL of 37% formaldehyde to the flask containing dissolved agarose. Gently swirl to mix and allow to cool for 5–10 min.Pour 100 mL into the casting tray and let the gel solidify at room temperature for ~1 h in the dark by loosely covering with aluminum foil.Prepare mRNA samples by mixing 500 ng of in vitro–transcribed mRNA, 1 μL of 1 mg/mL ethidium bromide, 1 μL of RNA loading dye (see Recipes), 5 μL of freshly-prepared RNA sample buffer (see Recipes), and nuclease-free water to 15 μL total volume.Prepare the RNA ladder by mixing 1 μL of Millennium RNA size marker (1 μg/μL), 1 μL of 1 mg/mL ethidium bromide, 1 μL of RNA loading dye (see Recipes), 3 μL of RNA sample buffer (see Recipes), and 4 μL of nuclease-free water.Heat ladder and samples at 70 °C for 5 min; then, place the ladder and samples on ice for 2 min.Remove combs by gently pulling up vertically. Remove and rotate the casting tray so that the wells are near the cathode. Fill the gel tank and completely cover the agarose gel with 1× MOPS buffer .Load ladder and samples on the gel.Run at 60 V (constant) (or 5–10 V/cm gel width) in the dark  for 3–4 h (when the dye front reaches 2 cm from the bottom of the gel) or until the desired resolution.Image gel via UV transillumination to confirm a single band is running at the expected molecular weight (polyadenylating mRNA will add to the expected molecular weight).Note: Non-polyadenylated in vitro–transcribed mRNA will run as a single, crisp band at the expected molecular weight on a denaturing agarose gel. The same in vitro–transcribed mRNA that is polyadenylated will appear ~100–200 nt heavier as a slightly broader band. An additional control reaction lacking the Poly(A) polymerase can be included to better define the poly(A) tail length. Large smears spanning far beyond the expected molecular weight indicate RNA degradation, rolling circle transcription due to the presence of non-linear template DNA, nondenatured RNA secondary structure, or errors during RNA clean up steps. We have also found that making fresh, day-of-use RNA sample buffer is critical.In vitro translationPre-cool the Sorvall Discovery M120 SE micro-ultracentrifuge and S100-AT3 rotor to 4 °C prior to setting up in vitro translation reactions. The S100-AT3 rotor can hold up to 20 samples.Note: For each experimental mRNA, at least six samples should be prepared and translated: three replicates that are translated and not puromycin-treated and three replicates that are translated and puromycin-treated (step B3). Each of the six samples is mixed with a translated normalizing control mRNA-containing reaction (step B2), then diluted and layered on top of individual sucrose cushions (step C1).In vitro translation of normalizing control mRNA:be sure to use the ssRNA setting; see Software). Using its molecular weight from above and the online Prism GraphPad Molarity Calculator (see Software), calculate the mass required for 3 nM in 10 μL. This is the mass of mRNA required in the complete 10 μL in vitro translation reaction for 3 nM mRNA . Dilute the purified mRNA in nuclease-free water such that 2 μL of RNA contains the mass calculated above, resulting in a final concentration of 15 fmol/μL. Other similar online tools are available and would suffice.Dilute normalizing mRNA to 15 fmol/μL in nuclease-free water and keep on ice. To do so, use the plasmid DNA sequence from the first transcribed nucleotide of the T7 RNA polymerase promoter to the restriction endonuclease cut site and determine the corresponding RNA sequence using the online Transcription and Translation Tool (see Software). Then, calculate the molecular weight of the control mRNA reporter with the online Oligo Calc: Oligonucleotide Properties Calculator , 0.1 μL of 1 mM amino acid mixture minus leucine, 0.1 μL of amino acid mixture minus methionine, 0.2 μL of 25 mM Mg(OAc), 0.4 μL of 2.5 M KCl, 0.2 μL of RNase inhibitor, and 4 μL of nuclease-free water. Perform in vitro translation for 15 min at 30 °C in a thermal cycler.Immediately place samples on ice.To each sample, add 4 μL of 5 mg/mL CHX (see Recipes) . Gently mix by inversion 20 times and collect contents by a short centrifugation spin. Samples should now be 14 μL. Keep samples on ice until step C1.Note: Cycloheximide is added in step B2 to robustly inhibit elongation and preserve ribosome-bound mRNAs when added to experimental samples that contain puromycin in step B3.In vitro translation of experimental mRNA:Dilute experimental mRNA(s) to 15 fmol/μL (as in step B2a) in nuclease-free water and keep on ice.On ice in a PCR tube, set up the following 10 μL translation reaction: mix 2 μL of 15 fmol/μL in vitro transcribed mRNA, 3 μL of Flexi RRL (nuclease-treated), 0.1 μL of 1 mM amino acid mixture minus leucine, 0.1 μL of amino acid mixture minus methionine, 0.2 μL of 25 mM Mg(OAc), 0.4 μL of 2.5 M KCl, 0.2 μL of RNase inhibitor, and 4 μL of nuclease-free water. If translating multiple experimental reporters or many biological replicates, prepare a master mix with all components except for the mRNA; add 8 μL of the master mix to 2 μL of 15 fmol/μL in vitro–transcribed mRNA.Perform in vitro translation for 15 min at 30 °C in a thermal cycler.Immediately place samples on ice.To each sample, add 2 μL of nuclease-free water or 0.6 mM puromycin (see Recipes) . Gently mix by inversion 20 times and collect contents by a short centrifugation spin.Place puromycin-containing samples on a thermal cycler and incubate at 30 °C for 30 min. Keep samples without puromycin  on ice.Place all samples on ice and immediately add 2 μL of 10 mg/mL CHX (see Recipes) . Gently mix by inversion 20 times and collect contents by a short centrifugation spin. Keep samples on ice.All translation reactions should now be 14 μL.Ribosomal pelleting through low-speed sucrose cushionPrepare samples for low-speed ribosomal pelleting through a sucrose cushion.Mix a 14 μL normalizing control mRNA-containing reaction tube and a 14 μL experimental mRNA-containing reaction tube for a total sample volume of 28 μL. Keep samples on ice.Add 28 μL of ice-cold 2× ribosome dilution buffer . Each sample should now be 56 μL.Gently mix by inversion 20 times and collect contents by a short centrifugation spin. Keep samples on ice.Prepare sucrose cushions and overlay samples.Label 7 mm × 20 mm polycarbonate thick-walled tubes. Mark a single spot on the rim of each tube . Place tAdd 130 μL of ice-cold 35% buffered sucrose  to the bottom of each tube.Very carefully, overlay all 56 μL of the diluted sample from step C1 on top of the sucrose cushion.Low-speed centrifugationWithout disturbing the sample–sucrose interface, place the tubes into the pre-chilled S100-AT3 rotor. Be sure to position the marked spot facing outward and toward the back of the rotor. This spot will indicate the side of the tube where the ribosome pellet will be located after centrifugation . Using pCarefully place the rotor into the pre-cooled Sorvall Discovery M120 SE micro-ultracentrifuge.g for 1 h at 4 °C. Use an acceleration setting of 9 (fastest setting) and a deceleration setting of 5 (middle setting).Centrifuge samples at 50,000× Carefully, remove the tubes from the rotor and place them on ice using the homemade ice bucket for the 7 mm × 20 mm tubes. Using pointed tweezers for this step is helpful.Using a pipettor, remove and discard the supernatant without disturbing the pellet. The glossy clear pellet should be at the bottom edge of the tube below the mark that was facing outward and toward the back of the rotor during centrifugation.Resuspend the pellet in TRIzol.i. First, add 200 μL of TRIzol to a new, labeled, nuclease-free microcentrifuge tube.ii. Add 100 μL of TRIzol to the ribosome pellet in the 7 mm × 20 mm tube and mix 20 times by gently pipetting up and down. The pellet will dissociate from the tube wall when TRIzol is first added and will float in solution until it ultimately dissolves.iii. Transfer this 100 μL to the microcentrifuge tube (now containing 300 μL).iv. Add another 200 μL of TRIzol to the 7 mm × 20 mm tube to wash off any remaining ribosomes by gently pipetting up and down 10 times.v. Transfer the 200 μL sample to the labeled microcentrifuge tube Pause point: Store samples at -80 °C.Mix samples end-over-end at room temperature for 15 min at 15 rpm. Collect contents by a short centrifugation spin. RNA extractions, cDNA synthesis, and RT-qPCRRNA extractionsThaw samples at room temperature if necessary. Add 100 μL of chloroform to each sample.Mix vigorously by hand for 1 min (do not vortex).g at 4 °C.Centrifuge for 15 min at 12,000× Without disturbing or touching the protein interface, carefully remove the top 200 μL clear aqueous layer and transfer it to a new, labeled, nuclease-free microcentrifuge tube.Add 1.5 μL glycogen (20 mg/mL) to each sample. Upon dispensing, wash the tip in the aqueous phase by gently pipetting up and down three times.To each sample, add 500 μL of 100% isopropanol. Gently mix by inversion 20 times.g at 4 °C.Centrifuge for 15 min at 12,000× Note: When pelleting RNA in microcentrifuge tubes, place the hinge upright facing the outside of the rotor. This will allow you to predict the location of the pellet at the bottom of the tube on the same side of the hinge.Aspirate off the isopropanol until ~100 μL remains in the tube, leaving the RNA pellet untouched. Remove the final ~100 μL with a P200 pipette. The pellet should be white but very small.Add 600 μL of ice-cold 70% ethanol. Vortex each sample for 1 s.g at 4 °C.Centrifuge for 15 min at 12,000× Aspirate off the ethanol until ~100 μL remains in the tube, leaving the RNA pellet untouched. Remove the final ~100 μL with a P200 pipette and finally a P10 pipette. Aspirate any remaining ethanol off the tube walls. Allow to air dry with the top open for 2–3 min at room temperature on the bench.Place tubes on ice, add 30 μL of nuclease-free water to the pellet, and let stand on ice for 2 min. Gently resuspend the pellet by pipetting up and down 20 times. Be sure to wash down the side of the tube (same side as the hinge) to ensure complete resuspension of the RNA pellet.cDNA synthesisOn ice, combine 16 μL of RNA and 4 μL of 5× iScript Reverse Transcription Supermix. Mix by gentle inversion 20 times and collect the contents by a short centrifugation spin. Store the remaining 14 μL of RNA at -80 °C.Using a thermal cycler, reverse transcribe using:i. 25 °C for 5 min (priming)ii. 46 °C for 20 min (reverse transcription)iii. 95 °C for 1 min (reverse transcriptase inactivation and RNA cleavage)iv. Hold at 4 °CPause point: Store samples at -20 °C.To each 20 μL cDNA sample, add 180 μL of nuclease-free water for a 1:10 dilution. Gently mix by inversion 20 times and collect contents by a short centrifugation spin. RT-qPCRDesign a 96-well plate layout scheme for all samples and negative controls. Each cDNA sample will be amplified with two primer sets: one targeting the normalizing control mRNA and the other targeting the experimental mRNA. A no-template negative control (where water is added instead of cDNA) should also be included for each primer set. Perform at least technical duplicates for each sample and primer set.Dilute qPCR primers by mixing 510 μL of nuclease-free water, 45 μL of 10 μM forward primer, and 45 μL of 10 μM reverse primer. See General notes below for the primer sequences we used for FFLuc and nLuc.Our typical RT-qPCR reaction is 15 μL. Each well will contain 7.5 μL of iTaq Universal SYBR Green Supermix (2×), 1.5 μL of the 1:10 diluted cDNA, and 6 μL of diluted primers. For both the normalizing control and experimental primer sets, create a master mix of iTaq Universal SYBR Green Supermix and diluted primers. Add 13.5 μL of this master mix to the appropriate wells. Then, carefully add 1.5 μL of the appropriate diluted cDNA samples. The no-template negative control contains 13.5 μL of appropriate master mix and 1.5 μL of nuclease-free water.Seal the plate with a microseal ‘B’ seal. Be sure not to touch the top of the plastic directly, but rather apply pressure to the seal by using a clean tissue wipe (or Kimwipe). Remove and discard the perforated edges.Centrifuge the plate in a plate centrifuge for ~30 s to pull the samples to the bottom of the wells.Place the plate into the Bio-Rad CFX Connect Real-Time System.Run the following RT-qPCR program:i. One cycle of 95 °C for 3 min.ii. Forty cycles of 95 °C for 10 s, 60 °C for 30 s, followed by a Plate Read.iii. Melt curve from 65 °C to 95 °C with an increment of 0.5 °C for 5 s and a Plate Read.Note: If using new primer sets, peel back the plastic film after a run and take out 10 μL to confirm the expected size amplicon on a 2% (w/v) agarose gel.For each experimental mRNA, at least six samples should be prepared and translated: three replicates that are translated and not puromycin-treated and three replicates that are translated and puromycin-treated (step B3). Each of the six samples is mixed with a translated normalizing control mRNA reaction (step B2), then diluted and layered on top of individual sucrose cushions (step C1). During RT-qPCR, each cDNA should be assayed in at least technical duplicates. Primer sets for both the normalizing control mRNA and experimental mRNA should be used in separate wells on the same plate.t-test with Welch’s correction. For nLuc as the experimental mRNA reporter, we typically observe a relative ~60% reduction in signal with puromycin treatment  to the signal of the normalizing control mRNA  to account for any error during RNA extraction and/or cDNA synthesis. Once the gene expression has been calculated by the CFX Maestro software, export the data and perform the remaining analysis in Excel. For each experimental reporter, group the without puromycin-treatment replicates and set to 100%. Then, determine the relative signal for each replicate with puromycin-treatment. Statistical comparisons can be made using an unpaired reatment .Two additional controls should be incorporated in step B3:1) A no-template negative control, where water is added instead of either reporter mRNA, is critical to ensure specificity of RT-qPCR primers and determine background levels of detection.2) To confirm that the low-speed sucrose cushions are working as expected to selectively pellet ribosome-bound mRNA, set up translation reactions with the experimental mRNA without puromycin, incubate for 15 min on ice instead of 30 °C (step B3c), and proceed as directed above. The ice-incubated sample should have very few, if any, ribosomes loaded on the experimental mRNA and should be minorly detected by RT-qPCR when compared to the 30 °C–incubated sample.Journal of Biological Chemistry, DOI: 10.1016/j.jbc.2022.102660. See Figure 6E and 6F.This protocol was validated in Scarpitti et al. (2022) These in vitro translation reaction conditions have been optimized to be in the dynamic linear range for time and mRNA input for a range of reporter mRNAs . These sg sucrose cushion in the Sorvall Discovery M120 SE micro-ultracentrifuge and S100-AT3 rotor. Increased centrifugal force resulted in loss of specificity for pelleting ribosome-bound mRNAs in a translation-dependent manner. Both variables may have to be re-optimized if using different translation extracts, if making substantial alterations to the translation reaction conditions, and/or if using a different rotor.We empirically optimized the 0.1 mM puromycin  for use with the in vitro translation reaction conditions described in this protocol. Increasing puromycin beyond 0.1 mM did not further reduce the amount of mRNA co-pelleted in a translation-dependent manner. We also optimized the 50,000× If testing ribosome stalling by an mRNA-binding protein , we recoLastly, while we have always used FFLuc as the normalizing control mRNA and nLuc as the experimental mRNA, we believe other reporters  would function just as well for either the normalizing control or experimental mRNA. We have previously cloned FFLuc from pGL4.13 into the dual promoter plasmid pCR II. The use of pCR II is not required, but pCR II/FFLuc under control of the T7 RNA polymerase promoter is available upon request. pcDNA3.1(+)/nLuc-3XFLAG, which contains a T7 RNA polymerase promoter upstream of nLuc, is available upon request and from Addgene.See below for reporter coding sequences and qPCR primer sequences.FireFly Luciferase ATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTAAnanoLuciferase ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAART-qPCR primersF_pGL4.13 RT-qPCR: GCAGTACCGGATTGCCCAAGR_pGL4.13 RT-qPCR: GTCGGGGATGATCTGGTTGCF_nLuc (pNL1.1) RT-qPCR: CAGCGGGCTACAACCTGGACR_nLuc (pNL1.1) RT-qPCR: AGCCCATTTTCACCGCTCAG


Bacillus cereus strain PC2 (GenBank accession No. MZ314010) and Streptomyces praecox strain SP1 (GenBank accession No. MZ314009). In-site bacterial and fungal isolates defined in the current study were proficient in cleaning wastewater of chlorpyrifos pesticide residues.Pollutants cause a huge problem for humans, animals, plants, and various ecosystems, especially water resources. Agricultural, domestic, and industrial waste effluents change the water quality and affect living microorganisms. Therefore, the current study aimed to identify possible microorganisms in wastewater as potential bioremediation agents of pesticide residues. Wastewater samples were collected from El-Khairy agricultural drainage, which receives agricultural and domestic wastes. Bacteria and fungi species were isolated as clean cultures. Wastewater samples were analyzed for pesticide residues via gas chromatography-mass spectroscopy (GC–MS) system. Results uncovered the presence of ten pesticides ranging from 0.0817 to 28.162 µg/l, and the predominant pesticide was chlorpyrifos. Along with that, about nine species  were relatively efficient in the removal of chlorpyrifos residues up to 2000 µg/l with removal percentages ranging from 24.16 to 80.93% under laboratory conditions. Two bacterial isolates proficiently degraded significant amounts of chlorpyrifos:  Therefore, the current study aimed to isolate indigenous bacteria and fungi and screen their potential as bioremediation agents of wastewater of pesticide residues.Acetonitrile HPLC-grade and culture media were purchased from local chemical providers. DNA purification kit (Germany) and PCR clean-up kit were from Maxim Biotech Inc. (USA). The internal standard (TPP) and extraction (Cat#5982–0650) and dispersive SPE clean-up (Cat#5982–5056) kits were purchased from Technoscient for Lab & Optical Product, Cairo, Egypt. Certified reference standard materials of pesticides were obtained from ULTRA Scientific Analytical Solutions  Table .Table 1L3/s  were collected by water sampler into cleaned and sterilized one-liter Pyrex borosilicate dark glass bottles  and plate count agar for isolation of the bacteria colonies  at 25 °C. Nine samples were collected; 3 from each collection point as replicates and 3 plates per replicate were planted from each sample under sterilized conditions and incubated at 37 °C. The complete growth of the microbe was reached after about 7 days of incubation. Each microorganism was transferred into a new Petri dish. The subculture of each microbe was repeated several times until a visually clean culture was obtained.4 (anhy) and NaCl, respectively, were thoroughly mixed for 1 min. Then the internal standard triphenyl phosphate (TPP) solution was added to tubes and shaken for 30 s. Then tubes were centrifuged at 1350 × g for 10 min . About 1 ml of supernatant (acetonitrile) was mixed by hand for 5 min with 25 mg PSA sorbent and 150 mg MgSO4 (anhy) and centrifuged for 5 min at 1350 × g. About 500 µl of each tube was filtered through 0.22-μm PTFE filters  into HPLC vials for GC–MS analysis.Pesticide residues in wastewater were extracted and cleaned up using a modified method of Anastassiades . Separation conditions were as reported by AOAC AOAC, , where tThe intra-day assay (repeatability) and inter-day assay (intermediate precision) of the used analytical technique were calculated according to Ermer . Also, tThe radial growth of separated and identified fungi and bacteria on media mixed with the prevalent insecticide in wastewater, chlorpyrifos (CPF), was examined. Five concentrations of CPF  were prepared in the growing media. Five plates of each microorganism were used as replicates per each concentration. Microorganisms were incubated with the CPF at 37 °C for 1 week, and then their radial growth was photographed and recorded. Then competence of growth fungi and bacteria on such media with the insecticide was calculated compared to control plates. This experiment was repeated six times. Then the performance of the potential bioremediation activity of organisms was examined using 500, 1000, and 2000 µg/l. The residues of CPF in media after the incubation time were measured using the GC–MS as described previously.The fungal isolates were cultured onto clean growth media until pure cultures were obtained and used for various evaluations and identification  following the manufacturer’s guidelines.E. coli, forward, 5ʹ AGG ACG TGC TCC AAC CGC A ʹ3, and reverse, 5ʹ AAC TGG AGG AAG GTG GGG AT ʹ3  was performed by Macrogen Company . The 16S rRNA gene nucleotide sequences of isolated bacterial strains were submitted to the GenBank database under accession numbers MZ314009 and MZ314010.http://www.ebi.ac.uk/clustalw)  . The pesticide residues and microbial isolates’ performance and growth were expressed as mean ± SD. Significant means were contrasted using Tukey’s honest significant difference test (HSD) (05) SAS, .The analytical method used in the analysis of pesticides was accurate and suitable based on obtained values of repeatability and intermediate precision Ermer, . ResultsAnalysis of pesticides residues revealed the presence of lenacil, chlorpyrifos, cypermethrin, bifenthrin, carbofuran, and permethrin at 0.721, 28.16, 4.14, 0.052, 7.881, and 0.208 µg/l, respectively, in samples collected from site A  were presented in Table Verification of the performance of isolated bacteria  and fungi  was examined by challenging their growth on media with 500, 1000, and 2000 µg/l of CPF Fig. . ResultsAspergillus terreus, Aspergillus foetidus var. pallidus, Aspergillus fumigatus var. ellipticus, and Aspergillus fumigates according to the database identification program of RCMB For Aspergilli  and Bacillus cereus (I). For further differentiation between the two bacterial strains, similar morphology, physiological, and biochemical traits were reported, except for starch hydrolysis and lactose production that were negative in B. cereus and positive in S. praecox. Also, B. cereus produced acid from sucrose, but S. praecox did not.The characteristics of the most efficient bacterial isolates (F and I) in degrading CPF were listed in Table Bacillus cereus PC2 (GenBank Acc# MZ314010) and Streptomyces praecox SP1 (GenBank Acc# MZ314009) were constructed via molecular identification. Results of DNA sequences of the 16S gene of F and I bacterial isolates were as the following for F, identified as Streptomyces praecox, with a sequence of the following:GACGGCCTTCGGGTTGTAAACCTCGGGCAGCAGGGAAGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGCCCCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGAAAGCCCGGGGCTTAAGGGGGGGTCTGCATTCGATACGGGCTAGCTAGAGTGTGGTAGCCCAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGTTGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTGGGGGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATThe 16S rRNA gene sequences of Bacillus cereus with a DNA sequence of the following:CAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTAThe second bacteria (I) was identified as S. praecox strain SP1 was compared with the other 16 species of Streptomyces in GenBank. The neighbor-joining phylogenetic tree revealed high homology between S. praecox strain SP1 and S. praecox strain 7445   within 24 h  as the main metabolite with greater water solubility compared to TCP. Streptomyces sp. AC5 and AC7 species, effectively biodegraded CPF insecticide. The AC5 removed over 90% of CPF (50 mg/l) after 72 h. of incubation, while the AC7 strain was less effective  , I (Bacillus cereus), and A (Aspergillus terreus) removed about 80.82, 80.93, and 75.59% of the 2000 µg/l of CPF in 1 week. Following the molecular identification of these species, they were registered in the GenBank as Streptomyces praecox strain SP1 and Bacillus cereus strain PC2 with accession Nos. MZ314009 and MZ314010, respectively.Pollution of water resources with pesticides might cause serious problems. Indigenous microorganisms help lessen the adverse effects of pesticide residues through degradation. Agricultural wastewater samples were analyzed for pesticide residues, and results showed the detection of lenacil, chlorpyrifos, cypermethrin, bifenthrin, carbofuran, tolfenpyrad, oxamyl, dicloran, simetryn, sulfotep, ethofumesate, and permethrin. Chlorpyrifos insecticide was the dominant compound in wastewater samples. Indigenous bacterial and fungal species were isolated, and their ability to degrade chlorpyrifos insecticide was examined. After 1 week of incubation of isolated bacteria  and fungi  with CPF, results revealed efficiency % of removal ranging from 31.41 to 90.82%. Specifically, F (


Salmonella entericaserovar Typhimurium. Eight unique sRNAs were selected for deletion primarily based on their genomic location and/or putative targets. Quantitative and qualitative analyses confirm one of these, sRNA1186573, is required for efficient biofilm formation inS. entericafurther highlighting the significance of sRNAs duringSalmonellastress response.Small RNAs (sRNAs) are short noncoding RNAs of ~50-200 nucleotides believed to primarily function in regulating crucial activities in bacteria during periods of cellular stress. This study examined the relevance of specific sRNAs on biofilm formation in nutrient starved Salmonella entericais rod-shaped, motile, non-spore-forming gram-negative bacilli responsible for causing gastrointestinal disease and thousands of deaths across the world every year. Salmonella, like many other bacteria, forms biofilm when subjected to stressAcinetobacter baumanii, which is a classic biofilm formerE.coliandS. enterica:bssRandcsgD Small RNA (sRNA) are non-coding RNA molecules that are less than 200 nucleotides in length and have been found to control gene expression in many regulatory circuits in bacteria, such asSalmonella entericaserovar Typhimurium, we selected eight unique sRNAs for deletion primarily based on their genomic location and/or putative targets then performed qualitative and quantitative assays of biofilm formation on each mutant and controls . After 24-h of incubation at room temperature in a nutrient deficit condition (as nutrient starvation is an inducer of biofilm formation). In agreement with Congo Red assay results, standard microplate biofilm UV absorbance assays confirmed that the ability of the sRNA1186573 deletion mutant to form biofilms during nutrient deprivation was almost entirely lost, whereas the ability of the other sRNA deletion mutants to form biofilms did not significantly differ from wild type .To examine the relevance of specific sRNAs on biofilm formation inS. enterica, further highlighting the significance of sRNAs during Salmonella stress response. Finally of note, although RT-qPCR analyses confirm that sRNA1186573 deletion does not disruptcsgDgene expression, it is tempting to speculate that this sRNA may be involved with the post-transcriptional regulation ofcsgD, as csgD protein directly contributes to curli expression inSalmonella entericaIn conclusion, our results confirm that sRNA1186573 is required for efficient biofilm formation inPre-selection of sRNAs and mutant generationSalmonella entericaserovar TyphimuriumA total of 8 sRNAs  inThe primers used for generating mutants are mentioned below:sRNA924744 F: CACATTCACCGCTTACACAGGTCTGAACAAGGGGAGGCGAGTGTAGGCTGGAGCTGsRNA924744 R: AAGGCTCCAGTATATTTTTAAAGGATTTTTGGCATAATGAACATATGAATATCCTCCTsRNA1170414 F: GTAGTAATAGCGGTAGTTCCCCGGCAGTGATGGTCACTCAGTGTAGGCTGGAGCTGCsRNA1170414 R: AATGATGAGAGCTTTTAAGATGACAAGACCACCACCGGCGACATATGAATATCCTCCsRNA1186573 F: GTAATGGCTAGATTGAAAACAGTTAGTGTAGGCTGGAGCTGCTTCsRNA1186573 R: CCCCATAAAATAAAGGCACCAGAAGTACTGACAGATGTTGCATATGAATATCCTCCTTsRNA176086 F: TGAATTTGACACTGCGCACAGGGCGAsRNA176086 R: ACGACCTGCTTCTGAGGCTTTCTCTTTsRNA2594511 F: AAATAAGATCCCGGCCAGCCTGATACsRNA2594511 R: CGTGAACTGGGGAACTGGAAAGATTTsRNA3551252 F: TTTTAATATCATTAAAATCAAAAGTATAGACATTCATAGCGTGTAGGCTGGAGCTGCsRNA3551252 R: TGACTATACTTATTTGAGATACAAAAACAGCGCAAGAGTGCATATGAATATCCTCCTsRNA4130247 F: ATCTTGTGCTATTGGCAAAACCTATGGTAACTCTTTAGGTGTGTAGGCTGGAGCTGCsRNA4130247 R: TCGTCCAAGTGCAGCCCCGCACGGTGGGATAATAATCACCACATATGAATATCCTCCTsRNA4720054 F: CACAAAACTTATGGATTTATGCGTATAATCCGCGGCGCAAGTGTAGGCTGGAGCTGCsRNA4720054 R: CGTTATTGTGTCACTGTCTTACACACCGGTAAGACAGCAGACATATGAATATCCTCCThe primers used for confirming the mutants are here below:sRNA924744 F: GAATCCCCAGCAAACCAAG; sRNA924744 R: GCAGGCATAGTGATGATTTCCsRNA1170414 F: CTATGGAGATCGCGAATGGT; sRNA1170414 R: GAATGTCCGTACAGGGTGTTGsRNA1186573 F: AGGCACCAGAAGTACTGACAGA; sRNA1186573 R: ACGGCTATTTCAACCCACAGsRNA176086 F: GACATATCATATTTAAAACGCAACA; sRNA176086 R: CGCGATGTTCTGCCATAATsRNA2594511 F: TCTTCGTTGAGTCGCCTTT; sRNA2594511 R: CGTAAATAAATGCCTGGAAGGsRNA3551252 F: ACCATCCCGACAGACAA; sRNA3551252 R: TTGGAAGTGAAACCTCTGCATsRNA4130247 F: AGCCAAGATGCAAGAATAGACA; sRNA4130247 R: CCACGCTAATCACGACCAsRNA4720054 F: TTACTTACCGGAGGCGACAT; sRNA4720054 R: GAAAATTCTCCATCGCGGcsgDgene expression are as follows:The primers used to confirm that sRNA1186573 deletion does not disruptCsgD_F_qPCR: GGTCAGCGGATTACAGGGTA; CsgD_R_qPCR: TCGCGATGAGTGAGTAATGCBiofilm formation assaySalmonella Typhimurium: (i) rdar , (ii) pdar , (iii) bar , and (iv) saw et al.Both qualitative (Congo-Red agar test


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a +sense single-strand RNA virus. The virus has four major surface proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), respectively. The constitutive proteins present a high grade of symmetry. Identifying a binding site is difficult. The virion is approximately 50–200 nm in diameter. Angiotensin-converting enzyme 2 (ACE2) acts as the cell receptor for the virus. SARS-CoV-2 has an increased affinity to human ACE2 compared with the original SAR strain. Topological space, and its symmetry, is a critical component in molecular interactions. By exploring this space, a suitable ligand space can be characterized accordingly. A spike protein (S) computational model in a complex with ACE 2 was generated using silica methods. Topological spaces were probed using high computational throughput screening techniques to identify and characterize the topological space of both SARS and SARS-CoV-2 spike protein and its ligand space. In order to identify the symmetry clusters, computational analysis techniques, together with statistical analysis, were utilized. The computations are based on crystallographic protein data bank PDB-based models of constitutive proteins. Cartesian coordinates of component atoms and some cluster maps were generated and analyzed. Dihedral angles were used in order to compute a topological receptor space. This computational study uses a multimodal representation of spike protein interactions with some fragment proteins. The chemical space of the receptors  suggests the relevance of the receptor as a drug target. The spike protein S of SARS and SARS-CoV-2 is analyzed and compared. The results suggest a mirror symmetry of SARS and SARS-CoV-2 spike proteins. The results show thatSARS-CoV-2 space is variable and has a distinct topology. In conclusion, surface proteins grant virion variability and symmetry in interactions with a potential complementary target . The mirror symmetry of dihedral angle clusters determines a high specificity of the receptor space. Designing an antibody against the SARS-CoV-2 spike protein S is challenging due to its nonspecific nature, mainly conferred by each spike protein monomer . The spiRecent research and protein sequences designed to fit spike protein S failed to bind. A lack of specificity is a result of high mobility and flexibility combined with additional maximum solvent exposure of the monomer domains . A mass effect should virtually resolve this lack of specificity; in other words, by increasing the mass of the desired paratope, that will interact with the epitope (S protein), an alternative unavailable from the receptor–ligand interaction point of view . Judging from the antigen–antibody point of view, epitope amino acid (Aa) sequences located on the antigen must fit complementarily with the Aa chain  of the paratope (antigen variable (VL) and constant regions (CR)) .f) of one argument from a given domain is a polynomial function if a polynomial exists. Like any other functions, polynomial functions can be represented by a graph  in H2n. Stated precisely, this is a bilinear form. This is a symmetric form for n even , in which case the signature of M is defined to be the form’s signature, and an alternating form for n odd . These can be referred to uniformly as ε-symmetric forms, where ε = (−1)n = ±1, respectively, for symmetric and skew-symmetric forms . These fθ is the determinant, and cosθ represents the bound angle value in Å, and if cosθ11, cosθ22, cosθ33, and cosθ44 are considered zero, the determinant matrix can be written as follows  × [−165 − (−130)] = (−0.35) × (−35) = 1225. The convergence of the two functions is determined via the following relationship:On the function interval −0.5 ≤ x ≤ −0.15, function f1 = −14x6) and (f1(x))/(−x6) is computed: (f1(x))/〖−x〗6 =10 + 8/x + 6/x2 − (0.0002)/x3 − (0.0556)/x4 − (0.146)/x5 + (110.3)/x6 also (f2(x))/(−x6) = 14 + 10/x − 7/x2 +5/x3 + (0.0265)/x4 − (3.0432)/x5 + (165.5)/x6. Because A is a constant, the limits of the two functions are 10 and 14, respectively. While the limits have positive values , both of the functions have the following convergence domain: −∞ < x < +∞.The Riemann theorem shows that via permutation of a series of terms, the sum can be made equal to any number given before. In the case of functions f1(x) and f2(x), these numbers are 10 and 14. So, the functions are convergent.y-axis is described by = (x(t), θ, y(t)). In Cartesian coordinates, these becomes (y(t)cos(θ), y(t)sin(θ), z(t) and (x(t)cos(θ), x(t)sin (θ), y(t)) [The surface of revolution in Euclidean space is a surface created by a rotating curve around an axis of rotation . Here, t), y(t)) ,46. ProtRamachandran plots are a tool for visualizing energetically allowed domains for backbone dihedral angles ψ against ϕ of amino acid residues in proteins. Dihedral angle values are annular (0 degrees is the same as 360 degrees). Ramachandran plots warp from right to left and bottom to top . The ω aThe property space of both spike proteins demonstrates similar characteristics of both molecules. Solvent exposure of both molecules shares common characteristics. Spike protein SARS and SARS-CoV-2 solvent exposure are similar .Binding site identification of potential therapeutic targets is crucial in every drug design process as the expected spike protein has various binding sites. Also, many binding sites in various spike protein regions are described in the literature. An efficient vaccine can be designed by identifying the energetically and structurally favorable binding site. In the present study, the ligand’s interaction with its receptor (spike protein) is a general process of applying various binding sites.Furthermore, the mirror symmetry of SARS-CoV-2 compared with SARS can potentially suggest potential ligands that can be used as vaccines. Symmetric molecules of SARS-proven ligands can be considered in such a way. Also, as seen in The design of an appropriate paratope that can lead to a COVID-19 antibody in in silico methods was used together with secondary data derived from the literature. In order to find a suitable paratope, a set of very low (VL) random sequences were used to assess the chemical space of epitope–paratope interactions see also .Firstly, a target for a potential antibody was established. Spike protein S was chosen as a target due to its volume, shape, and frequency at the external membrane of COVID-19 compared to proteins E, M, and N, respectively. Computational studies and published data show that spike S protein is a valid target ,50,51,52Also, an Aa sequence was chosen as an epitope. The Aa sequence was chosen from the literature  ,54.A set of 47 PDB VL chains was chosen from the literature to explore the paratope space by evaluating the epitope–paratope interactions using distinct computational techniques.https://www-cohsoftware.ch.cam.ac.uk, accessed on 1 July 2023). For example, the following sequence was obtained for 1A8J using Paratome: SYEGSDF. After checking that the sequence was of the outer external region and could contact the antigen, the probabilistic contribution of each Aa was computed using Parapred, in this case yielding the following values: S 0.1649, Y 0.486472, E 0.713605, G 0.32135, S 0.469778, and F 0.0840889. In order to take into account the epitopes’ first light chains, interactions with the light chains were studied. The PSOPIA online server was used [Firstly, a light chain of 47PDB structures was used. Paratopes were detected for each structure using the Paratome online engine . Resultswas used . PSOPIA was used .iFrag, a protein–protein interaction prediction server, was used to compute and quantify the interaction between epitopes and paratopes . First, Spike protein monomer:AYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVNCTFEYVSFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKGNYNYLYRKPFERDIYFPLQSYGFQPTNVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTSQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSACEII monomerSTTEELAKTFLETFNYEAQELSYQSSVASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQTYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFEIn order to characterize the VL sequences, a cluster analysis was performed using molecular descriptors. The properties, after the cluster was computed, are the following: sum of atomic polarizabilities, number of hydrogen bond acceptors, number of acidic atoms, number of aromatic atoms, number of H donor atoms, the sum of the number of H bond acceptors and donors, number of heavy atoms, information content, medium information content, number of carbon atoms, number of hydrogen atoms, number of nitrogen atoms, number of oxygen atoms, Balaban index, difference in bonded atom polarizabilities, number of rotable single bonds, number of aromatic bonds, number of bounds, number of heavy bounds, number of single bounds, atomic connectivity index, atomic valence connectivity index, number of chiral centes, number of unconstrained chiral centers, density, diameter (most considerable vertex eccentricity in a graph), SlogP, molar refractivity, topological polar surface area, vertex adjency information, volume surface area, van der Waals acceptor surface area, molecular weight, winner path, Weiner polarizability, Zagreb index, surface rugosity, total energy, angular energy, electronic energy, non-bonded energy, solvation energy, strain energy, van der Waals energy, and globularity. As seen in The following PDB models were used: 6VXXfor SARS-CoV-2spike protein, 6CVR for SARS spike protein, 5X29for SARS-CoV-2 envelope protein, and3I6Gfor SARS-CoV-2 spike protein. Furthermore, the PDB models were energetically minimized, charges corrected, and protonated at pH 7.4, a salt concentration of 0.9 nmol/L, and at 315 K. The model preparations were performed using Schrodinger2009 and MOE 2009software packages ,58. ShapIn order to reduce the amount of data and to simplify the results, monomers for each protein were prepared computationally, as stated before. The trimers and pentamer PDB structures were energetically minimized, and charges were corrected using the AMBER force field available in MOE 2009 software package. For each monomer structure, the energy was minimized, charges corrected, and structures protonated at a physiological pH. Dihedral (torsion angles) for each monomer were computed using the Chemoffice 2008 software package.In probing the chemical space, some structural information, from simple dihedral angles to complex tertiary and quaternary organizations, was obtained computationally using Schrodinger 2009 software packages. Dihedral angles were represented using a scatter plot for each monomer . While an abrupt cut-off was observed in data, and an abrupt variation in data was noted, a logarithmic scale was used. Also, as discussed in the introductory part, by normalizing the data, clusters are more easily observed . A logarhttps://www.wolframalpha.com/, accessed on 1 July 2023) interface was used to perform a cluster analysis of all dihedral angles. To compare their function domains, the function table dn/dxn (f(x)) for n = 1 … 4 was calculated for all four monomers. Surface plots were computed using the radius of gyration of each logarithmic trend line. Here, r2, while exploring a trend and not a correlation between two phenomena, is not significant. A histogram was computed using dihedral angle values on 300 intervals for all three monomers. Using logarithmic trendline equations, a derivate equation was computed for all three protein monomers to represent the dynamic of dihedral angles as a function graphically.In order to expand data dimensionality, a Ramachandran plot diagram was computed for all four monomers in order to characterize the dihedral angles of the structures using the Schrodinger 2009 software package. The Wolfram Alpha software (n/dxn (−0.059 log(x) + 6.1779) for n = 1 … 4; for M (COVID-19) protein monomer table: dn/dxn(−1.51011 log(x) + 6.1779) for n = 1 … 4; for E (COVID-19), protein monomer table: dn/dxn(−0.842 log(x) + 62.544) for n = 1 … 4; and for S (COVID-19) protein monomer table: dn/dxn(−0.443 log(x) + 9.7581) for n = 1 … 4.Radar plots were used to emphasize the dihedral angles of protein monomers. Furthermore, the surface of revolution for protein S for SARS and for M, E, and S for SARS-COV-2 were computed to retrieve the 3D dimensionality of monomers’ dihedral angles. Surfaces were generated using the following formulas: for S (SARS) protein monomer table: dThe dimensionality of the COVID-19 interaction space is defined by its interaction with its core receptor. The interaction space has a finite dimensionality defined quantitatively by the polynomial discriminant. Judging by its 2D dimensionality, the interaction space has a negative and positive domain through which epitope–paratope interactions are defined.SARS and SARS-CoV-2 spike proteins have similar solvent exposure and mirror symmetry regarding their dihedral angle chemical space. Both proteins have a similar solvent exposure even if their dihedral angle conformational space has mirror symmetry. Mirror symmetry explains to some extent the chemical inaccessibility of the spike protein SARS-CoV-2 as a receptor and the evasive but permanent chemical bonding in which it is involved.


Foxc1, Foxc2, or both in mice worsen I/R‐induced intestinal damage by causing defects in vascular regrowth, expression of chemokine CXCL12 and Wnt activator R‐spondin 3 (RSPO3) in blood ECs (BECs) and LECs, respectively, and activation of Wnt signaling in ISCs. Both FOXC1 and FOXC2 directly bind to regulatory elements of the CXCL12 and RSPO3 loci in BECs and LECs, respectively. Treatment with CXCL12 and RSPO3 rescues the I/R‐induced intestinal damage in EC‐ and LEC‐Foxc mutant mice, respectively. This study provides evidence that FOXC1 and FOXC2 are required for intestinal regeneration by stimulating paracrine CXCL12 and Wnt signaling.Intestinal ischemia underlies several clinical conditions and can result in the loss of the intestinal mucosal barrier. Ischemia‐induced damage to the intestinal epithelium is repaired by stimulation of intestinal stem cells (ISCs), and paracrine signaling from the vascular niche regulates intestinal regeneration. Here, we identify FOXC1 and FOXC2 as essential regulators of paracrine signaling in intestinal regeneration after ischemia–reperfusion (I/R) injury. Vascular endothelial cell (EC)‐ and lymphatic EC (LEC)‐specific deletions of  The transcriptional activity of FOXC1 and FOXC2 in lymphatic and blood endothelial cells in the small intestine contributes to vascular repair and intestinal regeneration after ischemia‐reperfusion injury by regulating CXCL12 and R‐spondin3 signalling, respectively. Endothelial cells (ECs) present in the blood and lymphatic vessels are crucial participants in the vascular‐dependent processes that restore damaged tissue because they control the secretion of paracrine factors from both the vessels themselves and nearby cells  and other nonepithelial stromal cells express Wnt ligands in the ISC niche , is a homeostatic chemokine expressed in many cell types such as stromal cells, ECs, and fibroblasts in various tissues  defective repair of intestinal BECs and LECs, (ii) reduced expression of CXCL12 and RSPO3 in intestinal BECs and LECs, respectively, and (iii) decreased activation of the Wnt/β‐catenin pathway in ISCs. Importantly, treatment with either CXCL12 or RSPO3 partially rescues the defects in intestinal repair and regeneration associated with EC‐ and LEC‐Foxc1/c2 deficiency. Chromatin immunoprecipitation (ChIP) assays reveal that both FOXC1 and FOXC2 proteins bind to the regulatory elements of the CXCL12 and RSPO3 loci in BECs and LECs, respectively. Together, our data show a new role for FOXC1 and FOXC2 as key transcriptional regulators of paracrine signaling in the intestinal blood/lymphatic vessels during postischemic intestinal repair/regeneration, and our findings may have important implications for the treatment of ischemic bowel disease by modulating the vascular paracrine signaling pathways.In this study, we report that FOXC1 and FOXC2 in intestinal BECs and LECs contribute to vascular repair and intestinal regeneration after I/R injury by regulating the expression of paracrine signaling factors. Inducible, EC‐ and LEC‐specific, single and compound mutant mice for Foxc1 and Foxc2 mRNA expression in intestinal BECs and LECs was evaluated via quantitative PCR (qPCR) of BECs and LECs isolated from the small intestine in adult mice , which were used as a relative control , LYVE1 (LEC marker), and FOXC1 Fig . In the Foxc2‐CreERT2 knock‐in mice  by oral gavage for 5 consecutive days and subjected to the I/R injury 12 day post tamoxifen treatment. FOXC2‐GFP+ cells were detected mainly in the LECs in sham‐ and I/R‐intestines , which is characterized by gut microbiota induced intestinal inflammation and injury and intestinal ischemia arising from derangements in the intestinal microcirculation angiogenesis have been generally unsuccessful because global single and compound Foxc1/Foxc2‐mutant mice die perinatally with severe cardiovascular abnormalities  mice, which (after the mutation is induced) are referred to as EC‐Foxc‐DKO mice  and lymphatic (CD31+LYVE1+) vessels to confirm Cre‐mediated recombination in intestinal BECs and LECs  and their control littermates. Recovery from intestinal damage was impaired in both EC‐Foxc1‐KO and EC‐Foxc2‐KO mice 24 h after I/R injury  were measured under homeostasis and at an early time point (3 h) after I/R injury via qPCR. No significant difference was found in the levels of these regulators between sham‐operated mice  vs. the control mice but a significant increase vs. the sham‐operated EC‐Foxc‐DKO mice. Furthermore, EC‐specific loss of Foxc1/c2 significantly reduced the proliferative response of intestinal epithelial cells to I/R injury as assessed via immunostaining of EpCAM  and BrdU  by breeding conditional‐null Foxc1fl/fl and Foxc2fl/fl mice  intestinal epithelial cells  cells  regrow earlier than lacteals (LECs) in the villous stroma  analyses of distal jejuna from control and EC‐Foxc‐DKO mice 18.5 h after I/R injury. Dimensionality reduction and clustering analysis identified 22 transcriptionally distinct cell clusters  after intestinal I/R injury  and human umbilical vein endothelial cells  by retro‐orbital injection 30 min before intestinal ischemia. Quantification of Chiu scores was then performed 24 h after I/R injury. In control mice, the rescue of intestinal mucosal damage was not significant after RSPO3 treatment at I/R‐24 h. However, in EC‐Foxc‐DKO mice, RSPO3 treatment could partially rescue the mucosal damage  and complement also contribute to intestinal I/R injury , as well as the lacteal length of EC‐Foxc‐DKO mice 24 h after I/R injury  and stimulating Wnt signaling in the crypts of the intestinal epithelium.jury Fig . Given emice Fig . Moreovement Fig . The numISCs Fig  and celljury Fig . However.5 h Fig . TogetheFoxc1/Foxc2 expression is crucial for the repair of the intestinal mucosa, BECs, and LECs after I/R injury and that the EC‐ and LEC‐Foxc‐DKO mutations in mice impair canonical Wnt/β‐catenin signaling in ISCs at the crypt base. Furthermore, our scRNA‐seq data indicate that RSPO3 expression is attenuated in LECs and stromal cells of the EC‐Foxc‐DKO mice after intestinal I/R injury, which is at least partially attributable to impairments in intestinal regeneration because ISC activity appears to be crucially dependent on Wnt/β‐catenin signaling in the subepithelial cellular microenvironment in the ISC niche , the extent of Foxc downregulation in the EC‐ and LEC‐specific mutant mouse lines may not be equal. However, the degree of impairments in intestinal repair is consistently greater in the EC‐Foxc‐DKO mutant line than in the LEC‐Foxc‐DKO mutant line, suggesting that Foxc1 and Foxc2 are required in both BECs and LECs for intestinal tissue repair. Equivalent experiments were also performed with adult mice carrying EC‐ (and LEC‐) specific KO mutations of each individual Foxc gene to determine the similarities and differences between the phenotypes associated with each deletion. While our qPCR and scRNA‐seq analyses show that expression levels of Foxc1 in intestinal ECs are higher than those of Foxc2, the phenotypic differences between EC‐ (or LEC‐) Foxc1‐KO and EC‐ (or LEC‐) Foxc2‐KO mice are not particularly distinct. Although the reason(s) for the phenotypic similarities remains unclear, recent evidence indicates that Foxc2 is essential for the maintenance of intestinal LECs and that treatment with antibiotics to deplete gut microbiota rescues the phenotype of LEC‐specific Foxc2 mutants, including lymphatic dilation in the small intestine ‐specific Foxc genes in mice impair the regulation of RSPO3 in LECs  with Cdh5‐CreERT2;Foxc1fl/fl (EC‐Foxc1‐KO), Cdh5‐CreERT2;Foxc2fl/fl (EC‐Foxc2‐KO), Cdh5‐CreERT2;Foxc1fl/fl;Foxc2fl/fl (EC‐Foxc‐DKO), Vegfr3‐CreERT2;Foxc1fl/fl (LEC‐Foxc1‐KO), Vegfr3‐CreERT2;Foxc2fl/fl (LEC‐Foxc2‐KO), Vegfr3‐CreERT2;Foxc1fl/fl;Foxc2fl/fl (LEC‐Foxc‐DKO) males, respectively, as described previously , mTmG/+;Vegfr3‐CreERT2;Foxc1fl/fl;Foxc2fl/fl (mTmG/LEC‐Foxc‐DKO) and mTmG/+;Foxc2‐CreERT2 mice were generated by crossing mTmG females  with EC‐Foxc‐DKO, LEC‐Foxc‐DKO and Foxc2‐CreERT2 males, respectively. Genotyping of mice was performed by Transnetyx Inc.et al, For adult mice, Tamoxifen  was dissolved in corn oil (Sigma #C8267) at 40 mg/ml by shaking at 37°C for 3–4 h. 7–8‐week‐old male adult mice were treated with 150 mg/kg Tm by oral gavage once daily for 5 consecutive days. For neonatal mice, each individual was treated with Tm  by oral gavage once daily from postnatal day 1 (P1) to day 5 (P5) , followed by dehydration in 30% sucrose, and OCT  embedding. Ten or 15 μm cryosections were cut and immunostained with CD31 and/or LYVE1 antibody  surgery as previously described  by intraperitoneal (i.p.) injection 2 or 18.5 h  before tissue dissection (at I/R‐24 h and I/R‐18.5 h respectively).et al, Distal jejunum was selected for this study because this segment has the most severe mucosal damage after intestinal I/R surgery compared with other segments according to pilot experiments. Different time points  were chosen for different analysis purposes. For histological analysis, transcardial perfusion was performed on the adult mice with cold PBS followed by 4% PFA after anesthesia. Distal jejunum was harvested and cut longitudinally to expose the lumen. After several washes with PBS, the intestine was postfixed in 4% PFA at 4°C for 4 h (for frozen or whole‐mount samples) or for O/N (for paraffin‐embedded samples). For qPCR and western blot on whole tissue lysates of the small intestine, blood was removed by transcardial perfusion with cold PBS. Distal jejunum was harvested, opened longitudinally, and washed with cold PBS, then snap‐frozen in liquid nitrogen for RNA isolation and protein extraction. For the tissue collection for neonatal intestinal whole‐mount staining, the neonates were euthanized at P7 after Tm treatment from P1 to P5. The proximal jejuna were collected, washed with cold PBS, and fixed in 4% PFA at 4°C for 4 h, then subjected to the whole‐mount staining protocol. Proximal jejunum was collected from neonatal mouse due to the ease of operation and similar lacteal length/blood capillary network length ratio between proximal and distal jejuna . Based on the H&E staining, Chiu Score (Chiu 3 in PBS) for 2 h at 4°C, and then incubated with the indicated primary antibodies  according to the manufacturer's instructions.For IHC‐P, 4 or 15 μm paraffin sections were deparaffinized, rehydrated, subjected to antigen retrieval, permeabilized with PBST, blocked with blocking buffer containing 5% donkey serum in PBST for 30 min at room temperature (RT), and incubated with indicated antibodies Table  in blockH&E staining images were acquired using an Olympus Vanox AHBT3 Research Microscope  and an Apple iPhone 12 Pro Max. Fluorescent images were acquired using a Zeiss AxioVision fluorescence microscope, a Nikon A1 Confocal Laser Microscope, or a Nikon AXR confocal microscope with the software of Zeiss AxioVision SE64 Rel. 4.9.1 or NIS‐Elements Viewer, respectively. Images were processed and analyzed with Adobe Photoshop, Imaris, and Fiji (ImageJ) software. Imaris imaging software was used to create videos Movies  and EV2 et al, et al, The mouse lacteal permeability was assessed as previously described  in 100 μl PBS  to get the CD45−LYVE1+ LECs. The CD45−LYVE1− cell suspension was incubated with Dynabeads labeled with CD31 antibody (BD #553369) to get the CD45−LYVE1−CD31+ BECs. Finally, the sorted Epis, LECs, and BECs were used for RNA isolation and qPCR analysis. For the detection of gene deletion in ECs in EC‐Foxc‐DKO mice, CD45−CD31+ ECs (including BECs and LECs) were isolated by using Dynabeads labeled with CD45 antibody (depletion of CD45+ cells) followed by Dynabeads labeled with CD31 antibody (positive selection of CD45−CD31+ cells).Epithelial cells (Epis), blood endothelial cells (BECs), and lymphatic endothelial cells (LECs) were isolated from the distal jejunum for further qPCR analysis as previously described (McCarthy A RNeasy Mini Kit (Qiagen #74104) was used for RNA extraction from cells. TRIzol™ Reagent (Invitrogen #15596026) was used to isolate RNA from whole distal jejunum. The concentration of RNA was determined using NanoDrop™ 2000 Spectrophotometers (Thermo Scientific). cDNA was synthesized using an iScript reverse transcriptase kit (Bio‐Rad #170‐8891). qPCR was performed on triplicates of cDNA samples by using QuantStudio® 3 Real‐Time PCR System (Applied Biosystems), Fast SYBR reaction mix (Applied Biosystems), and gene‐specific primer sets. 18S (for mouse samples) and PPIA (for human cells) were used as internal standards for mRNA expression in mouse samples. Primer sequences are provided in Table The frozen intestinal tissue was grinned using mortar and pestle chilled with liquid nitrogen, followed by lysis in RIPA buffer  containing protease inhibitors (Roche #4693116001). After centrifugation, the supernatant of the tissue lysates was collected and mixed with 5× Protein Loading Buffer. Equal amount of total protein for each sample was loaded and run on an SDS–PAGE gel. Samples were transferred to 0.45 μm nitrocellulose (Invitrogen) and western blotted with the antibodies listed in Table Foxc‐DKO I/R‐18.5 h. Briefly, mice were anesthetized by isoflurane. Blood was removed by cardiac perfusion with cold PBS. The distal jejunum was then dissected, washed with cold PBS, and cut into small pieces. The tissue was processed for scRNA‐seq upon dissociating into single‐cell suspension in a digestion buffer as mentioned above for 35 min at 37°C, followed by filtration through a 70 μm and a 40 μm cell strainer. Cells were washed with washing buffer  for three times and resuspended in PBS with 0.04% BSA at a concentration of 1,200 cells/μl (according to 10× Genomics Document #CG00053 Rev B) before being passed through a 30 μm MACS SmartStrainer. The cell viability was tested by using the Cellometer Auto 2000 Cell Viability Counter . The cell sample was processed for scRNA‐seq only when the cell viability was more than 70%. Average cell viability for samples was determined to be 80.96%.For single‐cell RNA sequencing, mouse distal jejunums were collected 18.5 h after intestinal I/R surgery. Two mice were used for each group: control I/R‐18.5 h and EC‐Single‐cell 3′ gene expression libraries were constructed by using the Chromium Next GEM Single‐Cell 3' Reagent Kits v3.1  according to the manufacturer's manual CG000204 Rev D. The single‐cell libraries were assessed for quality  and then run by using paired‐end 50 bp sequencing on the Illumina HiSeq 4000 platform . Ten thousand cells were targeted for each sample with a sequencing depth of 20,000 read pairs per cell.Foxc‐DKO mice using CellRanger. The matrix files were then utilized for data processing and downstream analysis using the BIOMEX browser‐based software platform and its incorporated packages developed in R  using the CellRanger toolkit . Gene expression matrices were then generated from both control and EC‐FindClusters function in Seurat . Marker set analysis was then performed in BIOMEX on highly variable genes to identify the top 10 gene markers expressed in each initial cluster using a similar methodology described previously  and previously reported the data  package  were then auto‐scaled and summarized by principal component analysis (PCA), followed by visualization using Uniform Manifold Approximation and Projection ) to reduce the data into a two‐dimensional space. Graph‐based clustering was then performed in BIOMEX to cluster cells according to their respective gene expression profile using a methodology similar to the Plotly software was used for UMAP and volcano plot visualization.BIOMEX implementation of the et al, https://www.ebi.ac.uk/gxa/sc/home) and was exported to check Lgr5 expression in mouse small intestinal ECs.The scRNA‐seq data for mouse small intestinal ECs from the publication entitled “Single‐Cell Transcriptome Atlas of Murine Endothelial Cells” . Putative sites in the human genome were then searched against the Genome Reference Consortium Mouse Build 38 (mm10) genome using the Evolutionary Conserved Region (ECR) Browser (https://ecrbrowser.dcode.org) and rVista 2.0 tools to identify conserved and aligned putative binding sites between mouse and human sequences. Conserved and aligned putative FOX‐binding site sequences are underlined and bolded within the human RSPO3 and CXCL12 ECRs shown below:Putative FOX‐binding sites in the TATTTACAAAACGTCTTGGAATGAGAATGAGCTGCTTGTGGTTCCTGTGGCTGATTCAGGGATGGTTTCCTACAGGCAGAGGATGCTGGTCAACCGAATGACCTCTCTGTAACTAACCCGTGCACCCCTGTGGTAAGGCTGTTTGGTCTTATAGGTACCTCTTCTAACTAAGCTTGGAGGGATTTGTTTTTGTGGTAAAGAACTTAGTAATAACCAAACGTCACTGTAAAGACAGATTTAATAATGTTAAGGTCCATCAGAGCCTACTCCTTCTACTACCAACAAGAGAAGCCAGAAATACACTGGGATGCCTTTAGATTCCTGTGCATCAATCTTTCTTTCTCTAAGGATTATGGTCTTGAAATGGATAATGATAGATTCCAAACACATGGAAATCTCTTGCCCCTTTTACTTTTTAGGATCTTTGCAAGCTTACAATATGTACACGTTTTCTGTAAGTCACCAATGCTGAGTTACTGGCATGAAAAATGACCCTGTTACTTGGAAAGTAGTTTCACTTACAAGTCCCCCAGGCCCTGTAATGTCTAAACCTCCTGTGCCACTTTATGTGACTACCCCGCCCCCACAGAGGAGCATGCACAGGAAAAGCAGACTTCCCTTCCCCCACACATTTCCTTAGTTATAAATATAGATTTATGAGATACTTTAATGTTCTAAAACAAATGAAAACCACCCAAGAGGAGCCTCACCAAACCTGAGGTTGTCCAGATTGCATTGACTAAGATTAAGTAAAAGATCATTCATCTCCAGAGGTCATGCAATTAATCTCAGAGTGGGAGTTAAAGCAATGACTAAGCAGAAAAGGAAGCCAAATACAAGCTCGTAACAAAAGGTGCTGGGGCTCCAACATCAAGGAACTTGTTATTTCCTTTTTATTTATTTATTTTTTTTTAATAGACCTAAAACACTCATTCCTTACTACTGGTTTCTTTGGGTCCTAAAATTCCACTTGGTTAGGTCAGCTATTTTCCATGACTATTTTTGATACGGTCAAACAAATACAAAGAATAAGCTTTTAAAAAACAATACCAGTAAATGGGGACACATTATATTGAATAAGGGTATTGTTAGCCAAATTCTAAGATTCATCTTAAATTGTTTTCTTATAAGAATTGTGTATTTACCATTTTAAAAATCACTATTATTTTAAAACACTTAGAAAGTGAACATTTGAAAATGATGTGCCTTTGGATGCTCTGTAATGTTAAGCAGATCCAGACATAAAGACAAAAGTAAATTCCAGAGTATTTTTGTAGCCATGGAATCACCATAAAAAGGGGTTTTTGACCCCAATGTTACCGTAACATTGTCTTCAGCATTTCATATTTAATTACAGTAGATTACTCACCAATATATGTTTACTTCTCTGCATTTAATGAGCAGTTGTTAACATGACTGAAACCATCTGATGATTTTTACCAAATGGAAAAATCTGCCTACAGGGGCAATAAAATAAATATTCAGAATAGAGAGAGGCAGTCATAAAAGACATTACCCGGTTGTAAACGGAGGCGGGTGGTGGTGATCTATTACCCCTGCCTCGGCAGCTTTCAACAGAGTTCTGGAATTCCAGGAGGGGCCCTGACCCAAGGCAATATTTACTTCTGCGGCTTCTTCATCAGGTCAGCATGGGTATAATTCTGTCTACCAGTTGACTGGAGCTGAGGTTTCGAGCAGGAAGTGCAAACCCTGAGTGCTTATAACTCAGGAGGAGTGAGGCACCCCTTCCCAGAGTATGCCAAGAAAAGCACATTGTACTGTCCTGGCTGCAGGGGTGAGGCCCCTGCACACCCAGGCCATTATCAGCTTTGTGCCCTGGCCAACAGCGCCTCTGGTTGGTGCATTTGTCAGCTGTATTTTACCCTAGAGCTCTGGGAGGCTCATCCTTTTTTGGTATACCACCACGTGGAGAGAGCAGAGTTTTAATAGTGTGGCTGCATCAAAACCTTCACCTTTCTCTGCTGAAGGAATGGCCTTCTCTTATGGGCAGGGAGGGTTTCCTAGGGAAAGCCCACCCAGGCAGGAGATGAGGAGAGCAGCATCTGAGCACACTTCATCCCACAGTGCCCATCCCATGAGTATCCTCCATAAATTACAAAGAAAGAAAAAAAATAGGGAAAAAACAAACCTTTATTTCTCGTAAATAAATTCCCACATACAGTAGGACGTTTATACCATGAAACAATTAGCATTTTATTGCTAGTGCATATAATGTCACATTTGATACAATTTTAGTACAAGTGAAAAAATACACTGTGGCTAACATTGAAAAGCTGCAATCACATTTATATATCATATATATTTCTTTACAAATTGCCAGTAGTTTGAGATAATAGAGAAGTATAAACTACTGACATTCATATGGCTCCACTTCAAATATATGAATTGTTCGACTATAAATATATTTTGAAATACATTTGTTTTCTAAAGAAACGTAAAAAAAAATGTGCACAAAAATATATATAAAAAAATGCCTTGCAAAAAGTTACAAATACCACCAGGACCTTCTGTGGATCGCATTTATGCATGGAAATGTCACCTTGCCAACAGTTCTGATTGGAACCTGAAACCCTGCTGTGGCTTCAGGAGGGGGTAGTGGCAAGATGATGGTTTATTCACTGATTTTTTCGCTTCTGATTTCGGAAACCTCAGAGTTTGTTAGTGCCTC CATGGCATACATAGGCTGGGTTAAAAAAAAAAAAAAAAGATCCAAAAACTTGAGCTGCAGATCTAATCTGCTCGTGAGAAAAGCCCATACACTGTCACACATGGGCTGTGAGAAGGGGTCTCAGACACCTGACTGCAGGCAGGCTTAACTATATAAACCAGAAACGTCTATAAGCTCCATCACTAACAACTAATGAATTTTATTTCAGRspo3 and Cxcl12 ECRs corresponding to the human RSPO3 and CXCL12 ECRs are shown below:As comparison, in mouse, the conserved and aligned putative FOX‐binding sites (underlined and bolded) within the mouse sequences of ATAAATAAACTGAGGAAATGTGTGGGGGAAGGGAGGGCTGCTTTTCCTGTGCATGTTCTTCTGTAGGGTGGGGCAGTCCTATAAAGTGGGTGAGGAGGGTTTGCCATTATGGGACCTGAGGGATTTGTACCAGAAACTCCTTTCCAAGGAAGGTCATTTTTTATGACAGGGGACTAGGGGTGGGGGAGTGCTTTAGACATGGACACTATTAATCTGTATTTGCAATGATGTCTGATAATTAAGCTCTTTATTGCAGAAACAAAGCCCTGCAGTCTTACGATGCCACACCAAGAAGCAGAGAACAGGAGGAGAACCATGGTCTGGGAAATAAACTTCAAAAGTCCCATATTAATAATTTTTCTCCTCCAGCAAGGCCACACCTAAGGGCTCCATCTCTCCCCCAGATATTATCACCTACTAGCTAGGGATCAAGTATTCAAAACATGAGCCGGGAAGAATTATTTCATATTTCAAGTATAATATTAGTCTGTAATCACAGTGATAATTGGTTATTAAGCTCTTCATTGGAACAACAAAGCCCTCCAGGCACAGTCTGAAATGATACCCATAACAAAGATCCTTCCTGTTGGGGTATAGGAGTTAGCTCCATAGAGGTCATTCAGTTGACCCGCTTCCCTGGCCTGCAGGAAACCATCCCTGAATAAGCCACAGGAACCACAAGCAGCTCGCTCTCATTCCAAGATGTTTCTATTTATTATATTAGTGAATACTATACTACAATTAGATATGAAATGCTGAAGACAATGTTAAGGTGAGAGTGGGTTCACAAACCTCTATATTGGTAGCTTCAGAATTGCAAAAACATTTGTGGAATATTGGTTTTGTCTCTATGCCTGGATCTGTTTAACATAACATAGTATCCACAGCACACTCTTTCAGACAGGGACTTTGTGAATCCTCTAAAACAACCATCATTTCTAAATGCAACAATAAGGAGTATTTTAGGTTTATTCAGGAGGAGGAAAAAAAAAAGGAAATAGCTCCTTGATGTTGGAGGCCAGGCCCCTCTTGTTATGGGCTTGCATGCTGGCTTTCTTTTCTGCTTAATCATCGCCTGAACTCCCACTCTAAGATTGCTTACATAACCCTGGGAGATGAATGATCTTCTACTTAATTTCAGCCAGTGCAATCTGGACAACCTCAGGTTGGATTGGTGAGGCTCCTCTTGGGTGGTTTTCACTTGCTTTAAGAACATTAAAATATCTCATAAATCTAGTAAATATTGCCTTGGGTCAGATCCTCTCCTGGAATCCCAGAACTGTTGAAAGCTGCCAAGGCAGGGGTAATAGATCACCACGACCCGCCTCTGTTTACAACCGGGTAATGCCTTTTACGACCGCCTCTCTCTGTCCTGAATATTTATTTTATTGCCCCTGTAGGGAGATTTTTCCATTTGGTAAAAATCATCAGATGGTTTCAGTCATGTTAACAACTGTTCATTAAACGCAGAGGGTAAACAGAGATGGAAAGGTTTGTTTCTTTCCTTTTTCTCTATTTATTTTTAACTTATGGAGAAAACTCAGGGAGTGAACCCAGGGAGGGAGGCATGTTCAGATACCAGCCATCAGTACCGTCTGCCCAAGTACAAACAAGCTGTATATCCCACTGACAATGGCCTGGTTATGTGGGACCCTCACCTTGACAGCCAGGACGGTACCATGAGCTTTTCTTGGCACACCCTGGAAAGGGTGCCTTACTCTTCCTGAGTTATGAGTGCTTAGGGCTCTGGCTTCCTGCTCAAAACCTCAACTCCAGTCAACTGGTGGACAGAATTATACCCACAATGACCTGCCGAAAAAGCCACAGAATATTTGTAGTCAATTCATATATTTGAAGTGGAGCCATAGTAATGCCAGTAGATATCTCTATGATCTTGAGCTACTGGCAACTTGTAAAGAAATATATATGACATATAAATGTATTGTAGCTTTCCGGTGTCAGCCACGGTGTATTTTTCCACTTGGAATGAAATTGTATCAACTGTGACATTATATGCACTAGCAATAAAATGCTAATTGTTTCATGCTGTAAACCTCCTACCGTATGTGGGAATTTATTTACCTGAAATAAAATCTACTAGTTGTTAGATGGAGTGCACATACATTTCTGAAGATGGAGAAAAACAGGTGTGCCTGCTGATCAGGTGCTGTGGGCTGAAGCCACAGTGGGGATTCTGGGTTCCAATCAGAAATGGAGACAAGATAAAACTTGCATACATTCTTATGATCACAGACGGCCCTGGTGGTTTTTGGTAACTATTTACAAGGCATTTTTTTACATATATTTTTGTGCACTTTTTATGTTTCTTTGGAAGACAAATGTATTTCAGAATAet al, CXCL12 promoters  according to the prediction of JASPAR, as well as the binding sites at the ECRs of CXCL12 and RSPO3 mentioned above. The ChIP primers are listed below:The ChIP experiment was performed as previously described  and LPS (5 mg/kg) to perturb the normal intestinal colonization process; (ii) gavage with formula every 3 h ; and (iii) exposure to brief episodes of hypoxia (60 s in 100% N2) followed immediately by cold stress (10 min at 4°C) twice daily. This protocol induces intestinal injuries ranging from epithelial injury to transmural necrosis resembling human NEC, which typically develop after 36 h and has been widely used to study NEC pathogenesis  per sample were analyzed for the fluorescent intensity (FI) of β‐catenin within the ISC by ImageJ. For quantification of FI of FOXC1 and FOXC2 in intestinal BECs and LECs, about 40 ~ 50 cells were quantified by ImageJ. For analysis of BEC and LEC proliferation and apoptosis, confocal Z‐stacks were acquired using a 20× objective from about 4 ~ 8 different fields of 15 μm paraffin sections for each sample. Area of blood vessels (CD31+LYVE1−) and lymphatic vessels (CD31+LYVE1+) were measured using ImageJ software. Then, the number of BrdU+ or TUNEL+ cells per 0.1 mm2 vessel areas was calculated and compared between groups. Measurements for the length of blood capillaries and lacteals were performed as previously described  package . All experimental protocols and procedures used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) at Northwestern University.Can Tan: Conceptualization; data curation; formal analysis; validation; investigation; visualization; methodology; writing – original draft; writing – review and editing. Pieter R Norden: Data curation; formal analysis; visualization; writing – original draft. Wei Yu: Data curation; formal analysis; investigation; visualization; methodology. Ting Liu: Data curation; investigation; methodology. Naoto Ujiie: Data curation; formal analysis; investigation. Sun Kyong Lee: Investigation. Xiaocai Yan: Investigation. Yaryna Dyakiv: Investigation. Kazushi Aoto: Resources. Sagrario Ortega: Resources. Isabelle G De Plaen: Resources; methodology. Venkatesh Sampath: Resources; funding acquisition. Tsutomu Kume: Conceptualization; supervision; funding acquisition; investigation; writing – original draft; project administration; writing – review and editing.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Movie EV1Click here for additional data file.Movie EV2Click here for additional data file.Source Data for Expanded View and AppendixClick here for additional data file.PDF+Click here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 4Click here for additional data file.Source Data for Figure 5Click here for additional data file.Source Data for Figure 6Click here for additional data file.Source Data for Figure 7Click here for additional data file.Source Data for Figure 8Click here for additional data file.Source Data for Figure 9Click here for additional data file.Source Data for Figure 10Click here for additional data file.