Management of COVID-19 and other epidemics requires large-scale diagnostic testing. The gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains reverse transcription quantitative PCR (qRT-PCR) analysis, which detects viral RNA more sensitively than any other method. However, the resource use and supply-chain requirements of RT-PCR have continued to challenge diagnostic laboratories worldwide. Here, we establish and characterize a low-cost method to detect SARS-CoV-2 in clinical combined nose and throat swabs, allowing for automation in high-throughput settings. This method inactivates virus material with sodium dodecylsulfate (SDS) and uses silicon dioxide as the RNA-binding matrix in combination with sodium chloride (NaCl) and isopropanol. With similar sensitivity for SARS-CoV-2 viral targets but a fraction of time and reagent expenditure compared with commercial kits, our method also enables sample pooling without loss of sensitivity. We suggest that this method will facilitate more economical widespread testing, particularly in resource-limited settings.  •Homebrew is a fast, cheap, validated RNA extraction method for SARS-CoV-2 testing•Homebrew RNA extraction only requires glassmilk, NaCl, SDS, isopropanol, and ethanol•Homebrew is scalable and allows for sample pooling without loss of sensitivity Large-scale diagnostics will be required long term for COVID-19 and future pandemics. Gold-standard PCR requires RNA extraction for increased sensitivity; however, supply chains and costs of commercial kits challenge many laboratories worldwide. We have established a sensitive, low-cost, automatable RNA extraction method in which samples can be pooled. Homebrew employs glassmilk and common laboratory reagents to increase diagnostic capacity in resource-limited settings. Diagnostics will be required long term for COVID-19 and future pandemics. Page and Scourfield et al. present a sensitive, low-cost, and automatable RNA extraction method for detection of SARS-CoV-2 in clinical samples employing glassmilk and common laboratory reagents to increase diagnostic capacity in resource-limited settings. Even in countries where combined vaccination and non-pharmacological strategies to contain the virus have decreased hospitalization and death rates, new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are causing local outbreaks. Therefore, widespread testing and contact tracing will remain as key tools for control of SARS-CoV-2 for the foreseeable future.As the world approaches the 21The gold standard for SARS-CoV-2 diagnostics consists of viral RNA extraction from combined nose and throat swab samples followed by qRT-PCR (quantitative reverse transcription and polymerase chain reaction). Standard RNA extraction for SARS-CoV-2 diagnostics is usually carried out with commercial kits, typically based on solid-phase reversible immobilization (SPRI) of nucleic acids. While these kits are optimized to purify high-quality RNA for extremely sensitive downstream applications, in resource-limiting situations, their high cost per sample and requirement for dedicated supply chains and expertise can be a barrier to more widespread testing. Purification-free methods, such as SalivaDirect, hid-RT-PCR, SwabExpress, or others , reduce Prevention of outbreaks therefore requires highly sensitive methods that allow early detection. Thus, to control transmission but also for variant detection, RNA isolation and qRT-PCR testing will continue to be irreplaceable.While assessing different commercial kits for SARS-CoV-2 , we encoWe set out to establish an alternative RNA extraction method for SARS-CoV-2 testing that would free our setting from supply-chain shortages. Nucleic acids are known to reversibly bind silica in the presence of chaotropic salts and non-basic pH conditions . While mMost commercial RNA extraction kits employ guanidinium thiocyanate (GITC) as chaotropic and protein-denaturing salt to induce binding of nucleic acids to silica. However, the global shortage of GITC combined with its toxicity prompted us to test the capacity of different chaotropic salts to support silica binding. Both NaI and NaCl could substitute for GITC to support similar binding of viral RNA to GM A and S1ATo improve viral particle solubilization from combined nose and throat swabs concomitant with inactivation , we compChaotropic salt-mediated nucleic acid precipitation and SPRI binding is improved in the presence of low dielectric constant solvents, such as isopropanol, . Blood only marginally affected N2 detection, but not N1, and all samples remained positive A.Having established the individual components and limits of the GM protocol, we considered it crucial to reduce handling time and plastic usage to enable faster and cheaper high-throughput testing. High-throughput facilities employ commercial or open-source automated stations that rely on magnetic bead isolation . To thisIncreased throughput for testing can also be achieved via sample pooling . This tyAs a final comparison, we employed 100 clinical specimens and compared GM and automated CB with QIAamp C. Our daIn summary, we present a scalable, inexpensive, and simple method that uses reagents widely available in laboratories worldwide to detect viral genetic material from combined nose and throat swabs with high sensitivity.Glassmilk-based isolation was initially described as a cheap alternative for plasmid preparations . HoweverGAPDH and IL8 mRNAs by qPCR with a limit of 250,000 cells per reaction, below which Ct values remained too high ; and Service Delivery for King’s College Hospital. All samples were combined nose and throat swabs having already been tested at King’s College Hospital or KCL TEST as part of a potential service development. All samples were anonymised and assessed after being diagnosed as SARS-CoV-2 positive by the hospital or negative by KCL TEST. Samples were chosen based on Ct values in order to represent a broad range of values. Positive swabs were inactivated in a Category 3 facility employing 90°C 10 mins in a dry bead bath .Important note: work under fume hood. Suspend silicon dioxide 325 mesh (Sigma 342890) in 40mL 10% HCl from a 37% HCl stock diluted in water in a 50mL tube, agitate suspension for 4 hrs on a tube roller and centrifuge at 2000 g. Remove supernatant and resuspend the silica pellet with 40mL water. Centrifuge and repeat the water wash five times . Make sure the silica is fully resuspended after each wash so residual HCl is not ‘trapped’ in the pellet mass. After the final wash, weigh the pellet and resuspend in water at 700 mg/mL. The pH of the silica should be between 7 and 8.https://www.cdc.gov/coronavirus/2019-ncov/downloads/Viral-Transport-Medium.pdf), i.e. 2% FBS 100μg/mL Gentamicin 0.5 μg/mL Amphotericin B in Hanks Balanced Salt Solution (HBSS).Viral transport medium was prepared as per CDC instructions   were added to each sample, the mixture was inverted, vortexed and incubated at room temperature for 5 minutes. The sample was then centrifuged for 15 seconds in a bench-top microfuge at 4600 x g and the supernatant poured off. The silica pellet was resuspended in 500 μL of 70% ethanol before being centrifuged for 15 seconds at 4600 x g (wash 1). The supernatant was poured off and the silica pellet was resuspended again in 500 μL of 70% ethanol and centrifuged for 15 seconds at 4600 x g (wash 2). After another spin at 4600 x g (15 sec), all remaining ethanol in the supernatant was carefully removed with a pipette and the silica pellet was air-dried at 65°C for 5 minutes. Finally, RNA was eluted by resuspending the silica pellet in 50μL of nuclease-free water. The tube was centrifuged for 15 seconds and the supernatant containing RNA was transferred to a fresh 1.5mL tube.All steps as above, except that the GM_MB contained 10 μL 50 mg/mL carboxylate modified magnetic SpeedBeads (Merck GE45152105050250). To collect beads manually, we employed a magnetic rack . Beads were dried for 5 minutes at room temperature.RNA extraction with QIAamp Viral RNA Mini kit (QIAGEN) was conducted as per the manufacturer’s instructions. To allow comparison with the above method, 100 μL of heat-inactivated sample was used as input material and 50 μL of nuclease-free water was used to elute RNA from the column.Plasmid DNA purified in RT-qPCR reactions were carried out using TaqMan Fast Virus 1-Step Master Mix . Primer-probe sets for used SARS-CoV-2 detection were the CDC-recommended sets N1, N2 and RNaseP Emergency Use Authorisation kit . RT-qPCR reactions containing 5 μL master mix, 1.5 μL pre-mixed primer-probe, 8.5 μL water and 5 μL purified RNA were run on a QuantStudio 5 (Applied Biosystems/ThermoFisher Scientific) using the “Fast” cycling mode. RT-qPCR for cellular RNAs in P-values are p-adjusted values from the multiple comparison tests. P-value was deemed significant when p-adjusted was < 0.05. ∗: P-adjusted < 0.05; ∗∗ P-adjusted < 0.01; ∗∗∗: P-adjusted <0.001; ∗∗∗∗: P-adjusted < 0.0001.Statistics were performed on the Ct values obtained by amplification using the N1 primer-probe sets in


Dendrite morphogenesis plays an essential role in establishing the connectivity and receptive fields of neurons during the development of the nervous system. To generate the diverse morphologies of branched dendrites, neurons use external cues and cell surface receptors to coordinate intracellular cytoskeletal organization; however, the molecular mechanisms of how this signaling forms branched dendrites are not fully understood.C. elegans in several mutants of actin regulatory proteins, such as the WAVE Regulatory Complex (WRC) and UNC-34 (homolog of Enabled/Vasodilator-stimulated phosphoprotein (Ena/VASP)). We examined the direct interaction between the WRC and UNC-34 and analyzed the localization of UNC-34 in vivo using transgenic worms expressing UNC-34 fused to GFP.We performed in vivo time-lapse imaging of the PVD neuron in C. elegans. Specifically, local increases in width (“swellings”) give rise to filopodia to facilitate a “rapid growth and pause” mode of growth. In unc-34 mutants, filopodia fail to form but swellings are intact. In WRC mutants, dendrite growth is largely absent, resulting from a lack of both swelling and filopodia formation. We also found that UNC-34 can directly bind to the WRC. Disrupting this binding by deleting the UNC-34 EVH1 domain prevented UNC-34 from localizing to swellings and dendrite tips, resulting in a stunted dendritic arbor and reduced filopodia outgrowth.We identify a stereotyped sequence of morphological events during dendrite outgrowth in the PVD neuron in We propose that regulators of branched and linear F-actin cooperate to establish dendritic branches. By combining our work with existing literature, we propose that the dendrite guidance receptor DMA-1 recruits the WRC, which polymerizes branched F-actin to generate “swellings” on a mother dendrite. Then, WRC recruits the actin elongation factor UNC-34/Ena/VASP to initiate growth of a new dendritic branch from the swelling, with the help of the actin-binding protein UNC-115/abLIM. Extension of existing dendrites also proceeds via swelling formation at the dendrite tip followed by UNC-34-mediated outgrowth. Following dendrite initiation and extension, the stabilization of branches by guidance receptors further recruits WRC, resulting in an iterative process to build a complex dendritic arbor.The online version contains supplementary material available at 10.1186/s13064-021-00154-0. Neurite outgrowth and arborization are essential for the establishment and function of neural circuits. In particular, the morphology of dendrites is key for neuronal signal transmission and integration. Dendrite morphogenesis is often guided by extracellular cues recognized through a number of cell surface receptors, such as Semaphorins, DSCAMs, and protocadherins, which in turn instruct activities of various intracellular molecules leading to cytoskeletal reorganization , 2. SimiDrosophila, lamellipodia formation during ventral closure of C. elegans embryogenesis, and collective axon extension in mice [One major signaling axis for neurite outgrowth is the activation and membrane recruitment of the actin nucleation promotion factor, the WAVE Regulatory Complex (WRC), a pentameric complex comprising Sra1/Cyfip1, Nap1/Hem-2, Abi, HSPC300, and WAVE/Scar . After b in mice –14. It iDrosophila axon patterning [Drosophila larval sensory da neurons [One hypothesis is that complex processes like neuronal morphogenesis integrate structurally distinct actin networks built by different actin regulators. For example, in dendritic spines of hippocampal pyramidal neurons, patches of loosely bundled linear actin filaments constitute the dendritic spine neck, whereas branched actin networks drive the expansion of spine heads . Furthertterning . Finally neurons . TherefoC. elegans PVD neuron, the development of dendritic arbors requires two distinct, but cooperative, steps of actin assembly that involve the WRC, UNC-34 (Ena/VASP), and the actin-binding protein UNC-115 (abLIM). As a recently developed model system for studying dendritic morphogenesis, the PVD neuron elaborates complex dendritic arbors by extending a primary dendrite along the length of the worm, followed by orthogonal secondary, tertiary, and quaternary dendrites, which together create stereotyped “menorah”-like structures [In this work, we provide evidence in support of this hypothesis showing that in the ructures . A multiructures –23. Thisructures . HoweverHere, we show that new dendrite branch points are established first as local “swellings” along dendrites, from which filopodia sprout to enable rapid and efficient outgrowth of dendritic branches. The formation of swellings is mediated by the WRC, while the subsequent extension of filopodial branches is mediated by UNC-34/Ena/VASP and UNC-115/abLIM. Our data suggest that a direct interaction between the WRC and UNC-34 provides the mechanism by which two distinct actin assembling processes are coupled to drive dendrite arborization.To identify distinct morphological events during the development of PVD dendritic arbors, we performed in vivo live imaging of the PVD dendrite at the third larval stage (L3) of wild-type worms. At this stage, the dendrite elaborates its tertiary and quaternary branches  previously implicated in neurite outgrowth. Ena/VASP proteins play a critical role in polymerizing linear F-actin to initiate and extend filopodia [C. elegans PDE neuron, and deletion of the actin-binding VHD domain greatly reduced these effects [unc-34 and null mutants of unc-115 both exhibited a strong loss of quaternary branches  plays a critical role in PVD dendrite development through its direct interaction with the guidance receptor complex that mediates PVD outgrowth [gex-2, gex-3, and wve-1 in C. elegans, respectively) all showed severely truncated dendritic arbors at the L4 stage  of Abi and WVE, and the EVH1 domain of Ena/VASP. Such an interaction was previously observed between human and  species .C. elegans WRC (ceWRC), we recombinantly purified MBP -tagged full-length (FL) Abi and WVE, MBP-tagged fragments of Abi and WVE, and a GST-tagged EVH1 domain of UNC-34 for pull-down assays  plates using OP50 Escherichia coli as a food source and maintained according to standard procedure unless otherwise noted . Worms wC. elegans were prepared by microinjection into the gonad [Pmyo2 > mCherry (2 ng/ µl) as a co-injection marker. C. elegans strains used in this study are listed in Table Expression vectors were generated in the pSMdelta vector backbone (a derivative of pPD49.26).Transgenic he gonad  using PmC. elegans. For images of mature dendrite morphology, L4 stage animals were anesthesized using 10 mM levamisole in M9 buffer and mounted on 3% agarose pads. Worms were then imaged on a 3i spinning disk microscope with a CSU-W1 spinning disk (Yokogawa) using a C-Apochromat 40x / 0.9 NA water immersion objective. Images were acquired as z-stacks  to cover the entire PVD dendritic arbor, and maximum-intensity projections were used for subsequent analyses.All images were acquired at room temperature in live For dynamic imaging of developing dendrite morphology, L3 worms were mounted onto a glass-bottom imaging dish (MatTek) as follows: Worms were picked using an eyebrow pick into a droplet of 5 mM levamisole on the glass bottom, and then covered with a 3% agarose pad. Worms were then imaged on a spinning disk microscope with a CSU-X1 spinning disk (Yokogawa) using a 40 × objective. Images were acquired every 90 s as z-stacks  to cover the entire PVD dendritic arbor, and maximum-intensity projections were used for subsequent analyses.For dynamic imaging of UNC-34:GFP, L3 worms were mounted onto a glass-bottom imaging dish as described above. Worms were imaged on a spinning disk microscope with a CSU-X1 spinning disk (Yokogawa) using a 63x/1.4NA objective. Images were acquired every 15 s as z-stacks  to image a developing menorah, and maximum-intensity projections were used for subsequent analyses in Fiji.Quantification of morphological features was done as follows: Filopodia formation was measured by manually counting the number of filopodia formed within a 12-min timeframe and normalized to the number of existing dendrites at the beginning of the timeframe. Swelling formation was measured by manually counting the number of swellings present at the end of the same 12-min timeframe and normalized to the number of existing dendrites at the end of the frame.unc-34 mutants. Swellings were usually accompanied by a local increase in morphology marker intensity.Filopodia were visually identified as protrusions no wider than 0.54 µm that extended at least 0.8 µm between 1.5-min frames. Swellings along dendrite shafts were visually identified as local increases in branch width of at least 30%, and occupying 0.54—1.4 µm of the shaft. Swellings at dendrite tips were allowed to be larger than 1.4 µm to accommodate the expanded tip swellings in Quantification of growth speed was done as follows: Length of individual dendrites was tracked for at least 13 min at 1.5-min time intervals. Positive increases in length between frames were identified as positive growth events for quantification. The standard deviation (SD) of such positive growth events was measured for each dendrite, and is presented in Figs. UNC-34:GFP enrichment to swellings before branch initiation was measured as follows: GFP intensity was measured every 15 s at the location of a future branch initiation event starting 1.75 min before growth, and normalized to the average starting intensity across all measured dendrites. Statistical comparison was done between the -1.75 min (baseline) and -1.0 min timepoints.UNC-34:GFP enrichment to dendrite tips was measured as % increase in GFP intensity at the tip of a growing dendrite as compared to GFP intensity along the shaft of the dendrite. Values from 4–10 branches were averaged per worm, and average values from a single worm were used for statistical analysis.p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05 in all graphs. All statistical analyses and graph construction were performed using Prism 8.0 software .All data are displayed as the mean ± standard error of the mean (SEM). Statistical comparisons were conducted using two-tailed Mann–Whitney tests (to test for differences between two groups) or Brown-Forsythe ANOVA with Dunnett’s multiple comparisons test (to test for differences between three or more groups). Sample sizes are indicated for each figure. ****, R (Sigma) cells after induction with 0.5 mM IPTG at 10 °C or 18 °C for 16 h. GST-UNC-34 EVH1 was purified through Glutathione Sepharose beads , followed by anion exchange chromatography using a Source 15Q column  at pH 7.0. MBP-WVE and MBP-Abi proteins were purified through amylose beads (New England Biolab). Trimeric subcomplexes containing WVE, Abi, and HSPC300 were assembled and purified by following previously described protocols [GST-UNC-34 EVH1, MBP-WVE, MBP-Abi, and MBP-HSPC300 were expressed in Arctic Express (DE3) RIL (Agilent) or BL21 (DE3) T1rotocols , 23. BriGST pull-down assays were performed as previously described . BrieflyAdditional file 1. Example branch time-lapse imaging of wild-type dendrite growth using a myr-GFP morphology marker. Images are taken every 90 seconds. Arrowhead tracks tip of an individual dendrite during outgrowth; asterisks indicate “swellings” along the dendrite. Scale bar is 5 µm.Additional file 2. Example branch time-lapse imaging of unc-34(null) dendrite growth using a myr-GFP morphology marker. Images are taken every 90 seconds. Yellow asterisks indicate persistent swellings that fail to generate filopodia and resemble lamellipodia-like growth cones. Scale bar is 5 µm.Additional file 3. Example branch time-lapse imaging of unc-115(null) dendrite growth using a myr-GFP morphology marker. Images are taken every 90 seconds. Yellow asterisks indicate persistent swellings that fail to generate filopodia. Scale bar is 5 µm.Additional file 4. Whole worm time-lapse imaging of wild-type dendrite growth using a myr-GFP morphology marker. Images are taken every 90 seconds. White arrowheads highlight three of many regions exhibiting quaternary dendrite growth. Magenta arrowhead highlights a secondary dendrite showing “rapid growth and pause” mode of growth, including swelling formation during pauses. Scale bar is 10 µm.Additional file 5. Whole worm time-lapse imaging of gex-3(null) dendrite growth using a myr-GFP morphology marker. Images are taken every 90 seconds. Arrowheads point out dendrite growth events, although overall the dendritic arbor is much more static. Scale bar is 10 µm.Additional file 6. Example branch time-lapse imaging of UNC-34:GFP localization using a myr-mCherry morphology marker. Images are taken every 15 seconds. White arrows indicate enrichment of UNC-34:GFP at tips of growing quaternary dendrites. White arrowheads indicate enrichment of UNC-34:GFP at branching sites prior to new branch initiation. Scale bar is 5 µm.Additional file 7. Example branch time-lapse imaging of UNC-34ΔEVH:GFP localization using a myr-mCherry morphology marker. Images are taken every 15 seconds. White asterisk highlights outgrowth of a quaternary dendrite with large, lamellipodia-like growth cone that lacks UNC-34ΔEVH:GFP enrichment. Scale bar is 5 µm.Additional file 8. Example branch time-lapse imaging of wild-type rescue experiment: UNC-34:GFP is expressed in an unc-34(null) worm. Dendrite growth is imaged using a myr-GFP morphology marker. Images are taken every 90 seconds. Yellow arrowheads indicate several filopodia events that emerge from swellings. Scale bar is 5 µm.Additional file 9. Example branch time-lapse imaging of UNC-34ΔEVH rescue experiment:  UNC-34ΔEVH:GFP is expressed in an unc-34(null) worm. Dendrite growth is imaged using a myr-GFP morphology marker. Images are taken every 90 seconds. Yellow asterisks indicate persistent swellings that fail to generate filopodia and resemble lamellipodia-like growth cones. Scale bar is 5 µm.Additional file 10: Figure S1. Formation of swellings during dendrite outgrowth in mutants lacking filopodia. As in Fig. unc-34 mutant (B), and unc-115 mutant (C) dendrites.


C. elegans, the bilateral Q neuroblasts and their descendants undergo long-range migrations with left/right asymmetry. QR and its descendants on the right migrate anteriorly, and QL and its descendants on the left migrate posteriorly, despite identical patterns of cell division, cell death, and neuronal generation. The initial direction of protrusion of the Q cells relies on the left/right asymmetric functions of the transmembrane receptors UNC-40/DCC and PTP-3/LAR in the Q cells. Here, we show that Q cell left/right asymmetry of migration is independent of the GPA-16/Gα pathway which regulates other left/right asymmetries, including nervous system L/R asymmetry. No extracellular cue has been identified that guides initial Q anterior versus posterior migrations. We show that collagens DPY-17 and SQT-3 control initial Q direction of protrusion. Genetic interactions with UNC-40/DCC and PTP-3/LAR suggest that DPY-17 and SQT-3 drive posterior migration and might act with both receptors or in a parallel pathway. Analysis of mutants in other collagens and extracellular matrix components indicated that general perturbation of collagens and the extracellular matrix (ECM) did not result in directional defects, and that the effect of DPY-17 and SQT-3 on Q direction is specific. DPY-17 and SQT-3 are components of the cuticle, but a role in the basement membrane cannot be excluded. Possibly, DPY-17 and SQT-3 are part of a pattern in the cuticle and/or basement membrane that is oriented to the anterior–posterior axis of the animal and that is deciphered by the Q cells in a left–right asymmetric fashion. Alternatively, DPY-17 and SQT-3 might be involved in the production or stabilization of a guidance cue that directs Q migrations. In any case, these results describe a novel role for the DPY-17 and SQT-3 collagens in directing posterior Q neuroblast migration.Cell adhesion molecules and their extracellular ligands control morphogenetic events such as directed cell migration. The migration of neuroblasts and neural crest cells establishes the structure of the central and peripheral nervous systems. In  C. elegans, the Q neuroblasts and their descendants undergo long-range migrations in a left–right (L/R) asymmetric fashion  and peripheral nervous system . In iewed in ). The biiewed in ,3,4,5. Qiewed in ,3,4,5.While similar in origin and development, QL and QR have an inherent L/R asymmetry that controls anterior–posterior (A/P) migration ,6. In QLC. elegans, including internal organ asymmetries of the gut and gonad, which is often reversed in gpa-16 mutants , ayIs9[Pegl-17::gfp] LGIII: dpy-17, dpy-31(e2919), emb-9, hse-5(tm472 and ok2493) LGIV: dpy-13(e184), unc-129(ev554), epi-1, col-99(ok1204) LGV: sqt-3, dbl-1(nk3), lqIs58[Pgcy-32::cfp]. LGX: him-4, let-2, lqIs97[Pscm::mCherry]. The temperature-sensitive lethal emb-9 and let-2 alleles were grown at permissive 15 °C and shifted to nonpermissive 25 °C as late embryos. gpa-16(it143) mutants were grown at 25 °C.Genetics: ayIs9[Pegl-17::gfp] or lqIs97[Pegl-17::mCherry] transgene and standard epifluorescence microscopy.L1 synchronization and Q cell imaging: L1 larvae were synchronized by hatching as previously described ,15. GravScoring of AQR and PQR migration: AQR and PQR, neuronal descendants of QR and QL, respectively, migrate the longest distances of Q descendants into the head and tail of the animal. AQR migrates anteriorly to a region near the anterior deirid and the posterior pharyngeal bulb. PQR migrates posteriorly past the anus into the phasmid ganglia in the tail. AQR and PQR positions in young adult animals were scored as previously described ,15. The QL and QR left–right asymmetry of migration does not involve GPA-16. The Q cells are born in the embryo as the sisters of the V5 epidermal seam cells. Before migration, they reside between the V4 and V5 seam cells in the posterior of the animal (gpa-16(it143) temperature-sensitive mutants, L/R asymmetric organ placement is frequently reversed, including L/R gut–gonad asymmetric placement.e animal . Migratie animal A–C [1,4,gpa-16(it143) mutants at 25 °C, initial QL and QR protrusions and migrations were unaffected. QL protruded and migrated posteriorly, and QR protruded and migrated anteriorly (n = 50 for each). Furthermore, the Q descendants, AQR and PQR, migrated normally, even in animals with reversed gut–gonad L/R asymmetry  (COL24A1) [dpy-17(e164), a predicted null mutant, displayed defects in AQR and PQR migration. AQR sometimes migrated posteriorly, and PQR sometimes migrated anteriorly (dpy-17(e1295) mutant displayed similar defects, and the hypomorphic dpy-17(e1345) mutant showed fewer defects (COL24A1) . dpy-17(teriorly . The dpy defects .dpy-17. In dpy-17(e164), 6/100 QL migrated anteriorly and divided over V4  (COL21A1). Genetic and biochemical studies suggest that DPY-17 and SQT-3 interact in the same collagen structures, possibly in hetero-oligomeric trimers [sqt-3 mutants e2924 and e2906 showed AQR and PQR reversals to a level similar to dpy-17(e164) (sqt-3(e2924) is likely a null allele [sqt-3(sc63) mutation results in a dominant left-handed Roller phenotype but showed no AQR or PQR migration reversals alone ( trimers . sqt-3 m17(e164) . sqt-3; sqt-3(e2924) double mutants displayed AQR and PQR migration defects that were not significantly different than either single mutant alone (dpy-17(e164) double mutants with dominant sqt-3(sc63) displayed significantly increased PQR migration defects compared to dpy-17(e164) alone  might have a dominant interfering effect on PQR migration that is revealed in the absence of DPY-17.nt alone , suggestone 31%; , suggestdpy-10, sqt-1, and dpy-13 showed no AQR or PQR migration defects (dpy-31(e2919) and dbl-1(nk3) predicted null mutants displayed no AQR or PQR migration defects, nor did unc-129(ev554), which is predicted to encode a TGFβ-like molecule that controls axon guidance [Mutations in the cuticle collagen genes  defects , suggestguidance  ; unc-40(n324) double mutants, defective anterior migration of PQR was significantly enhanced (dpy-17(e1295); unc-40(n324) showed a similar significant effect, but hypomorphic dpy-17(e1345); unc-40 did not (sqt-3(e2924); unc-40(n324) double mutants also showed significant enhancement of PQR migration defects. These results suggest that DPY-17 and SQT-3 act in parallel to UNC-40/DCC in posterior migration.In  did not . sqt-3(edpy-17(e164); ptp-3(mu245) doubles showed significantly enhanced PQR anterior migration defects, but to a lesser extent than dpy-17; unc-40. Defective AQR posterior migration was enhanced in dpy-17; ptp-3 (p = 0.052), an effect not observed in dpy-17; unc-40. These results indicate that DPY-17 might act in parallel to UNC-40 in posterior PQR migration. Interaction with PTP-3 was more complex, with DPY-17 potentially acting in parallel for both posterior PQR migration and anterior AQR migration.EPI-1/lamininα5 and HIM-4/hemicentin affect the ability of AQR and PQR to migrate. EPI-1 and HIM-4 are extracellular matrix constituents known to control cell migration [epi-1 mutants displayed high-frequency defects in AQR and PQR migration, but few directional defects (epi-1 affected the ability of AQR and PQR to execute their migration but did not affect direction of migration. epi-1(rh233) did not enhance directional migration defects of unc-40(n324) (unc-40(n324), 48% of misdirected PQRs migrated completely anteriorly to position 1, whereas only 13% did so in epi-1(rh233); unc-40(n324) double mutants, p < 0.0001). him-4 mutants displayed low-frequency failures in AQR and PQR migration. One allele of three analyzed displayed one misdirected PQR in position 3 in the midbody. One allele, him-4(rh319), significantly enhanced PQR defects of unc-40 and ptp-3, but another, him-4(e1266), did not significantly affect AQR and PQR migration defects of unc-40 or ptp-3. Together, these data suggest that EPI-1 is required for the ability of cells to migrate but is not involved in the direction of migration. HIM-4 also controls the ability of cells to migrate but might also have a role in PQR direction of migration, although the effect is not as strong as DPY-17.igration ,33,34,35 defects . In othe40(n324) . Rather,EMB-9/collagenIVα5 controls PQR directional migration.emb-9 mutants display a variety of developmental defects, including defective migration of the gonadal distal tip cells [emb-9 mutants displayed low-frequency reversals of PQR migration, as well as incomplete AQR migration  family of collagens [col-99 mutants displayed no defects in AQR or PQR migration  displayed low-frequency defects in AQR and PQR directional migration but did not significantly enhance unc-40 or ptp-3  is a dominant right-hand Roller [e itself  might pre itself . Possible itself ,55. Howee animal . Roller slinking ,56. IndeDPY-17 and SQT-3 interact genetically with the UNC-40/DCC and PTP-3/LAR receptors. UNC-40/DCC and PTP-3/LAR are transmembrane receptors that act in the Q cells and control initial direction of migration [dpy-17 and sqt-3 enhance PQR directional migration defects of unc-40 in a manner similar to ptp-3, suggesting that DPY-17/SQT-3 might act in the PTP-3 pathway. However, dpy-17 also enhanced PQR defects of ptp-3, suggesting it functions at least partially in parallel to PTP-3 as well. Possibly, DPY-17 acts in both pathways or defines a third pathway in posterior migration. dpy-17 also enhanced AQR defects of ptp-3, a phenotype not observed in dpy-17; unc-40 doubles. DPY-17 might act in parallel to PTP-3 in anterior AQR migration, potentially in mutual inhibition of UNC-40/DCC, resulting in increased posterior migration of AQR. The L/R asymmetric function of UNC-40/DCC and PTP-3/LAR might control how the Q cells respond to the DPY-17/SQT-3 pattern , as well as permissive substrate required for migration . This reinforces the notion that DPY-17 and SQT-3 have specific roles in directing cell migration, and that general perturbations to the ECM do not cause directional defects.Factors regulating heparan sulfate proteoglycans control Q cell direction of migration. The heparan sulfate epimerase HSE-5 is predicted to catalyze the epimerization of d-glucuronic acid (GlcA) to l-iduronic acid (IdoA) in the heparan side chains of heparan sulfate proteoglycans. HSE-5 was previously shown to control directional QR and QL migration and AQR/PQR migration [hse-5 displayed complex genetic interactions with unc-40 and ptp-3, consistent with the possibility of HSE-5 acting with multiple HSPGs in different aspects of AQR and PQR migration.igration ,47. hse-unc-52, which encodes the basement membrane HSPG UNC-52/perlecan, with functional consequences on body wall muscle development [mec-8 mutants displayed low-frequency defects in AQR/PQR migration direction but did not enhance unc-40 or ptp-3 significantly. However, mec-8 suppressed PQR migration defects of ptp-3 similar to hse-5. Possibly, UNC-52 controls AQR/PQR directional migration. Hypomorphic unc-52 alleles, caused by mutations in exons affected by mec-8, did not cause AQR or PQR defects [unc-52 null mutants arrested before gcy-32::cfp expression in AQR and PQR (data not shown). No single or double mutant in genes encoding HSPGs resulted in AQR/PQR directional defects [The RNA processing factor MEC-8 regulates the alternative splicing of elopment . mec-8 m defects , and unc defects , but it The results presented here show that the collagens DPY-17 and SQT-3 instruct A/P migration of the Q neuroblasts. An intriguing idea is that DPY-17 and SQT-3 might be part of a pattern in the A/P axis of the ECM that instructs Q migration, either in the basement membrane or in the cuticle. Alternately, they could be involved in the production of an as-yet unidentified guidance cue that instructs Q migrations. In any event, these results demonstrate that collagens are involved in providing instructional guidance information to neuroblasts in vivo.


C. amycolatum strains isolated from healthy women. Previous studies have shown that these strains produced metabolites that significantly increased the antagonistic activity of peroxide-producing lactic acid bacteria against pathogenic and opportunistic microorganisms and had strong antimicrobial activity against opportunistic pathogens. Analysis of the C. amycolatum genomes revealed the genes responsible for adaptation and survival in the vaginal environment, including acid and oxidative stress resistance genes. The genes responsible for the production of H2O2 and the synthesis of secondary metabolites, essential amino acids and vitamins were identified. A cluster of genes encoding the synthesis of bacteriocin was revealed in one of the annotated genomes. The obtained results allow us to consider the studied strains as potential probiotics that are capable of preventing the growth of pathogenic microorganisms and supporting colonisation resistance in the vaginal biotope.The vaginal microbiome of healthy women contains nondiphtheria corynebacteria. The role and functions of nondiphtheria corynebacteria in the vaginal biotope are still under study. We sequenced and analysed the genomes of three vaginal  The vaginal microbiome is an open complex multicomponent system in dynamic equilibrium . The vagGardnerella vaginalis, or various species of Peptostreptococcus spp., Prevotella spp., Pseudomonas spp., Streptococcus spp. and/or Corynebacterium spp. Such changes in the structure of the vaginal microbiome are not considered a pathological disorder [However, using culture-independent methods based on sequencing of the 16S (rRNA) gene, researchers demonstrated that a significant proportion 7–33%) of healthy women lack lactobacilli in their vagina ,6. It is–33% of hdisorder .Corynebacterium genus contains approximately 130 different species of diverse and ecologically significant microorganisms. The well-known typical representatives of this genus are the pathogenic species C. diphtheriae, C. ulcerans, and C. pseudotuberculosis [The rculosis , and therculosis . For therculosis ,12. Indirculosis . There arculosis .Staphylococcus epidermidis constitute the main part, approximately 80% of the vaginal microbiota, in prepubescent girls [Nondiphtheriae corynebacteria in vaginal biotopes were found in women regardless of age and microecological status ,17,18. Nnt girls . The numnt girls . Despitent girls ,22.Corynebacterium amycolatum was isolated for the first time by Collins and Burton from clinical specimens in 1988 [C. amycolatum is rather frequently isolated from vaginal biotopes of healthy women, and features a high probiotic potential. Particularly, we isolated three strains of corynebacteria from the vaginal contents of healthy women. All of them were identified as C. amycolatum. Metabolites of these strains greatly increased the antagonistic activity of peroxide-producing lactobacilli against pathogenic and opportunistic microorganisms and had strong antimicrobial activity against opportunistic pathogens such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa [Corynebacterium amycolatum have not yet been previously described. in 1988 . Based oruginosa ,25. ThesC. amycolatum ICIS 5, ICIS 9 and ICIS 53 were previously isolated from vaginal smears of healthy women of reproductive age. The strains are deposited in the Collection of Microorganisms of the Institute for Cellular and Intracellular Symbiosis UrB RAS  under the same accession names. The phenotypic characteristics of these isolates have been previously described in detail [v/v) glycerol before experiment. The isolates were grown in tryptic soy broth (TSB) at 37 °C for 24 h.Strains of n detail ,25. The Overnight bacterial cultures were used for extraction genomic DNA with the phenol-chloroform method. DNA libraries were prepared and sequenced at the Center of Shared Scientific Equipment “Persistence of microorganisms” at the Institute for Cellular and Intracellular Symbiosis UrB RAS . The Nextera XT DNA library preparation kit  was used according to the manufacturer’s instructions. High-throughput sequencing of the DNA libraries was carried out in the MiSeq sequencer  using the MiSeq reagent kit v3 2 × 300 cycles . The reads were quality-trimmed with the Trimmomatic tool . De novohttp://eggnog.embl.de/version_3.0/, accessed on 19 December 2021) [Functional annotation of the genomes was carried out by the RAST server (Rapid Annotation using Subsystem Technology)  and NCBIer 2021) . The bioer 2021)  and Antier 2021)  were useer 2021) . The preer 2021) . The CRIer 2021) .C. amycolatum strains ICIS 5 (WGS Project: SSOR01), ICIS 9 (MTPT01) and ICIS 53 (MIFV01), draft genomes of nine C. amycolatum strains currently available in the NCBI database  [https://www.ncbi.nlm.nih.gov/, accessed on 19 December 2021). Sequences were aligned by MUSCLE [Phylogenetic analysis was conducted based on the 16S rDNA sequences retrieved from draft genomes of the database , includier 2021) . The 16Sy MUSCLE  with Uniy MUSCLE  using deC. amycolatum strains currently available at NCBI was determined by calculating the average nucleotide identity (ANI) and orthologous average nucleotide identity (OrthoANI) using OAT (version v. 0.93.1) software [Genome similarity of the strains ICIS 5, ICIS 9 and ICIS 53 and other software .C. amycolatum ICIS 5, ICIS 9 and ICIS 53, respectively. The strains C. amycolatum ICIS 9 and ICIS 53 were deposited in the culture collection of the All-Russian Collection of Microorganisms at the G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms  under registration no. VKM Ac-2843D and VKM Ac-2844D, respectively.The annotated genome sequences were deposited in the GenBank database as sequencing project PRJNA339674 with accession numbers SSOR00000000, MTPT00000000 and MIFV00000000 for As shown in http://www.ncbi.nlm.nih.gov/genome/annotation_prok (19 December 2021)), and 2195 coding sequences, including 2062 proteins (CDSs), 47 pseudogenes, complete rRNAs  and 53 tRNAs, were identified. The identified coding proteins were classified into 26 functional categories based on COG classification  Prokaryotic Genome Annotation Pipeline (PGAP)  and 53 tRNAs . The ideThe draft genome of strain ICIS 53 was composed of 2,460,257 bp, with an N50 length of 170,410 bp, an L50 of 4, and a G + C content of 59.0%. The final assembled genome consisted of 41 contigs. Genome annotation identified 2173 coding sequences, including 2076 proteins, 34 pseudogenes, 5, 1, and 1 complete rRNAs  and 53 tRNAs . The ideC. amycolatum from the NCBI database. The, C. amycolatum clade with a sister branch represented by Corynebacterium xerosis ATCC 373T was clearly separated from another clade containing all other species of Corynebacterium spp.  between ICIS 5, ICIS 9 and ICIS 53 ranged from 96.79% to 97.87%, and the average nucleotide orthology (OrthoANI) ranged from 96.89% to 97.93%  [C. amycolatum strains, ANI values ranged from 94.26% to 98.02%, OrthoANI values ranged from 94.3% to 98.1%.ANI values between our three strains and a type SK 46 strain of  95–96%) ,42. RelaCorynebacterium spp. must have evolved mechanisms of adaptation. In the studied genomes, we identified a large number of genes encoding proteins involved in stress response. These stresses included pH, temperature, osmotic pressure, nitrosative and oxidative stress. The detailed analysis of genes coding for proteins involved in stress response in the genomes of ICIS 5, ICIS 9 and ICIS 53 is shown in The vaginal ecosystem is an aggressive environment for most microorganisms. The acidic environment in the vagina creates a natural filter; as a result, most pathogens and opportunistic microbes die. In order to survive and successfully colonise this ecosystem, microorganisms of the genus +/H+ antiporters are membrane proteins that play a major role in pH and Na+ homeostasis of cells [ICIS 5, ICIS 9 and ICIS 53 contain 7 genes that encode F0F1-ATPase. Membrane-bound ATP synthases (F0F1-ATPases) of bacteria serve two important physiological functions. The enzyme catalyses the synthesis of ATP from ADP and inorganic phosphate utilizing the energy of an electrochemical ion gradient. On the other hand, under conditions of low driving force, ATP synthases function as ATPases, thereby generating a transmembrane ion gradient at the expense of ATP hydrolysis . Such acof cells . The anaof cells . Furtherof cells ,47. We aof cells ,49.Corynebacterium glutamicum to antibiotics, alkylating agents, ethanol and heavy metals [A large number of genes associated with oxidative stress were identified in the studied genomes. They can play a crucial significance for survival and adaptation of the bacteria in the vaginal niche. The genomes contain genes that encode catalase, thiol peroxidase and glutathione peroxidase, which are antioxidant protective enzymes capable to detoxify reactive oxygen species ,51,52. Ty metals ,60. The C. amycolatum [In order to survive and successfully colonise the vaginal biotope, nonpathogenic corynebacteria must have the ability to produce secondary metabolites with antimicrobial activity and determine their competitive advantage . In the ycolatum . The corAll enzymes are encoded by a single gene, except isoprenyl transferase (undecaprenyl diphosphate synthase), which is encoded by two gene copies. Isoprenyl transferase catalyses the condensation of isopentenyl diphosphate (IPP) with allylic pyrophosphates, generating different types of terpenoids . FarnesyBacteriocins are antimicrobial peptides ribosomally produced in bacteria, either processed or not by additional post-translational modification (PTM) enzymes, and exported to the extracellular medium ,75. Of tClostridia and some human multidrug-resistant bacterial pathogens [Sactipeptides are a new class of synthesized in ribosomes and post-translationally modified peptides (RiPPs). Sactipeptides are known as antibiotics with narrow spectrum capable to inhibit athogens . The revathogens . The pre2O2 is one of the key factors in maintaining vaginal biotope homeostasis [Heostasis . In the eostasis , these eMicroorganisms that colonize various biotopes of the human body form multi-species communities and represent a kind of “organ”, which, in turn, affects the functioning of all organs and systems that play an important role in maintaining the health of the host. Using intestinal microbiota as an example, commensal bacteria have been shown to be important sources of vitamins and amino acids. In addition to their nutritional/physiological properties, many of these vitamins are also involved in the development and functioning of host immune cells, as there is a direct link between biosynthetic biosynthesis intermediates derived from commensal bacteria and immune cells that directly recognise them ,81. The C. amycolatum strains for antibiotic resistance genes and found genes encoding resistance to antibiotics in only two strains, ICIS 5 and ICIS 9. The genome of strain ICIS 5 contained genes encoding resistance to chloramphenicol and aminoglycosides , terpenes and sactipeptides. The analysis of C. amycolatum genomes revealed common genes for all three strains; for example, genes encoding adaptation and survival in the vaginal environment, as well as unique genes determining strain specificity. The presence of an AOI encoding sactipeptide in the genome of strain ICIS 9 suggests that this strain may produce the sactipeptide. However, this assumed feature should be checked further through isolation and characteristics of this peptide.We presented a comparative study of draft genomes for three


Xist RNA plays an essential role in X chromosome inactivation (XCI) in female mammals. It coats the X chromosome in cis and mediates the recruitment of many proteins involved in gene silencing and heterochromatinization. The molecular basis of how Xist RNA initiates chromosomal silencing and what proteins participate in this process has been extensively studied and elucidated. Its involvement in the establishment and maintenance of the X-inactivated state is, however, less understood. The XistIVS allele we previously reported is peculiar in that it can initiate XCI but fails to establish the inactive state that is stably maintained and, therefore, may provide an opportunity to explore how Xist RNA contributes to establish a robust heterochromatin state. Here we demonstrate that ectopic splicing taking place to produce XistIVS RNA disturbs its function to properly establish stable XCI state. This finding warrants the potential of XistIVS RNA to provide further insight into our understanding of how Xist RNA contributes to establish sustainable heterochromatin.Non-coding  Xist RNA is one of the most extensively studied lncRNAs  associates with chromatin and regulates chromatin state.  lncRNAs , which b lncRNAs  and indu lncRNAs . In the  lncRNAs , which c lncRNAs . Xist RNXist and its association with the chromosome in cis upon induction of differentiation  have been extensively used for the study of XCI as they carry two active X chromosomes, one of which undergoes chromosome-wide silencing with monoallelic upregulation of ntiation . Functio embryos  but alsoXist RNA . A more Xist RNA . GeneticXist RNA .Xist, IVSXist, which initiates XCI in the extraembryonic tissues upon paternal transmission, but the XCI state initiated by XistIVS is not stable, resulting in derepression of X-inactivated genes in the extraembryonic tissues  0.9 kb downstream of the major transcription start site of Xist as a control allele for the analysis of antisense regulation by Tsix  females and C57BL/6J (B6) males. JB4, one of the established ESCs, was adapted to grow on a gelatin-coated dish with conventional ESC medium containing 2i  for some passages. For induction of differentiation, ESCs were cultured with N2B27 medium on gelatin-coated dishes for up to 7 days.Embryonic stem cells used in this study were established from E3.5 blastocysts according to Eif2s3x, a 938-bp fragment present downstream of a termination codon of Eif2s3x was amplified by PCR using primers, Eif2s3x-F (5′-actctgtaga caaggctggc-3′) and Eif2s3x-R (5′-TTCTGTAGGGAGAATTGG CC-3′), on B6 genomic DNA and cloned into pBluescriptII-SK(+), in which the SpeI site present in MCS had been destroyed. This plasmid was linearized at the SpeI site present in the Eif2s3x fragment, and an IRES neo cassette was subsequently cloned in an appropriate orientation to generate p3′EifIRESneo. For construction of a targeting vector for Hprt, a 763-bp fragment containing an ATG start codon of Hprt was amplified by PCR using primers, gHprt-F1 (5′-agacgacagagggcctgggggctgc-3′) and gHprt-R1 (5′-ttgtagagctgggcctctcccagga-3′), on JF1 genomic DNA. This plasmid was used for inverse PCR using primers Hprt-invR(Spe) (5′-gggaaacttactagtCGGCAAAAAGC GGTCTGAGGAGGAAGC-3′) and Hprt-invF(Pst) (5′-gggaaact tctgcagCGACCCGCAGTCCCAGCGTCGTGgtga-3′), and the amplified fragment was circulized by self-ligation. The resultant plasmid was digested by PstI and SpeI and used to clone a CAG-Zeo-pA cassette to produce pHprt_invF/R-Zeo.For construction of a targeting vector for XistCAGIVS2lox allele, a 0.9-kb IVS fragment amplified on genomic DNA carrying the XistIVS allele by PCR according to Xist promoter with the CAG promoter  linearized by BbsI to generate pX330-EifgRNA1, pHprt-gRNA, and pX330-Xist(-20). sgRNA sequences are as follows: Eif2s3x (5′-ATTTATAGCTGCTACTAGTA-3′), Hprt (5′-TGACGGGACCGGTCGGCTCG-3′), and Xist (5′-GATCAGTTAAAGGCGTGCAA-3′).Eif2s3x locus, which is known to escape XCI, and the Hprt locus were selected for the site to introduce an IRESneo cassette and a CAGzeo-pA cassette, respectively. JB4 ESCs were used at P8 to introduce an IRESneo cassette at the Eif2s3x locus, and 5 × 105 cells were transfected with 1 μg of pX330-EifgRNA1 and 1 μg of p3′EifIRESneo using FuGENE HD Transfection Reagent  and seeded on two gelatin-coated 60-mm dishes. Twenty-four hours later, selection was started with conventional ES medium containing 2i/LIF and 200 μg/ml of G418 for 8 days. Of the 40 colonies isolated, 12 turned out to harbor expected homologous recombination at one of the two Eif2s3x alleles. One of the 12 clones, termed JB4/EI7, which were confirmed to have an IRESneo cassette integrated on the X chromosome derived from B6, was used for targeting a CAGzeo-pA cassette at the Hprt locus on the X chromosome derived from JF1. Five hundred thousand cells were transfected with 1 μg of pHprt-gRNA and 1 μg of pHprt_invF/R-Zeo, as above. Selection was carried out using conventional ES medium containing 2i/LIF and 25 μg/ml of Zeocin for 10 days. Of the 40 colonies screened by PCR, one was found to harbor a correct homologous recombination at the Hprt locus on the X chromosome derived from JF1 and termed JB4/EI7HZ2. JB4/EI7HZ2 retained two X chromosomes in 99% of the population after 70 days of culture in the presence of G418 and Zeocin.The XistCAGIVS2lox and XistCAG16in2lox, respectively. To identify which of the B6 or JF1 alleles was targeted, a relevant region containing a single-nucleotide polymorphism (SNP) was amplified by PCR and analyzed by restriction digestion and sequencing. A floxed PacECFP-pA cassette was removed by transient expression of Cre recombinase to convert XistCAGIVS2lox and XistCAG16in2lox into XistCAGIVS and XistCAG16in, respectively.Five hundred thousand JB4/EI7HZ2 cells were transfected with 1 μg of pX330-Xist(-20) and 1 μg of either pCAG-CΔM20-IVS#5 or pCAG16in#3, as above. Selection was carried out using conventional ES medium containing 2i/LIF and 2 μg/ml of puromycin for 7–10 days. Twelve of 48 and 24 of 96 colonies isolated turned out to harbor Xist and Atrx probes were prepared using pXist_SS12.9 and a BAC clone P23-260I15, respectively, as previously described  or Carnoy’s fixative as previously described . For PFAescribed .Xist and Atrx probes overnight at 37°C, followed by washes for 5 min twice at 37°C in each of 2xSSC/50% formamide and 2xSSC/0.05% Tween 20 and subsequent counterstaining with Hoechst 33258.Whole-mount RNA-FISH was carried out according to Xist, probes were prepared according to Xist prepared according to For allele-specific RNA-FISH for XhoI, whose restriction site is present in the fragment derived from wild-type Xist RNA but destroyed in that derived from XistIVS and Xist16in . One-fiftieth of the reaction was used as a template for PCR using a primer set, R700P2, and F1063AS . The PCRXist16in .XistIVS on XCI in the embryonic lineage, we established wild-type and XistIVS/XistIVS ESCs from blastocysts obtained from a cross between XistIVS/+ females and XistIVS/Y males. They were induced to differentiate for 5 days and examined for Xist expression by RNA-FISH. While Xist was monoallelically upregulated in about 40% of wild-type cells, the majority of XistIVS/XistIVS cells exhibited two pinpoint signals and none contained the Xist cloud  6.5 for whole-mount RNA-FISH. Since the Xist1lox allele is functionally null and does not form the Xist cloud  by visual inspection. XistIVS and Xist1lox/XistIVS embryos examined at E6.5 for the expression of Xist and another X-linked Atrx gene by whole-mount RNA-FISH. In the +/XistIVS embryo, the Xist cloud was detected in the epiblast as well as the visceral endoderm layer surrounding the epiblast. Atrx was monoallelically expressed in these tissues, suggesting that one Atrx allele was silenced by XCI. In contrast, the Xist cloud was detected in the visceral endoderm but not in the epiblast of Xist1lox/XistIVS embryos. A pinpoint Xist signal detected was, however, often juxtaposed to a pinpoint signal of Atrx. Since even if RNA was transcribed from the Xist1lox allele, it was truncated due to the insertion of an IRESEGFP-pA cassette in exon1 and would not be detected by the Xist probe used, and the pinpoint signals detected by the Xist probe should represent RNA transcribed from the XistIVS allele, suggesting a defect of the XistIVS allele to undergo upregulation. It was, therefore, likely that XCI was not induced in the epiblast of Xist1lox/XistIVS embryos, resulting in developmental failure of the epiblast. Taken together with the result in female ESCs homozygous for XistIVS, we concluded that the XistIVS allele was defective in upregulating its transcription in the epiblast lineage. This contrasts with the fact that the XistIVS allele is upregulated and capable of inducing XCI in the extraembryonic lineages and suggests some differences in the mechanism of Xist upregulation between the embryonic and extraembryonic lineages.To investigate the effect of st cloud . Extensist cloud , RNA-FIS embryos , in whicXistIVS allele to be upregulated, we attempted to replace the endogenous Xist promoter with the CAG promoter (B6) and the Zeocin resistance gene on the X derived from JF1 (XJF1) and, therefore, stably maintained both X chromosomes in the presence of G418 and Zeocin in culture medium . We isolated several lines harboring the XistCAGIVS2lox allele on either X chromosome and chose two lines, one targeted on the XB6 (IVS-2L-B47) and the other on the XJF1 (IVS-2L-J19), for further analyses. A floxed PacECFPpA cassette was removed by transient expression of Cre recombinase in IVS-2L-B47 and IVS-2L-J19 lines to obtain IVS-B47#24 and IVS-J19#3, respectively, in which the XistCAGIVS2lox allele was converted into the XistCAGIVS allele.To circumvent the defect of the promoter  in F1 hypromoter . ESCs usXistCAGIVS2lox (IVS-2L-B47 and IVS-2L-J19) or XistCAGIVS (IVS-B47#24 and IVS-J19#3) were allowed to differentiate for up to 7 days in N2B27 medium without G418 and Zeocin, and expression of Xist and X-linked Atrx was examined by RNA-FISH. Given our previous studies of the targeted Xist alleles generated in a similar scheme, XistCAG2L and CAGΔ5′L–2Xist   . Expressreported . FollowiXist RNA forming the Xist cloud in differentiating female ESCs, we employed allele-specific RNA-FISH using labeled B6- and JF1-specific oligo probes that differentially hybridized to Xist RNA derived from the B6 and JF allele, respectively , and their parental line containing a floxed PacECFPpA cassette, 16in-2L-B11 and 16in-2L-B38, respectively, were allowed to differentiate in N2B27 medium and examined for Xist and Atrx expression by RNA-FISH. In all cases, the proportion of cells containing the Xist cloud gradually increased over time  bind in the vicinity of the exon-exon junction. It is, therefore, reasonable to expect that such proteins would bind to the processed XistIVS RNA but not to Xist16in RNA. The IVS2 sequence was located about 0.2 kb downstream of the A-repeat, which is essential for the silencing function of Xist RNA  as well as monoallelic expression of either allele makes it difficult to compare the quantity of the RNA produced from the respective wild-type and XistCAGIVS allele. To circumvent this problem, we are currently attempting to derive a unique cell population with monoallelic expression of either allele or that with biallelic expression of both by inducing differentiation of ESCs into neural stem cells and subsequent cloning.We expected that when female ESCs heterozygous for Xist RNA  and mediXist RNA  and RBM1Xist RNA . We are Xist mutant allele so far generated compromise the initiation process of XCI, XistIVS RNA is peculiar in that it can initiate XCI but fails to maintain the XCI state. This is most probably due to the failure of the establishment of robust heterochromatin. It is likely that proteins recruited by Xist RNA contribute to not only the initiation of XCI but also the establishment of a sustainable heterochromatin state. XistIVS RNA would provide a unique opportunity to explore such factors involved in the latter process and how Xist participates in the establishment of the chromatin state required for the stable maintenance of the X-inactivated state.Since most of the The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the committee for the Care and Use of Laboratory Animals of Kindai University.TS and TN designed the study. RM, TN, SI, and TS performed the experiments and analyzed the data. TS wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The collagen architecture in the extracellular matrix (ECM) is highly remodeled in high grade serous ovarian cancer (HGSOC). Many of these tumors begin in the fallopian tubes (FT) before metastasizing to the ovaries and it is important to study ECM alterations in carcinogenesis. Here, we used Second Harmonic Generation (SHG) microscopy to classify changes in the collagen fiber morphology in normal FT, and precursor pure p53 signatures and serous tubal intraepithelial carcinoma (STICs) in tissues with no HGSOC. Using a machine learning approach based on image features, we were able to discriminate the tissue groups with good classification accuracy. We additionally performed mass spectrometry analysis of normal and HGSOC tissues to associate the differential expression of collagen isoforms with fiber morphology alterations. This work provides new insights into ECM remodeling in early stage HGSOC and suggests the combined use of SHG microscopy and mass spectrometry as a new diagnostic/prognostic approach.Background: The collagen architecture in high grade serous ovarian cancer (HGSOC) is highly remodeled compared to the normal ovary and the fallopian tubes (FT). We previously used Second Harmonic Generation (SHG) microscopy and machine learning to classify the changes in collagen fiber morphology occurring in serous tubal intraepithelial carcinoma (STIC) lesions that are concurrent with HGSOC. We now extend these studies to examine collagen remodeling in pure p53 signatures, STICs and normal regions in tissues that have no concurrent HGSOC. This is an important distinction as high-grade disease can result in distant collagen changes through a field effect mechanism. Methods: We trained a linear discriminant model based on SHG texture and image features as a classifier to discriminate the tissue groups. We additionally performed mass spectrometry analysis of normal and HGSOC tissues to associate the differential expression of collagen isoforms with collagen fiber morphology alterations. Results: We quantified the differences in the collagen architecture between normal tissue and the precursors with good classification accuracy. Through proteomic analysis, we identified the downregulation of single α-chains including those for Col I and III, where these results are consistent with our previous SHG-based supramolecular analyses. Conclusion: This work provides new insights into ECM remodeling in early ovarian cancer and suggests the combined use of SHG microscopy and mass spectrometry as a new diagnostic/prognostic approach. High Grade Serous Ovarian Cancer (HGSOC) is a highly metastatic disease, defined genetically by mutations in the tumor suppressor genes Tp53, BRCA I, and BRCA II, and DNA copy number alterations . While tSerum tests CA125 and HE4) do not have sufficient specificity and sensitivity for early reliable diagnosis  and HE4 ,7. As a For this purpose, we utilized the high-resolution collagen specific, optical modality of Second Harmonic Generation (SHG) microscopy to probe all levels of collagen structure (molecular through fiber). Importantly, we previously developed machine learning algorithms to differentiate between normal and high-risk ovarian stromal tissues as well as cancer sub-types based on the 3D collagen fiber morphology patterns . We wereWe have also documented sub-resolution macro/supramolecular changes (protein helix attributes) and fibril organization (size and packing) in the aberrant tissue classes ,11. CollAs the majority of HGSOC cases originate through precursors in the fallopian tube (FT) secretory epithelium ,13,14,15In contrast, STICs are associated with high p53 intensity and the acquisition of cellular changes, where the morphology becomes more disorganized. Malignant disease in the FT (and primary ovary) that develops from the STICs has additional morphological alterations along with a high proliferative index . SimilarWe recently used SHG microscopy to investigate the collagen fiber structure in concurrent STIC and HGSOC fallopian tube tissues . InteresHere, we extended these studies to pure p53 and STIC precursors to determine if early changes in the collagen fiber morphology are detectable along with the p53 molecular changes. It is also important to complete these studies in the absence of concurrent HGSOC as these lesions can result in the transformation of distant collagen through a field effect mechanism ,19,20,21N = 3) and tumor fallopian tube tissues (N = 3) were analyzed via mass spectrometry, while archived fallopian tube tissues (N = 12) were analyzed via SHG microscopy. In this retrospective study, archived fallopian tubes and ovarian tissues from the University of Wisconsin Carbone Cancer Center Tissue Bank and the University of Wisconsin Department of Surgical Pathology were analyzed under an IRB-approved protocol (protocol #2019-0211). Flash frozen normal  and sonicated using a probe sonicator . Protein extracts were reduced with 10 mM dithiothreitol (DTT) for 30 min at room temperature and alkylated with 50 mM iodoacetamide for another 30 min in the dark before quenching with DTT. The proteins were then precipitated with 80% (v/v) cold acetone (−20 °C) overnight. The samples were centrifuged at 14,000× g for 15 min after which supernatant was discarded.w/w) at 37 ℃ overnight. The digestion was quenched with 1% trifluoracetic acid, and the samples were desalted with Sep-Pak C18 cartridges . The concentrations of peptide mixture were measured by peptide assay . Ten micrograms of peptide were aliquoted for each sample and dried in vacuo.The pellets were rinsed with cold acetone and air-dried at room temperature. Eight molar urea was added to dissolve the pellets, and 50 mM Tris buffer was used to dilute the samples to a urea concentration <1 M. On-pellet digestion was performed with LysC/trypsin  at a 50:1 ratio -tandem mass spectrometry analysis. The samples were analyzed on a Q-Exactive quadrupole Orbitrap mass spectrometer  coupled to a Waters nanoAcquity Ultra Performance LC. Each sample was dissolved in 15 μL 4% acetonitrile (ACN) and 0.1% formic acid (FA) in water before loading onto a 75 μm inner diameter homemade microcapillary column, which was packed with 15 cm of Bridged Ethylene Hybrid C18 particles  and fabricated with an integrated emitter tip. Mobile phase A was composed of water and 0.1% FA, while mobile phase B was composed of ACN and 0.1% FA. m/z were performed at a resolving power of 70,000 with an automatic gain control (AGC) target of 1 × 106 and maximum injection time of 250 ms. The top 15 precursors were then selected for higher-energy collisional dissociation (HCD) fragmentation with a normalized collision energy of 30, an isolation width of 2.0 Da, a resolving power of 17,500, an AGC target of 1 × 105, a maximum injection time of 150 ms, and a lower mass limit of 120 m/z. The precursors were subject to dynamic exclusion for 45 s with a 10 ppm tolerance. Each sample was acquired in technical triplicates.LC separation was achieved across a 120-min gradient elution of 4% to 30% mobile phase B at a flow rate of 300 nL/min. Survey scans of peptide precursors from 300 to 1500 Data analysis. The raw files were searched against the UniProt Homo Sapiens reviewed database (February 2020) using MaxQuant (version 1.5.2.8) [1.5.2.8)  with tryPeptides that were found as reverse or potential contaminant hits were filtered out, and all other parameters were set as the default. ECM proteins were identified and classified by matching the results to Human Matrisome dataset . Proteint tests with a two-tailed distribution for binary comparison and hierarchical clustering analysis were conducted using Perseus. The volcano plot was generated using R packages. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [Two-sample Student’s pository  with theThe SHG laser scanning microscope used here has been described in detail previously and only the salient features are given here . The excThe SHG emission has an associated directionality resulting from the sub-resolution fibril structure , and we N = 37), p53 only signature (N = 7), and STIC lesion (N = 37). We also included a comparison with HGSOC tissues (N = 33) from confirmed cancer patients in some of the analyses.A total of 81 image stacks were analyzed and utilized for classification across three tissue groups: distal normal  was generated from the outputs of Gray Level Co-Occurrence Matrix (GLCM) analysis, two-dimensional fast Fourier transform (2D-FFT) methods, and the curvelet transform combined with the FIRE extraction algorithm (CT-FIRE) . FIJI wiThe 2D-FFT analysis was performed in FIJI using the Radial Profile Extended and the Oval Profile Plot plugins to characterize the alignment and radial exponential decay of the image power spectrum. We previously described the radial- and azimuthal-averaging procedures used in this approach . All curCT-FIRE was utilized to perform curvelet transform and fiber extraction to characterize individual fiber morphology features . As there was insufficient signal from the weaker backward channel for the CT-FIRE analysis, only the data readouts from the forward channel were analyzed. Lastly, the image coverage (packing coefficient) was quantified by creating a binary mask over a dynamic lower threshold and calculating the fraction of the resulting non-vanishing pixels. The methods and features used are summarized in The SAS software  was used to reduce the full feature set to the most significant metrics via forward selection at a significance level of α = 0.35 (STEPDISC procedure). After the features were selected, the truncated dataset was inputted to a linear discriminant analysis (LDA) performed with singular value decomposition and N-weighted priors. For this portion, 37 crops as normal, 6 as p53 signatures, and 37 as STIC were analyzed. 1 scores of the trained model were calculated for each class, where the latter quantity is the harmonic mean of precision (TP/(TP + FP)) and recall (TP/(TP + FN)), where TP, FP, and FN are true positives, false positives, and false negatives, respectively. An F1 score is an additional metric of classifier accuracy which accounts for class imbalance. Receiver Operating Characteristic (ROC) curves were generated under five-fold cross-validation to assess the binary classifier performance and to quantify the trade-off between the true positive rate (TPR) and false positive rate (FPR).The accuracies and FWe explored the changes in ECM and ECM-related proteins, with a focus on collagen isoform expression in human normal ovarian and tumor tissues via mass spectrometry-based proteomics approaches. In total, we identified 233 ECM proteins, where 25 were single α-chains comprising several collagen isoforms. We observed numerous proteins that were only found in either tumor or normal samples . For insAs shown in the cluster map in Locating and mapping pure p53 signatures and STIC lesions. To obtain tissues with p53 signatures and STIC lesions with no concurrent tumors, we focused on FT tissues obtained from gynecologic surgeries not related to HGSOC. An archival text-based survey of patients with STIC over a 5-year period (2013–2018) revealed 12 patient cases that met our criteria. Only 2 of the 12 had concurrent p53 signature lesions along with the STIC. None of these cases had any pathological characteristics for HGSOC. The low number of cases with pure p53 signature and STIC lesions is consistent with their reported low incidence in these cohorts.To confirm that these were pure precursors without the presence of cancer, routine histological stains (H&E and p53) were completed. The fallopian tube tissue slices for SHG imaging were unstained and, therefore, posed a challenge to accurately identify the normal regions, p53 signatures, and STICs. This issue was addressed by the H&E and p53 staining of an adjacent section of the same tissue, where these slides were used as a template to manually map and score the normal areas and precursors on the slides used for SHG imaging. This workflow was completed for all FT samples and is outlined in A trained gynecologic pathologist (P.W.) first identified the specific areas of normal tissue (green rectangle), p53 signature (red rectangle), and STIC (blue rectangle) a. For orN = 37; p53 signature, N = 6; STIC, N = 37) were analyzed using the image analysis protocols from our previous study [Our previous study indicated that a collagen coverage of less than 70% significantly altered the accuracy of the image analysis techniques, and many of these regions had sparser coverage. As a solution, representative image stacks for each channel were duplicated and cropped to 45 × 45 microns field of view (FOV), and we selected regions of interest (ROIs) with sufficient coverage. The p53 signatures and STIC lesions were small in spatial extent and localized in their respective tissue and yielded z-stacks comprised of 10 or fewer optical sections. Cropped images of each group , we included HGSOC as a point of comparison. In good agreement with a previous study , we foun2D-FFT methods were able to distinguish HGSOC as having higher alignment in the forward and backward channels, where both readouts of this metric should trend in the same direction. There was also a greater relative occurrence of high frequency  features in HGSOC, which is indicated by the higher time constant in its radial power spectrum b. ConverLinear Discriminant Analysis (LDA). While none of the individual metrics from In order to better differentiate between the precursor groups (and to prevent overfitting in the trained LD model), the feature space generated by the GLCM, 2D-FFT methods, CT-FIRE, and the packing coefficient was limited via forward selection up to a significance level of α = 0.35 (SAS/STEPDISC procedure). The most significant variables for discriminating between these groups  were then used to construct canonical variables and visualize the classes via a scatter plot .1 scores between ~65 to 91% and 9.1–66.0, respectively (N = 37 each). The corresponding AUROCs for these classifiers were somewhat low . Through this analysis, we were able to achieve accuracies and Fectively . In part1 score of 9.1 indicates a low number of true positives. To overcome the limitation of low N for the p53 signature group, we trained a classifier to distinguish between the distal normal and precursor regions, where the p53 signature and STIC were aggregated into a more general precursor group. Through a similar model using slightly different metrics, we achieved a high accuracy and F1 score , as well as an AUROC of 0.68. The scatter plot and ROC curve for this model are included in the Despite the high overall accuracy (~91%) for p53 signature classification, the corresponding FUsing SHG microscopy, we have previously shown that there are significant changes in the collagen fibrillar morphology in HGSOC in the ovary itself, as well as in the fallopian tubes ,17. MoreIt is further important to perform these investigations in tissues without HGSOC, as these lesions can induce collagen remodeling in distant regions through a field effect mechanism . HoweverWe also sought to determine if there were differences in the collagen expression patterns between normal ovarian tissues and HGSOC, and further, if these were related to the collagen morphology changes visualized by SHG microscopy. This is important as, while previous studies have suggested the up-regulation of several collagen isoforms in HGSOC  ,32, thesThis is potentially problematic as most available antibodies lack a high level of specificity for different isoforms   as the sTo begin making this connection, we utilized MS and found the decreased expression of numerous single α-chains from several different collagen isoforms in HGSOC. Of particular note, the expression of Col I and Col III chains were both downregulated, where the latter is not consistent with previous immunostaining data . This isInterestingly, we previously used detailed SHG analysis to show that there were large structural changes in the α-chains; however, these were not consistent with an increase in Col III expression . MoreoveWe did not expand the proteomic analysis to the p53 and STIC precursors as the tissues were insufficient in volume. However, given the current paradigm in HGSOC that the ovary is the metastatic site from the FT ,35, the Given the congruence in the SHG and the proteomic data suggesting differential collagen isoform expression in normal and HGSOC tissues, these observations further support the validity of performing the MS analysis on more available ovarian stromal tissues. We further suggest that the SHG analysis of the collagen in precursors is a true reflection of the biochemical alterations occurring during carcinogenesis and is an important surrogate measurement that allows for non-destructive analysis, preserving the rare precursors for IHC and transcriptomic analysis.1 scores, and ROC curves, which were further improved by grouping p53 signatures and STIC lesions together. Importantly, even with the small sample size, our analysis supports our hypothesis that collagen alterations in the FT occur prior to frank HGSOC. Based on our prior work , we expeWe note that, since the collagen alterations of early-stage disease are subtle, the classifier performance will be sensitive to under-sampling. We suggest that with a much larger specimen number, the performance could be improved to a standard that is suitable for future clinical applications. The sample thickness and coverage and limited collagen density leading to relatively weak SHG signal intensities were major limitations in this study. We suggest that higher accuracies should be achievable on thicker sections (~50–100 μm), which are readily imaged by SHG.Another limitation of the study at the moment is that there is no clear definition of the biochemical factors that affect the change in the collagen structure in HGSOC or in precursor lesions. The differential expression of collagen isoforms combined with specific post-translational processing may contribute to the change in collagen structure. The successful implementation of mass spectroscopy and transcriptomic analysis will be necessary to obtain the biochemical understanding of changes occurring in the collagen and the surrounding ECM. For example, other markers, such as fibronectin, laminin, and secreted MMPs, have been suggested to have altered regulation in HGSOC and could be added to the analysis.We foresee both long and short-term applications based on our observations and methodology. For the former, it may be possible to construct an SHG laser-scanning micro-endoscope to be used in conjunction with laparoscopy or hysteroscopy ,37. AnalThe scheme may be feasible for the in vivo detection of precursor lesions, especially since the majority of the significant variables for class discrimination came from the SHG backward channel, which would be the usable direction in an endoscopic configuration. In the shorter term, the ex vivo analysis of resected fallopian tube tissue from risk reduction surgery can be used either as a pre-screen or to complement the histology and also to identify MSbased proteomic correlations.The collagen fiber morphology is highly remodeled in HGSOC in the ovary or the fallopian tubes, where the fibers become more aligned relative to normal tissues and other tumor sub-types. However, for clinical applications, it is important to investigate these alterations in the precursors (p53 and STICs) due to the early metastasis of HGSOC. Unfortunately, there is a limited availability of pure precursor lesions without the presence of malignant tissue. Still, due to the high specificity and sensitivity to collagen morphology afforded by SHG microscopy, sufficient discrimination between distal normal regions and p53 and STIC precursors was attained in this limited study to demonstrate proof of concept. Moreover, mass spectrometry analyses showed concurrent proteomic changes in normal and HGSOC tissues, where many collagen single α-chains were downregulated. These results suggest that the combined use of MS proteomic and SHG microscopy analyses forms a basis for further in vivo and ex vivo explorations of HGSOC and its precursor lesions.


Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49 ng/uL, which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50  It is a very well-known fact that the global pandemic named COVID-19 caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hit the world in late December of 2019. It has enveloped major parts of the world resulting in a global emergency in most countries with more than 163,869,893 confirmed cases and 3,398,302 deaths as per the latest May 2021 COVID19 situation report of WHO , 5. The Thus, the present experiment was designed to test the sensitivity of standard qRT-PCR and reevaluated the sensitivity of pool results by addition of RNA purification step using isopropyl alcohol, which could improve the qRT-PCR performance for SARS CoV-2 virus screening.μL) VTM of each of the 5 samples was pooled in to form a single pool  from various health care centres of Varanasi, UP, India, were selected from July to August 2020 . Each sample was a combination of nasopharyngeal and oropharyngeal swabs in a 3 mL viral transport media (VTM) preserved under cold chain. A total of 500 samples were selected for the study that includes 100 positive and 400 negatives. The positive COVID-19 sample selected for the study includes Cq values ranging from 25-30, while negative samples were selected without amplification. These samples were pooled in to form 100 pools (each pool containing 5 samples) for RNA extraction. All the samples were received in VRDL, Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. 50 microlitre  was purified using the isopropyl alcohol method (modified RNA purification protocol) (μL) in eluted RNA, which was placed at -20°C for 15 minutes. Further, the tubes were centrifuged at 14000 RPM for 10 minutes (at 4°C). The supernatant was discarded, and 100 μL of absolute ethanol  was added and placed at room temperature for 5 minutes. Further, the supernatant was discarded very gently, and the tubes were allowed to dry in a horizontal position for 10 minutes. After drying, 30 μL of AVE (elution buffer) was added and mixed, while 50 μL of elution buffer was used in extraction using the column-based kit.All the samples for viral nucleic acid extraction were treated as per the manufacturer's instruction by column-based viral RNA/DNA Mini Kit ). Extraction control (EC) was also used at the time of RNA extraction in each batch, to check the validity of the extraction procedure. RNAs of 100 pooled samples extracted were divided into two parts; one part with 25 rotocol) . This RNC equipment . The sample absorbance was measured at 260 nm and 280 nm, and the 260/280 ratio was used to assess RNA purity along with the nucleic acid concentrations in ng/mL. As per recommended guidelines, RNA purity was considered adequate when the ratio of 260/280 was ~2.0 [Isolated purified and unpurified pool sample RNA concentrations/purity were measured using NanoDrop Onewas ~2.0 . Howeverwas ~2.0 .μL of extracted RNA (purified and nonpurified) was added to 20 μL of reaction master mix prepared using real-time fluorescent qRT-PCR kit (Invitrogen). SARS-CoV-2 qRT-PCR assay was performed using first-line screening E gene assays  and human ribonuclease P  gene target, which was included to confirm the authenticity of the specimen collection, RNA extraction, identification of PCR inhibitors, and also defined as internal positive control [5  control . Second- control . The priThe values of amplification (Cq) and fluorescence (RFU) were noted and compared for both sets (purified and nonpurified extracted RNAs).μL of 2 × reaction buffer containing 0.4 mM of each deoxyribonucleotide triphosphate (dNTP) and 3.2 mM magnesium sulphate, 0.5 μL of reverse transcriptase/Taq enzyme mixture provided with kit, 1 μL of E/RNase P gene primer-probe mix, and 6 μL DNase/RNase-free water. Thermal cycling conditions used for screening as well as for confirmatory assay were 55°C for 30 minutes for reverse transcription, followed by 95°C for 3 minutes and then 45 cycles of 95°C for 15 seconds, 58°C for 30 seconds [Superscript III one-step qRT-PCR system with Platinum Taq Polymerase  was used for the detection of COVID-19 targets. The reaction mixture composition used for SARS-CoV-2 detection was 12.5  seconds . The assThe raw values obtained from NanoDrop (A260/A280 ratio) were statistically analysed using chi-square and Mann–Whitney tests in SPSS software. The sensitivity and specificity of the tested samples were evaluated and presented in a 2 × 2 table.μL, while the unpurified set of RNAs presented with a range between 1.24 and 8.14 ng/μL. Also, the values of absorbance ratio (at 260/280 nm) of unpurified RNAs were reported between 0.9 and 4.8. In addition, the RNA concentration detected using NanoDrop in purified pooled RNA samples were 24.73 ± 5.60 ng/μL, while in unpurified pooled RNA it was detected to be 2.38 ± 1.09 ng/μL. The A260/A280 ratio for purified RNA was ∼2.0, whereas unpurified RNA presented a higher 260/280 ratio. The results showed that the A260/A280 ratio in unpurified and purified RNA samples was 1.58 ± 0.06 and 2.02 ± 0.02, respectively.A total of 100 pooled specimens of known SARS-CoV-2 positive, along with available negative samples, were selected to assess this proposed RNA purification method. Both sets of extracted RNAs were subjected to spectrophotometric analysis. The average concentration of purified set of pooled RNAs showed a value between 15.5 and 56.8 ng/Cq). Similarly, the RNase P gene was also not detected in 15% of the samples. The average Cq values of RNase P in the unpurified pool group was 29.46, while average values after purification were 27.44. However, 15% of the samples (15 pool samples RNA) were reported with very high Cq of RNase P ranges from 37.06 to 41.56, which was considered as inappropriate sample or repeat sampling. But, after purification, the Cq values of RNaseP were 34.05 to 35.37, i.e., in the recommended detectable range. Amplification was successful with lower Cq values after purification. Using modified RNA purification protocol with isopropanol, 75% of the total tested pools showed lower Cq values when compared with the unpurified RNA set. The difference of 2.3 to 4.5 low Cq was observed in purified RNA , while it was significantly amplified in purified set  and high purity index of 1.9 depicted remarkable amplification graphs in both the cases (in unpurified and purified sets of RNA). However, the samples that may contain low viral load and low quantity of RNA showed progressed amplification graphs in the purified set and can be successfully detected after purification. The overall sensitivity and specificity of pool samples before and after purification of extracted RNA was calculated and represented in a 2 × 2 table. The results showed that sensitivity with 85% and specificity found to be 100% among 500 samples. In addition, PPV  and NPV  were found to be 100% and 96.38%, respectively. The details of true or false positive or negative were tabulated in Cq values (near borderline ~36), making the lab person declare the respective pool as negative, which might have one or two suspected cases.The results also showed that the samples with a high concentration of RNA (50 ng/μL) as compared with individual RNA isolation (~250 μL). Besides, the purification step resulted in an improved quantity and quality of RNA for COVID-19 pool screening.The statistical analysis demonstrated that the RNA purification step with the isopropanol method significantly increased the amplification success rate of SARS-CoV-2 detection in pool testing. That could be due to the less sample volume in pool testing , which can only be copurified using modified RNA protocol reported by Das et al., 2006 . HoweverIn addition to the concentration of extracted RNA obtained from NanoDrop, sample quality can also be gauged by analysing the A260/280 ratio. It was reported that the pure nucleic acids mostly yield a 260/280 ratio of approximately 2 for the RNA sample. However, A260/280 ratio is dependent on the pH and ionic strength of the buffer used in the sample measurements. The ratio below the standards represents the acidic or basic solutions, which indicate protein, phenol, or other contaminants present after extraction that absorbs strongly at or near 280 nm. The purity ratio may vary from the expected values, which indicate the different RNA isolation procedure, which further required optimization before performing a qRT-PCR diagnostic assay to minimize the false-negative results.An alternative approach of RNA purification for pooled specimens was designed to obtain error-free results. Application of isopropanol-based purification after extraction of pooled-specimens RNA from commercial kits fetched a high-quality RNA from NP/OP COVID-19 suspected samples, and it could easily be performed in the laboratory. Extracted RNA of the pooled specimen with minimal impurities would be an ideal template for qRT-PCR assay. Thus, proper sampling of COVID-19 suspects, good laboratory practice (GLP) standard, and high-quality extraction procedures with purification using isopropanol would improve real-time RT-PCR findings and reduce inaccurate results.


Osteosarcoma is the most common skeletal malignancy and commonly metastasis to lung and bone. Here we report a case of osteosarcoma of the right knee with metastasis to the lower and inner quadrant of the breast along with axillary, mediastinal, retroperitoneal and inguinal lymphadenopathy with lung and liver metastasis. The diagnosis of breast metastasis was confirmed by ultrasonography-guided biopsy and immunohistochemistry (IHC). So this report highlights the rarest metastasis to breast and axillary lymph node from an osteosarcoma of the right knee primary. Osteosarcoma is the most common primary bone malignancy . OsteosaA 20-year-old female with unremarkable medical and surgical history presented to our institute with painful swelling over the right knee and breast lump. She had undergone patellectomy with repair three months prior at another hospital. The histopathological and immunohistochemistry (IHC) study report was suggestive of osteogenic sarcoma as it was positive for smooth muscle actin (SMA), S-100, CD68, and negative for caldesmon and desmin and CD34 Figures . CurrentOsteosarcoma is the most common skeletal malignancy . It mostThe incidence of breast metastasis from extra mammary malignancy is very low -14. AmonThere is no specific guideline for the management of this extremely rare case, so osteosarcoma with breast metastasis is treated the same as metastasis to other sites. The general treatment strategy for osteosarcoma is complete surgical excision and systemic chemotherapy, both neoadjuvant and adjuvant. Palliative chemotherapy should be considered in case of extensive metastasis. So the treatment protocol in our case is palliative chemotherapy due to extensive metastasis.Incidence of breast metastasis in osteosarcoma of the knee is very rare. Histopathology features and IHC study help to differentiate metastatic osteosarcoma to the breast from primary breast malignancy. Due to the rarity of cases, no specific diagnostic and management strategy has been found for these types of cases. So by reporting this extremely rare case of osteosarcoma of the right knee with breast and axillary lymph node metastasis, we recommend vigilance in metastatic work up and treatment of metastatic osteosarcoma.


This cross-sectional study examines the prevalence of restricted choice of treatment in control groups among physicians and investigators conducting oncology randomized clinical trials (RCTs). However, RCTs may be limited if the control group does not reflect the ongoing standard of care, and especially if the control group is inferior to standard care. A cross-sectional analysis of 95 consecutive Food and Drug Administration approvals of anticancer agents between 2013 and 2018 showed that 16 (17%) were based on RCTs with suboptimal control groups.Theoretically, this limitation can be overcome if the physician is allowed to choose the control-group treatment with access and freedom. Increasingly, trials refer to a control group of physician’s choice or investigator’s choice, which may be perceived as a real (unrestricted) choice among all treatment options. However, this choice can be restricted and ambiguously reported with 2 consequences. First, when the choice is restricted to a limited number of options, the wording is misleading for physicians considering it was unfettered and unlimited. Second, the restriction may penalize the control group, making it substandard by not allowing all treatment options in a specific setting.STROBE) reporting guideline. Because we used publicly available data, and this was not human subjects research, in accordance with 45 CFR §46.102(f), we did not submit this study to an institutional review board or require informed consent procedures.This cross-sectional study adhered to Strengthening the Strengthening the Reporting of Observational Studies in Epidemiology  choice, their evolution over time, whether the choice was real (unrestricted) or restricted. We systematically reviewed all reports on RCTs mentioning physician’s or investigator’s choice : in a trial investigating sacituzumab govitecan in patients with triple negative breast cancer, the physician’s choice was restricted to 4 options, not allowing platinum nor anthracyclines, 2 highly active agents, possibly leading to substandard outcomes.2 Also, as another potential limitation, we restricted our search to MEDLINE.One limitation of our study is that we did not assess for the prevalence of the included reports as compared with all published oncology RCTs. As an estimation, when they were 121 published RCTs in breast cancer between 2014 and 2017, we included 6 breast cancer trials .Physician’s or investigator’s choice have been used for decades in the literature; however, they are increasing over time. This is an important trend to be aware of in oncology RCTs. Through imprecise wording, potentially masking substandard control group, treating physicians may inaccurately think that the reported results can be generalized to their patients, whereas this may not be true. Our findings suggest that editors and regulators should demand clarification in the use of these terms within RCTs protocols and reports.