Landscape of semi-extractable RNAs across five human cell lines.

Phase-separated membraneless organelles often contain RNAs that exhibit unusual semi-extractability using the conventional RNA extraction method, and can be efficiently retrieved by needle shearing or heating during RNA extraction. Semi-extractable RNAs are promising resources for understanding RNA-centric phase separation. However, limited assessments have been performed to systematically identify and characterize semi-extractable RNAs. In this study, 1074 semi-extractable RNAs, including ASAP1, DANT2, EXT1, FTX, IGF1R, LIMS1, NEAT1, PHF21A, PVT1, SCMH1, STRG.3024.1, TBL1X, TCF7L2, TVP23C-CDRT4, UBE2E2, ZCCHC7, ZFAND3 and ZSWIM6, which exhibited consistent semi-extractability were identified across five human cell lines. By integrating publicly available datasets, we found that semi-extractable RNAs tend to be distributed in the nuclear compartments but are dissociated from the chromatin. Long and repeat-containing semi-extractable RNAs act as hubs to provide global RNA-RNA interactions. Semi-extractable RNAs were divided into four groups based on their k-mer content. The NEAT1 group preferred to interact with paraspeckle proteins, such as FUS and NONO, implying that RNAs in this group are potential candidates of architectural RNAs that constitute nuclear bodies.

Live-seq enables temporal transcriptomic recording of single cells.

Single-cell transcriptomics (scRNA-seq) has greatly advanced our ability to characterize cellular heterogeneity1. However, scRNA-seq requires lysing cells, which impedes further molecular or functional analyses on the same cells. Here, we established Live-seq, a single-cell transcriptome profiling approach that preserves cell viability during RNA extraction using fluidic force microscopy2,3, thus allowing to couple a cell's ground-state transcriptome to its downstream molecular or phenotypic behaviour. To benchmark Live-seq, we used cell growth, functional responses and whole-cell transcriptome read-outs to demonstrate that Live-seq can accurately stratify diverse cell types and states without inducing major cellular perturbations. As a proof of concept, we show that Live-seq can be used to directly map a cell's trajectory by sequentially profiling the transcriptomes of individual macrophages before and after lipopolysaccharide (LPS) stimulation, and of adipose stromal cells pre- and post-differentiation. In addition, we demonstrate that Live-seq can function as a transcriptomic recorder by preregistering the transcriptomes of individual macrophages that were subsequently monitored by time-lapse imaging after LPS exposure. This enabled the unsupervised, genome-wide ranking of genes on the basis of their ability to affect macrophage LPS response heterogeneity, revealing basal Nfkbia expression level and cell cycle state as important phenotypic determinants, which we experimentally validated. Thus, Live-seq can address a broad range of biological questions by transforming scRNA-seq from an end-point to a temporal analysis approach.

Role of detergents and nuclease inhibitors in the extraction of RNA from eukaryotic cells in complex matrices.

The potential for liquid biopsy samples to be used in place of more invasive tissue biopsies has become increasingly revalent as it has been found that nucleic acids (NAs) present in the blood of cancer patients originate from tumors. Nanomagnetic extraction has proven to be a highly effective means to rapidly prepare NA from clinical samples for molecular diagnostics. In this article, the lysis reaction used to extract RNA from the human epithelial melanoma cells have been optimized using silica coated superparamagnetic nanoparticles (SPM NP). The lysis buffer (LB) is composed of several agents that denature cells, i.e., surfactant and guanidinium isothiocyanate (GITC), and agents that inhibit the degradation of circulated nucleic acids (cfNAs). The surfactant Triton X-100 has been widely used in LB but has been placed on the European Union REACH list. We have compared the qRT-PCR sensitivity resulting from LBs composed of Triton X-100 to several sustainable surfactants, i.e., Tergitol 15-S-7, Tergitol 15-S-9 and Tween-20. Surprisingly, the inclusion of these surfactants in the LB was not found to significantly improve cell lysis, and subsequently the sensitivity of qRT-PCR. The role of the sample matrix was also examined by performing extractions from solutions containing up to 30 mg mL-1 serum albumin. The qRT-PCR sensitivity was found to decrease as the concentration of this protein was increased; however, this was linked to an increased RNase activity and not the concentration of the protein itself. These results lead us to recommend a reformulation of LB for clinical samples, and to conclude that sensitive qRT-PCR RNA analysis can be performed in serum with the timely addition of an RNase inhibitor.

Differentially Expressed Hepatic Genes Revealed by Transcriptomics in Pigs with Different Liver Lipid Contents.

The liver is the center for uptake, synthesis, packaging, and secretion of lipids and lipoproteins. The research on lipid metabolism in pigs is limited. The objective of the present study is to identify the genes related to lipid metabolism and oxidative stress in pigs by using transcriptomic analysis. Liver segments were collected from 60 Jinhua pigs for the determination of liver lipid content. The 7 pigs with the highest and lowest liver lipid content were set as group H and group L, respectively. Liver segments and serum samples were collected from each pig of the H and L groups for RNA sequencing and the determination of triglycerides (TG) content and high-density lipoprotein cholesterol (HDL) content, respectively. The HDL content in the serum of pigs in the H group was significantly higher than the L group (P < 0.05). From transcriptomic sequencing, 6162 differentially expressed genes (DEGs) were identified, among which 2962 were upregulated and 3200 downregulated genes with the increase in the liver content of Jinhua pigs. After GO enrichment and KEGG analyses, lipid modification, cellular lipid metabolic process, cholesterol biosynthetic process, fatty acid metabolic process, oxidoreduction coenzyme metabolic process, oxidoreductase activity, acting on CH-OH group of donors, response to oxidative stress, nonalcoholic fatty liver disease (NAFLD), sphingolipid metabolism, and oxidative phosphorylation pathways were involved in lipid metabolism and oxidative stress in Jinhua pigs. For further validation, we selected 10 DEGs including 7 upregulated genes (APOE, APOA1, APOC3, LCAT, CYP2E1, GPX1, and ROMO1) and 4 downregulated genes (PPARA, PPARGC1A, and TXNIP) for RT-qPCR verification. To validate these results in other pig species, we analyzed these 10 DEGs in the liver of Duroc×Landrace×Yorkshire pigs. Similar expression patterns of these 10 DEGs were observed. These data would provide an insight to understand the gene functions regulating lipid metabolism and oxidative stress and would potentially provide theoretical basis for the development of strategies to modulate lipid metabolism and even control human diabetes and obesity by gene regulations.

Chemical reversible crosslinking enables measurement of RNA 3D distances and alternative conformations in cells.

Three-dimensional (3D) structures dictate the functions of RNA molecules in a wide variety of biological processes. However, direct determination of RNA 3D structures in vivo is difficult due to their large sizes, conformational heterogeneity, and dynamics. Here we present a method, Spatial 2'-Hydroxyl Acylation Reversible Crosslinking (SHARC), which uses chemical crosslinkers of defined lengths to measure distances between nucleotides in cellular RNA. Integrating crosslinking, exonuclease (exo) trimming, proximity ligation, and high throughput sequencing, SHARC enables transcriptome-wide tertiary structure contact maps at high accuracy and precision, revealing heterogeneous RNA structures and interactions. SHARC data provide constraints that improves Rosetta-based RNA 3D structure modeling at near-nanometer resolution. Integrating SHARC-exo with other crosslinking-based methods, we discover compact folding of the 7SK RNA, a critical regulator of transcriptional elongation. These results establish a strategy for measuring RNA 3D distances and alternative conformations in their native cellular context.

Examining potential confounding factors in gene expression analysis of human saliva and identifying potential housekeeping genes.

Isolation of RNA from whole saliva, a non-invasive and easily accessible biofluid that is an attractive alternative to blood for high-throughput biodosimetry of radiological/nuclear victims might be of clinical significance for prediction and diagnosis of disease. In a previous analysis of 12 human samples we identified two challenges to measuring gene expression from total RNA: (1) the fraction of human RNA in whole saliva was low and (2) the bacterial contamination was overwhelming. To overcome these challenges, we performed selective cDNA synthesis for human RNA species only by employing poly(A)+-tail primers followed by qRT-PCR. In the current study, this approach was independently validated on 91 samples from 61 healthy donors. Additionally, we used the ratio of human to bacterial RNA to adjust the input RNA to include equal amounts of human RNA across all samples before cDNA synthesis, which then ensured comparable analysis using the same base human input material. Furthermore, we examined relative levels of ten known housekeeping genes, and assessed inter- and intra-individual differences in 61 salivary RNA isolates, while considering effects of demographical factors (e.g. sex, age), epidemiological factors comprising social habits (e.g. alcohol, cigarette consumption), oral hygiene (e.g. flossing, mouthwash), previous radiological diagnostic procedures (e.g. number of CT-scans) and saliva collection time (circadian periodic). Total human RNA amounts appeared significantly associated with age only (P ≤ 0.02). None of the chosen housekeeping genes showed significant circadian periodicity and either did not associate or were weakly associated with the 24 confounders examined, with one exception, 60% of genes were altered by mouthwash. ATP6, ACTB and B2M represented genes with the highest mean baseline expression (Ct-values ≤ 30) and were detected in all samples. Combining these housekeeping genes for normalization purposes did not decrease inter-individual variance, but increased the robustness. In summary, our work addresses critical confounders and provides important information for the successful examination of gene expression in human whole saliva.

Monitoring the maturation of the sarcomere network: a super-resolution microscopy-based approach.

The in vitro generation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC) is of great importance for cardiac disease modeling, drug-testing applications and for regenerative medicine. Despite the development of various cultivation strategies, a sufficiently high degree of maturation is still a decisive limiting factor for the successful application of these cardiac cells. The maturation process includes, among others, the proper formation of sarcomere structures, mediating the contraction of cardiomyocytes. To precisely monitor the maturation of the contractile machinery, we have established an imaging-based strategy that allows quantitative evaluation of important parameters, defining the quality of the sarcomere network. iPSC-derived cardiomyocytes were subjected to different culture conditions to improve sarcomere formation, including prolonged cultivation time and micro patterned surfaces. Fluorescent images of α-actinin were acquired using super-resolution microscopy. Subsequently, we determined cell morphology, sarcomere density, filament alignment, z-Disc thickness and sarcomere length of iPSC-derived cardiomyocytes. Cells from adult and neonatal heart tissue served as control. Our image analysis revealed a profound effect on sarcomere content and filament orientation when iPSC-derived cardiomyocytes were cultured on structured, line-shaped surfaces. Similarly, prolonged cultivation time had a beneficial effect on the structural maturation, leading to a more adult-like phenotype. Automatic evaluation of the sarcomere filaments by machine learning validated our data. Moreover, we successfully transferred this approach to skeletal muscle cells, showing an improved sarcomere formation cells over different differentiation periods. Overall, our image-based workflow can be used as a straight-forward tool to quantitatively estimate the structural maturation of contractile cells. As such, it can support the establishment of novel differentiation protocols to enhance sarcomere formation and maturity.

SMRT sequencing of the full-length transcriptome of Gekko gecko.

Tokay Gecko (Gekko gecko) is a rare and endangered medicinal animal in China. Its dry body has been used as an anti-asthmatic agent for two thousand years. To date, the genome and transcriptome of this species remain poorly understood. Here, we adopted single molecule real-time (SMRT) sequencing to obtain full-length transcriptome data and characterized the transcriptome structure. We identified 882,273 circular consensus (CCS) reads, including 746,317 full-length nonchimeric (FLNC) reads. The transcript cluster analysis revealed 212,964 consensus sequences, including 203,994 high-quality isoforms. In total, 111,372 of 117,888 transcripts were successfully annotated against eight databases (Nr, eggNOG, Swiss-Prot, GO, COG, KOG, Pfam and KEGG). Furthermore, 23,877 alternative splicing events, 169,128 simple sequence repeats (SSRs), 10,437 lncRNAs and 7,932 transcription factors were predicted across all transcripts. To our knowledge, this report is the first to document the G. gecko transcriptome using SMRT sequencing. The full-length transcript data might accelerate transcriptome research and lay the foundation for further research on G. gecko.

Deltamethrin interacts with Culex quinquefasciatus odorant-binding protein: a novel potential resistance mechanism.

BACKGROUND: Odorant-binding proteins (OBPs) play important roles in many physiological processes of mosquitoes. Previous high-throughput sequencing studies have revealed that some OBPs of Culex quinquefasciatus might be involved in the development of resistance to insecticides. METHODS: Based on the results of sequencing analyses, the OBP28 gene was selected for evaluation in this study. Three laboratory strains of Cx. quinquefasciatus [susceptible strain (SS), deltamethrin-resistant strain 1 (HN) and deltamethrin-resistant strain 2 (RR)] were first examined by using the Centers for Disease Control and Prevention bottle bioassay, after which the expression level of the OBP28 gene in the susceptible and deltamethrin-resistant strains was determined by real-time quantitative polymerase chain reaction. The OBP28 gene in deltamethrin-resistant strain RR was silenced using RNA interference technology. The expression level of OBP28 and the resistance level were tested in the silenced strain and control strain after microinjection of double-stranded RNA for a 48-h interference period. Four field-collected strains (henceforth 'field strains') of Cx. quinquefasciatus were also examined for their resistance to deltamethrin and levels of OBP28 expression. Finally, a correlation analysis between deltamethrin resistance and gene expression was carried out for all seven strains, i.e. the four field strains and the three laboratory strains. RESULTS: In the bioassay, the mortality of SS, HN and RR was 100%, 21.33% and 1.67%, respectively. The relative expression levels of OBP28 in strains HN and RR were 6.30- and 6.86-fold higher, respectively, than that of strain SS. After silencing of the OBP28 gene, the mortality of strain RR was 72.20% and that of the control strain 26.32%. The mortality of strain RR increased significantly after interference compared to that of the control strain. There was a negative correlation between OBP28 gene expression and mortality in adult mosquitoes after exposure to deltamethrin. CONCLUSIONS: To our knowledge, this study shows for the first time a correlation between the expression of a gene coding for OBP and insecticide resistance in mosquitoes. The potential resistance mechanism that was elucidated provides a new target gene for the surveillance of resistance in mosquitoes.

RNA isolation from micro-quantity of articular cartilage for quantitative gene expression by microarray analysis.

Isolation of quality RNA from articular cartilage has been challenging due to low cellularity and the high abundance of extracellular matrix and proteoglycan proteins. Recently developed methods for isolation of high quality RNA from cartilage are more applicable to larger cartilage specimens typically weighing at least 25 mg. While these methods generate RNA suitable for analysis, they are less successful with smaller tissue inputs. For the study of small focal defect cartilage specimens an improved RNA extraction method is needed. Here we report a protocol for direct RNA isolation from less than 3 mg of wet weight rabbit articular cartilage for quantitative microarray gene profiling. This protocol is useful for identifying differentially expressed genes in chondrocytes following focal cartilage repair and can potentially be adopted for gene expression analysis of cartilage biopsy specimens from human joints.

Single-copy Loss of Rho Guanine Nucleotide Exchange Factor 10 ( arhgef10) Causes Locomotor Abnormalities in Zebrafish Larvae.

OBJECTIVE: To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation. METHODS: Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 -/-, arhgef10 +/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively. RESULTS: WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 -/- zebrafish had more severe symptoms than arhgef10 +/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis. CONCLUSION: Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.

Experience of quantity and quality of DNA and RNA extraction from limited pediatric blood samples: A comparative analysis of automated and manual kit-based method.

INTRODUCTION: Optimal DNA and RNA quantity and purity is essential for downstream molecular biology experimentation and to avoid re-processing of sample. Despite availability of different kits and automated systems for nucleic acid isolation there is limited data on their performance evaluation, more so with pediatric blood samples, that are usually compromised in quantity. Hence, we evaluated the performance of automated QIAcube platform using pediatric blood samples in parallel with manual Qiagen extraction kits. MATERIALS AND METHODS: : A total of 500 samples were analyzed based on groups of PBMC and direct blood input. The isolated DNA and RNA were surveyed for quantity and quality tests by spectrophotometric and downstream analysis. RESULTS: : There was no significant difference in the DNA quantity (ng/ul) between manual and automated method based on similar sample input but quality (260/280) was significantly better with the QIAcube platform when direct blood and or PBMCs were used for extraction respectively (1.82 ± 004 Vs. 1.84.002; P-0.000008 and 1.859 ± 005 Vs. 1.843 ± 0.003; P-0.02). Moreover, the standard error mean was low for both quantity and quality in the QIAcube method suggesting uniformity. Comparison of quality assessment by spectrophotometer and qubit fluorimeter showed that QIAcube sheared DNA less (P- 0.038) as compared to manual method (P-0.013). Also, time taken to process the samples in QIAcube was 23% less than the kit-based method. CONCLUSION: Overall analysis of QIAcube platform suggests that it yields more better, uniform, and less-sheared quality of nucleic acid in a relatively less time as compared to manual extraction kits.

Assessing the quality of RNA isolated from human breast tissue after ambient room temperature exposure.

High quality human tissue is essential for molecular research, but pre-analytical conditions encountered during tissue collection could degrade tissue RNA. We evaluated how prolonged exposure of non-diseased breast tissue to ambient room temperature (22±1°C) impacted RNA quality. Breast tissue received between 70 to 190 minutes after excision was immediately flash frozen (FF) or embedded in Optimal Cutting Temperature (OCT) compound upon receipt (T0). Additional breast tissue pieces were further exposed to increments of 60 (T1 = T0+60 mins), 120 (T2 = T0+120 mins) and 180 (T3 = T0+180 mins) minutes of ambient room temperature before processing into FF and OCT. Total exposure, T3 (T0+180 mins) ranged from 250 minutes to 370 minutes. All samples (FF and OCT) were stored at -80°C before RNA isolation. The RNA quality assessment based on RNA Integrity Number (RIN) showed RINs for both FF and OCT samples were within the generally acceptable range (mean 7.88±0.90 to 8.52±0.66). No significant difference was observed when RIN at T0 was compared to RIN at T1, T2 and T3 (FF samples, p = 0.43, 0.56, 0.44; OCT samples, p = 0.25, 0.82, 1.0), or when RIN was compared between T1, T2 and T3. RNA quality assessed by quantitative real-time PCR (qRT-PCR) analysis of beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A (CYPA), and porphobilinogen deaminase (PBGD) transcripts showed threshold values (Ct) that indicate abundant and intact target nucleic acid in all samples (mean ranging from 14.1 to 25.3). The study shows that higher RIN values were obtained for non-diseased breast tissue up to 190 minutes after resection and prior to stabilization. Further experimental exposure up to 180 minutes had no significant effect on RIN values. This study strengthens the rationale for assessing RIN and specific gene transcript levels as an objective method for determining how suitable RNA will be for a specific research purpose ("fit-for purpose").

Simultaneous Isolation of High-Quality RNA and DNA From Postmortem Human Central Nervous System Tissues for Omics Studies.

Multi-omics approaches are increasingly being adopted to understand the complex networks underlying disease. The coisolation of high-quality nucleotides from affected tissues is paramount for the parallel analysis of transcriptomic, genomic, and epigenomic data sets. Although nucleotides extracted from postmortem central nervous system (CNS) tissue are widely used in the study of neurodegenerative disease, assessment of methods for the simultaneous isolation of DNA and RNA is limited. Herein, we describe a strategy for the isolation of high-quality DNA and RNA from postmortem human tissue from 7 CNS regions. Motor cortex, frontal cortex, hippocampus, occipital cortex, anterior cingulate cortex, cerebellum, and spinal cord tissues were obtained from 22 individuals diagnosed with motor neuron disease (MND) and 13 neurologically normal controls (n = 245 tissues). We demonstrated that the Qiagen AllPrep DNA/RNA kit consistently isolated DNA and RNA of high yield and quality from all 6 brain regions. Importantly, phenol-chloroform-based extraction was required to isolate high-yield RNA from spinal cord. RNA sequencing using RNA extracted from 6 CNS regions (n = 60) generated high-quality transcriptomes. Hierarchical clustering of data from motor cortex, using an MND susceptibility gene panel and marker genes of disease-associated microglia, demonstrated that MND-specific gene expression signatures could be detected in the transcriptome data.

A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue.

Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.

Nobiletin attenuates inflammation via modulating proinflammatory and antiinflammatory cytokine expressions in an autoimmune encephalomyelitis mouse model.

The aim of this study is to investigate the potential preventive and therapeutic effects of nobiletin by evaluating the expression of cytokines associated with inflammatory reactions in an autoimmune encephalomyelitis mouse model. A total of 60 male C57BL/6 mice aged between 8 and 10 weeks were used. Mice were divided into six groups (n = 10 mice per group): control, EAE, low-prophylaxis, high-prophylaxis, low-treatment and high-treatment. Experimental autoimmune encephalomyelitis (EAE) was induced by myelin oligodendrocyte glycoprotein (MOG) and pertussis toxin. Nobiletin was administered in low (25 mg/kg) and high (50 mg/kg) doses, intraperitoneally. The prophylactic and therapeutic effects of nobiletin on brain tissue and spinal cord were evaluated by expression of interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), interferon gamma (IFNγ), IL-6, IL-10 and transforming growth factor-beta (TGF-ß) using immunohistochemistry and real-time polymerase chain reaction (RT-PCR). Prophylactic and therapeutic use of nobiletin inhibited EAE-induced increase of TNF-α, IL-1ß and IL-6 activities to alleviate inflammatory response in brain and spinal cord. Moreover, nobiletin supplement dramatically increased the IL-10, TGF-ß and IFNγ expressions in prophylaxis and treatment groups compared with the EAE group in the brain and spinal cord. The results obtained from this study show that prophylactic and therapeutic nobiletin modulates expressions of proinflammatory and antiinflammatory cytokines in brain and spinal cord dose-dependent manner in EAE model. These data demonstrates that nobiletin has a potential to attenuate inflammation in EAE mouse model. These experimental findings need to be supported by clinical studies.

Single-copy Loss of Rho Guanine Nucleotide Exchange Factor 10 ( arhgef10) Causes Locomotor Abnormalities in Zebrafish Larvae / 生物医学与环境科学（英文）

OBJECTIVE@#To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation.@*METHODS@#Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 -/-, arhgef10 +/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively.@*RESULTS@#WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 -/- zebrafish had more severe symptoms than arhgef10 +/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis.@*CONCLUSION@#Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.

RNA modifications detection by comparative Nanopore direct RNA sequencing.

RNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. In recent years, a growing number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework that identifies modifications from these data. Our strategy compares an RNA sample of interest against a non-modified control sample, not requiring a training set and allowing the use of replicates. We show that Nanocompore can detect different RNA modifications with position accuracy in vitro, and we apply it to profile m6A in vivo in yeast and human RNAs, as well as in targeted non-coding RNAs. We confirm our results with orthogonal methods and provide novel insights on the co-occurrence of multiple modified residues on individual RNA molecules.

Robust Saliva-Based RNA Extraction-Free One-Step Nucleic Acid Amplification Test for Mass SARS-CoV-2 Monitoring.

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

Comparison of RNA-Based Next-Generation Sequencing Assays for the Detection of NTRK Gene Fusions.

Recently, the US Food and Drug Administration approved several targeted therapies directed against oncogenic fusions. One of the most effective such targeted therapies is Vitrakvi (larotrectinib), highly specific oral tropomyosin receptor kinase inhibitor indicated for the treatment of patients with any solid tumor harboring a fusion involving one of the neurotrophic receptor tyrosine kinase (NTRK) genes. Although several diagnostic approaches can be used to detect these NTRK fusions, RNA-based next-generation sequencing remains one of the most sensitive methods, as it can directly detect the transcribed end product of gene fusion at the mRNA level. In this study, performance characteristics of three RNA-based next-generation sequencing assays with distinct mechanisms and chemistries were investigated: anchored multiplex PCR, amplicon-based multiplex PCR, and hybrid capture-based enrichment method. Analytical sensitivity analysis shows that the amplicon-based multiplex PCR method has the lowest limit of detection. However, both hybrid-capture and anchored multiplex PCR methods can detect NTRK fusions with uncommon or novel fusion partners, which is challenging for the amplicon-based multiplex method. As for clinical sensitivity, all three methods were highly concordant in detecting NTRK fusions in patient samples. Additionally, they all presented equivalent high-level performance in specificity, suggesting that all three platforms can detect NTRK fusions in clinical samples with similar performance characteristics.