RNA Transcripts Research Articles

Dapagliflozin attenuates AKI to CKD transition in diabetes by activating SIRT3/PGC1-α signaling and alleviating aberrant metabolic reprogramming

BackgroundPatients with diabetes are prone to acute kidney injury (AKI) with a high mortality rate, poor prognosis, and a higher risk of progression to chronic kidney disease than non-diabetic patients. MethodsStreptozotocin (STZ)-treated type 1 and db/db type 2 diabetes model were established, AKI model was induced in mice by ischemia-reperfusion injury(IRI). Mouse proximal tubular cell cells were subjected to high glucose and hypoxia-reoxygenation in vitro. Transcriptional RNA sequencing was performed for clustering analysis and target gene screening. Renal structural damage was determined by histological staining, whereas creatinine and urea nitrogen levels were used to measure renal function. ResultsDeteriorated renal function and renal tissue damage were observed in AKI mice with diabetic background. RNA sequencing showed a decrease in fatty acid oxidation (FAO) pathway and an increase in abnormal glycolysis. Treatment with Dapa, Sitagliptin(a DPP-4 inhibitor)and insulin reduced blood glucose levels in mice, and improved renal function. However, Dapa had a superior therapeutic effect and alleviated aberrant FAO and glycosis. Dapa reduced cellular death in cultured cells under high glucose hypoxia-reoxygenation conditions, alleviated FAO dysfunction, and reduced abnormal glycolysis. RNA sequencing showed that SIRT3 expression was reduced in diabetic IRI, which was largely restored by Dapa intervention. 3-TYP, a SIRT3 inhibitor, reversed the renal protective effects of Dapa and mediated abnormal FAO and glycolysis in mice and tubular cells. ConclusionOur study provides experimental evidence for the use of Dapa as a means to reduce diabetic AKI by ameliorating metabolic reprogramming in renal tubular cells.

Immunological testing for female infertility: a modern view on the problem

Introduction. According to various estimates, in Russia 10 to 20 % of people of reproductive age are infertile. Changes in the immune system play a key role in the etiology and treatment of various infertility forms. The active introduction of immunological testing into clinical practice can potentially improve the results of infertility diagnostics and treatment.Aim: to analyze the current literature data on immunological testing in female infertility, as well as to assess its potential role in infertility diagnostics and treatment.Materials and Methods. There was conducted a search for publications in the electronic databases PubMed and eLibrary by using the following keywords and their combinations: "infertility", "immunology", "immune system", "immunological testing", "diagnostics", "treatment". The articles were evaluated in accordance with the PRISMA recommendations. Ultimately, 88 publications were included in the review.Results. Testing for antiphospholipid antibodies (APA) may be useful for women undergoing assisted reproductive technology (ART) therapy, as these antibodies increase the risk of pregnancy complications and thrombotic risks associated with ovarian stimulation, but studies assessing AFA effect on in vitro fertilization (IVF) outcomes have ambiguous results. The presence of antithyroid antibodies (ATA) may be associated with infertility, so their assessment is indeed important to determine treatment tactics. It has been suggested that antinuclear antibodies (ANA) may affect reproductive function by disrupting trophoblast cell development and interfering with RNA transcription, which may lead to lowered reproductive success. Studies have shown that patients with a positive ANA data have a lower incidence of pregnancy and a higher rate of miscarriages after IVF procedure. The human herpes virus type 6 (HHV-6) affects female fertility and is often the cause of spontaneous termination of pregnancy. B-cell lymphoma protein 6 (BCL-6) can serve as an important prognostic biomarker to identify individuals with endometriosis and related reproductive disorders, including idiopathic infertility. Evaluation of endometrial decidualization can be a useful tool to assess readiness for endometrial implantation and provide opportunities for targeted therapeutic interventions. The issue of testing for NK-cells in patients undergoing infertility screening remains controversial due to the difficulties of standardizing testing recommendations.Conclusion. To date, there is a limited number of reliable data on the role of various immunological tests in infertility diagnostics and treatment. With the exception of testing for AFA in patients with RPL, as well as for thyroid-stimulating hormone (TSH) and anti-thyroperoxidase antibodies (TPOAb) in patients undergoing therapy using various types of assisted reproductive technologies, the remaining immunological tests provide scant data to justify their routine use in clinical practice. The major limitations of existing studies are coupled to a small patient sample, as well as heterogeneity of inclusion criteria, patient groups and research methods.

Sensitive and Accurate Quantification of Enterovirus-D68 (EV-D68) Viral Loads Using Droplet Digital PCR (ddPCR).

Enterovirus-D68 (EV-D68) is a reemerging virus that has been associated with numerous outbreaks in children in the past 10 years. Most assays examining viral infection kinetics have relied on the use of quantitative RT-PCR (qRT-PCR) assays as an assay of choice. Though valuable, there are inherent limitations that introduce variability, thereby reducing its value when comparing results across the field. Unlike the qRT-PCR assay that uses a standard curve to determine the copy number of viral RNA, the droplet digital PCR assay (ddPCR) directly quantifies the absolute number of copies within a given sample, which in turn makes the assay highly sensitive and accurate. Here, we have developed an EV-D68-specific ddPCR assay that effectively quantifies EV-D68 RNA copies in both cells and supernatants within a dynamic range of 6.7 × 10-3 copies/μL to 1.2 × 104 copies/μL of the sample. The assay was highly specific for a broad range of EV-D68 isolates (Fermon, US/MO/14-18947, US/MO/14-18949, US/KY/14-18953, USA/2018-23088, USA/2020-23336 and EV-D68-infected human nasal turbinate samples from the 2022 outbreak) without cross-reactivity to other viruses such as Enterovirus-A71 (EV-A71), Human Parechovirus (HPeV)-1 and -2, Coxsackievirus (CV)-B1, Human Coronavirus (HCoV)-NL63, SARS-CoV-2, Influenza-A and B, Rhinovirus, and Respiratory Syncytial Virus (RSV)-A2, which are known to cause infection in children. The assay was able to readily quantify EV-D68 in infected cells and supernatants along with nasal turbinate samples collected from children during the 2022 outbreak. Our results suggest that the assay can be readily translated to accurately quantify viral loads in tissues and body fluids such as plasma and lung or nasal aspirates.

Genome-wide analysis of transcription-coupled repair reveals novel transcription events in Caenorhabditis elegans.

Bulky DNA adducts such as those induced by ultraviolet light are removed from the genomes of multicellular organisms by nucleotide excision repair, which occurs through two distinct mechanisms, global repair, requiring the DNA damage recognition-factor XPC (xeroderma pigmentosum complementation group C), and transcription-coupled repair (TCR), which does not. TCR is initiated when elongating RNA polymerase II encounters DNA damage, and thus analysis of genome-wide excision repair in XPC-mutants only repairing by TCR provides a unique opportunity to map transcription events missed by methods dependent on capturing RNA transcription products and thus limited by their stability and/or modifications (5'-capping or 3'-polyadenylation). Here, we have performed eXcision Repair-sequencing (XR-seq) in the model organism Caenorhabditis elegans to generate genome-wide repair maps in a wild-type strain with normal excision repair, a strain lacking TCR (csb-1), and a strain that only repairs by TCR (xpc-1). Analysis of the intersections between the xpc-1 XR-seq repair maps with RNA-mapping datasets (RNA-seq, long- and short-capped RNA-seq) reveal previously unrecognized sites of transcription and further enhance our understanding of the genome of this important model organism.

Identification and experimental validation of KMO as a critical immune-associated mitochondrial gene in unstable atherosclerotic plaque

BackgroundThe heightened risk of cardiovascular and cerebrovascular events is associated with the increased instability of atherosclerotic plaques. However, the lack of effective diagnostic biomarkers has impeded the assessment of plaque instability currently. This study was aimed to investigate and identify hub genes associated with unstable plaques through the integration of various bioinformatics tools, providing novel insights into the detection and treatment of this condition.MethodsWeighted Gene Co-expression Network Analysis (WGCNA) combined with two machine learning methods were used to identify hub genes strongly associated with plaque instability. The cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) method was utilized to assess immune cell infiltration patterns in atherosclerosis patients. Additionally, Gene Set Variation Analysis (GSVA) was conducted to investigate the potential biological functions, pathways, and mechanisms of hub genes associated with unstable plaques. To further validate the diagnostic efficiency and expression of the hub genes, immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were performed on collected human carotid plaque and blood samples. Immunofluorescence co-staining was also utilized to confirm the association between hub genes and immune cells, as well as their colocalization with mitochondria.ResultsThe CIBERSORT analysis demonstrated a significant decrease in the infiltration of CD8 T cells and an obvious increase in the infiltration of M0 macrophages in patients with atherosclerosis. Subsequently, two highly relevant modules (blue and green) strongly associated with atherosclerotic plaque instability were identified. Through intersection with mitochondria-related genes, 50 crucial genes were identified. Further analysis employing least absolute shrinkage and selection operator (LASSO) logistic regression and support vector machine recursive feature elimination (SVM-RFE) algorithms revealed six hub genes significantly associated with plaque instability. Among them, NT5DC3, ACADL, SLC25A4, ALDH1B1, and MAOB exhibited positive correlations with CD8 T cells and negative correlations with M0 macrophages, while kynurenine 3-monooxygenas (KMO) demonstrated a positive correlation with M0 macrophages and a negative correlation with CD8 T cells. IHC and RT-qPCR analyses of human carotid plaque samples, as well as ELISA analyses of blood samples, revealed significant upregulation of KMO and MAOB expression, along with decreased ALDH1B1 expression, in both stable and unstable samples compared to the control samples. However, among the three key genes mentioned above, only KMO showed a significant increase in expression in unstable plaque samples compared to stable plaque samples. Furthermore, the expression patterns of KMO in human carotid unstable plaque tissues and cultured mouse macrophage cell lines were assessed using immunofluorescence co-staining techniques. Finally, lentivirus-mediated KMO silencing was successfully transduced into the aortas of high-fat-fed ApoE-/- mice, with results indicating that KMO silencing attenuated plaque formation and promoted plaque stability in ApoE-/- mice.ConclusionsThe results suggest that KMO, a mitochondria-targeted gene associated with macrophage cells, holds promise as a valuable diagnostic biomarker for assessing the instability of atherosclerotic plaques.

Robust CXCL10/IP-10 and CCL5/RANTES Production Induced by Tick-Borne Encephalitis Virus in Human Brain Pericytes Despite Weak Infection.

Tick-borne encephalitis virus (TBEV) targets the central nervous system (CNS), leading to potentially severe neurological complications. The neurovascular unit plays a fundamental role in the CNS and in the neuroinvasion of TBEV. However, the role of human brain pericytes, a key component of the neurovascular unit, during TBEV infection has not yet been elucidated. In this study, TBEV infection of the primary human brain perivascular pericytes was investigated with highly virulent Hypr strain and mildly virulent Neudoerfl strain. We used Luminex assay to measure cytokines/chemokines and growth factors. Both viral strains showed comparable replication kinetics, peaking at 3 days post infection (dpi). Intracellular viral RNA copies peaked at 6 dpi for Hypr and 3 dpi for Neudoerfl cultures. According to immunofluorescence staining, only small proportion of pericytes were infected (3% for Hypr and 2% for Neudoerfl), and no cytopathic effect was observed in the infected cells. In cell culture supernatants, IL-6 production was detected at 3 dpi, together with slight increases in IL-15 and IL-4, but IP-10, RANTES and MCP-1 were the main chemokines released after TBEV infection. These chemokines play key roles in both immune defense and immunopathology during TBE. This study suggests that pericytes are an important source of these signaling molecules during TBEV infection in the brain.

Long noncoding RNA KCNMA1-AS2 regulates the function of colorectal cancer cells and sponges miR-1227-5p

BackgroundMany long noncoding RNAs (lncRNAs) with altered expression significantly influence colorectal cancer (CRC) progression and behavior. The functions of many lncRNAs in CRC are not clear yet. This study aimed to discover novel lncRNA entities and comprehensively examine and validate their roles and underlying molecular mechanisms in CRC.MethodsTissue samples, both tumourous and non-tumourous, from three CRC patients were submitted for sequencing. Following expression validation in samples from ten patients and four CRC cell lines. The lncRNA KCNMA1-AS2 was synthesized by In-vitro transcription RNA synthesis and the lncRNA was directly transfected into CRC cell lines to overexpress. Functional assays including MTT proliferation assay, Annexin-V/propidium iodide apoptosis assay, wound healing migration assay and cell cycle assays were performed to evaluate the effect of overexpression of KCNMA1-AS2. Furthermore, the binding of KCNMA1-AS2 to miR-1227-5p was confirmed using dual luciferase reporter assays and qPCR analyses. Subsequent bioinformatics analyses identified 58 potential downstream targets of miR-1227-5p across three databases.ResultsIn this study, we identified the lncRNA KCNMA1-AS2, the expression of which was down-regulated consistently in cancer tissues and CRC cell lines compared to non-cancerous tissues. The overexpression of lncRNA KCNMA1-AS2 led to significant reduction in CRC cell proliferation and migration, increase in cell apoptosis, and more cells arrested in S phase. Additionally, the interaction between KCNMA1-AS2 and miR-1227-5p was confirmed through dual luciferase reporter assay and qPCR analysis. It is also putatively predicted that MTHFR and ST8SIA2 may be linked to CRC based on bioinformatics analyses.ConclusionsLncRNA KCNMA1-AS2 exhibited distinct gene expression patterns in both CRC tissue and cell lines, impacting various cellular functions while also acting as a sponge for miR-1227-5p.The findings spotlight lncRNA KCNMA1-AS2 as a potential marker for diagnosis and treatment of CRC.

Conformational Dynamics of Influenza A Virus Ribonucleoprotein Complexes during RNA Synthesis.

Viral ribonucleoproteins (vRNPs) are the cornerstones of viral proliferation, as they form the macromolecular complexes that are responsible for the transcription and replication of most single-stranded RNA viruses. The influenza A virus (IAV) polymerase catalyzes RNA synthesis within the context of vRNPs where genomic viral RNA (vRNA) is packaged by the viral nucleoprotein (NP). We used high-speed atomic force microscopy and electron microscopy to study the conformational dynamics of individual IAV recombinant RNPs (rRNPs) during RNA synthesis. The rRNPs present an annular organization that allows for the real-time tracking of conformational changes in the NP-vRNA template caused by the advancing polymerase. We demonstrate that the rRNPs undergo a well-defined conformational cycle during RNA synthesis, which can be interpreted in light of previous transcription models. We also present initial estimations of the average RNA synthesis rate in the rRNP and its dependence on the nucleotide concentration and stability of the nascent RNA secondary structures. Furthermore, we provide evidence that rRNPs can perform consecutive cycles of RNA synthesis, accounting for their ability to recycle and generate multiple copies of RNA.

Unveiling systemic responses in kidney transplantation: interplay between the allograft transcriptome and serum proteins.

Immunity, as defined by systems biology, encompasses a holistic response throughout the body, characterized by intricate connections with various tissues and compartments. However, this concept has been rarely explored in kidney transplantation. In this proof-of-concept study, we investigated a direct association between the allograft phenotype and serum protein signatures. Time-matched samples of graft biopsies and blood serum were collected in a heterogeneous cohort of kidney-transplanted patients (n = 15) for bulk RNA sequencing and proteomics, respectively. RNA transcripts exhibit distinct and reproducible, coregulated gene networks with specific functional profiles. We measured 159 serum proteins and investigated correlations with gene expression networks. Two opposing axes-one related to metabolism and the other to inflammation-were identified. They may represent a biological continuum between the allograft and the serum and correlate with allograft function, but not with interstitial fibrosis or proteinuria. For signature validation, we used two independent proteomic data sets (n = 21). Our findings establish a biological link between the allograft transcriptome and the blood serum proteome, highlighting systemic immune effects in kidney transplantation and offering a promising framework for developing allograft-linked biomarkers.

Altered RNA export by SF3B1 mutants confers sensitivity to nuclear export inhibition

SF3B1 mutations frequently occur in cancer yet lack targeted therapies. Clinical trials of XPO1 inhibitors, selinexor and eltanexor, in high-risk myelodysplastic neoplasms (MDS) revealed responders were enriched with SF3B1 mutations. Given that XPO1 (Exportin-1) is a nuclear exporter responsible for the export of proteins and multiple RNA species, this led to the hypothesis that SF3B1-mutant cells are sensitive to XPO1 inhibition, potentially due to altered splicing. Subsequent RNA sequencing after XPO1 inhibition in SF3B1 wildtype and mutant cells showed increased nuclear retention of RNA transcripts and increased alternative splicing in the SF3B1 mutant cells particularly of genes that impact apoptotic pathways. To identify novel drug combinations that synergize with XPO1 inhibition, a forward genetic screen was performed with eltanexor treatment implicating anti-apoptotic targets BCL2 and BCLXL, which were validated by functional testing in vitro and in vivo. These targets were tested in vivo using Sf3b1K700E conditional knock-in mice, which showed that the combination of eltanexor and venetoclax (BCL2 inhibitor) had a preferential sensitivity for SF3B1 mutant cells without excessive toxicity. In this study, we unveil the mechanisms underlying sensitization to XPO1 inhibition in SF3B1-mutant MDS and preclinically rationalize the combination of eltanexor and venetoclax for high-risk MDS.

The problem of comorbidity in patients with HIV infection and hepatic cirrhosis in the intensive care unit

Objective: clinical and laboratory assessment of HIV-infected patients in the intensive care unit with liver cirrhosis that developed as a result of hepatitis of various etiologies.Materials and methods: a retrospective assessment of 91 medical records of patients with hepatic cirrhosis among 494 HIV-infected patients in the intensive care unit (ICU) was carried out in order to describe the clinical and laboratory features of the course of liver pathology against the background of HIV infection with secondary diseases. The subjects under observation were 46 (50.5%) men, 45 (49.5%) women, median age — 41 years.Results: patients were diagnosed with profound immunodeficiency (CD4+ lymphocyte count 100,000 copies of HIV RNA per 1 ml — in 45.1% of patients). Child-Pugh class C hepatic cirrhosis was diagnosed in 72.5% of patients. Cirrhosis in most cases developed as a result of toxic hepatitis in combination with chronic hepatitis C (CHC). More than half of the patients showed clinical and laboratory signs of decompensated hepatic cirrhosis. Combinations of two to five secondary infections were diagnosed in 52.8% of patients. The structure of secondary diseases was dominated by bacterial pneumonia, encephalitis and visceral candidiasis. 75.8% of patients did not receive ART, all patients did not receive antiviral therapy for chronic hepatitis.Conclusion: the mortality rate of patients with decompensated liver cirrhosis in the intensive care unit was 80.2% and was associated with the number of secondary diseases and the lack of antiretroviral therapy. Patients with a combination of HIV infection and viral hepatitis require earlier diagnosis and prescription of etiotropic therapy.

Exploring the impact of primer length on efficient gene detection via high-throughput sequencing

Reverse transcription (RT) is a crucial step in most RNA analysis methods. Optimizing protocols for this initial stage is critical for effective target detection, particularly when working with limited input RNA. Several factors, such as the input material quality and reaction conditions, influence RT efficiency. However, the effect of RT primer length on gene detection efficiency remains largely unknown. Thus, we investigate its impact by generating RNA-seq libraries with random RT primers of 6, 12, 18, or 24 nucleotides. To our surprise, the 18mer primer shows superior efficiency in overall transcript detection compared to the commonly used 6mer primer, especially in detecting longer RNA transcripts in complex human tissue samples. This study highlights the critical role of primer length in RT efficiency, which has significant potential to benefit various transcriptomic assays, from basic research to clinical diagnostics, given the central role of RT in RNA-related analyses.

Characterization of Caulimovirid-like Sequences from Upland Cotton (Gossypium hirsutum L.) Exhibiting Terminal Abortion in Georgia, USA.

In this study, we investigated the potential involvement of endogenous viral elements (EVEs) in the development of apical tissue necrosis, resulting in the terminal abortion of upland cotton (Gossypium hirsutum L.) in Georgia. The high-throughput sequence analysis of symptomatic and asymptomatic plant tissue samples revealed near-complete EVE-Georgia (EVE-GA) sequences closely related to caulimoviruses. The analysis of EVE-GA's putative open reading frames (ORFs) compared to cotton virus A and endogenous cotton pararetroviral elements (eCPRVE) revealed their similarity in putative ORFs 1-4. However, in the ORF 5 and ORF 6 encoding putative coat protein and reverse transcriptase, respectively, the sequences from EVE-GA have stop codons similar to eCPRVE sequences from Mississippi. In silico mining of the cotton genome database using EVE-GA as a query uncovered near-complete viral sequence insertions in the genomes of G. hirsutum species (~7 kb) but partial in G. tomentosum (~5.3 kb) and G. mustelinum (~5.1 kb) species. Furthermore, cotton EVEs' episomal forms and messenger RNA (mRNA) transcripts were detected in both symptomatic and asymptomatic plants collected from cotton fields. No significant yield difference was observed between symptomatic and asymptomatic plants of the two varieties evaluated in the experimental plot. Additionally, EVEs were also detected in cotton seeds and seedlings. This study emphasizes the need for future research on EVE sequences, their coding capacity, and any potential role in host immunity or pathogenicity.

Molecular basis for cA6 synthesis by a type III-A CRISPR-Cas enzyme and its conversion to cA4 production.

The type III-A(Csm)CRISPR-Cassystems are multi-subunit and multipronged prokaryotic enzymes in guarding the hosts against viral invaders. Beyond cleaving activator RNA transcripts, Csm confers two additional activities: shredding single-stranded DNA and synthesizing cyclic oligoadenylates (cOAs) by the Cas10 subunit. Known Cas10 enzymes exhibit a fascinating diversity in cOA production. Three major forms-cA3, cA4 and cA6have been identified, each with the potential to trigger unique downstream effects. Whereas the mechanism for cOA-dependent activation is well characterized, the molecular basis for synthesizing different cOA isoforms remains unclear. Here, we present structural characterization of a cA6-producing Csm complex during its activation by an activator RNA. Analysis of the captured intermediates of cA6 synthesis suggests a 3'-to-5' nucleotidyl transferring process. Three primary adenine binding sites can be identified along the chain elongation path, including a unique tyrosine-threonine dyad found only in the cA6-producing Cas10. Consistently, disrupting the tyrosine-threonine dyad specifically impaired cA6 production while promoting cA4 production. These findings suggest that Cas10 utilizes a unique enzymatic mechanism for forming the phosphodiester bond and has evolved distinct strategies to regulate thecOA chain length.

Analytical Validation of the Multitarget Stool RNA Test for Colorectal Cancer Screening

The multitarget stool RNA (mt-sRNA) test (ColoSense) is a noninvasive diagnostic test that screens for colorectal cancer and advanced adenomas in average-risk individuals aged 45 years and older. The mt-sRNA test incorporates a commercially available fecal immunochemical test, concentration of eight RNA transcripts, and participant-reported smoking status. As part of the CRC-PREVENT (Colorectal Cancer and Pre-Cancerous Adenoma Non-Invasive Detection Test) clinical trial, 12 analytical validation studies were conducted to assess analytical sensitivity, linearity, precision, interfering substances, cross-reactivity, carry-over, cross-contamination, and robustness. Analytical validation of the mt-sRNA test demonstrated limit of blank, limit of detection, and limit of quantification of <0.6, <0.7, and ≤2.5 copies/μL for all markers, respectively. The mt-sRNA test demonstrated linearity between 2.5 and 2500 copies/μL, and <20% coefficient of variation, and/or ≥95% concordance with regard to precision, interfering substances, carry-over, cross-contamination, and robustness. There was no significant impact of cross-reactivity from non-colorectal cancer diseases. These data provide a framework for laboratories to complete analytical validation for RNA-based panels that require premarket approval as a class III medical device from the US Food and Drug Administration.

A multiplex RT-qPCR assay for simultaneous detection of cucurbit viruses from individual whitefly and plant samples.

Whiteflies (Bemisia tabaci) are a significant pest of cucurbits and vectors many viruses leading to substantial economic losses. Modern diagnostic tools offer the potential for early detection of viruses in the whiteflies before crop production. One such tool is the multiplex reverse transcriptase quantitative PCR (RT-qPCR) probe-based technique, which can detect multiple targets in a single reaction and simultaneously quantify the levels of each target, with a detection limit of 100 copies per target. In this study, a multiplex RT-qPCR-based detection system capable of identifying one DNA virus and three RNA viruses in whiteflies: cucurbit leaf crumple virus (CuLCrV), cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and squash vein yellowing virus (SqVYV) was developed. To ensure the reliability of the assay, an internal gene control as the fifth target to monitor false-negative results was incorporated. This newly developed molecular diagnostic tool possesses several advantages. It can detect up to five desired targets from a single whitefly RNA sample, even at concentrations as low as 1 ng/µl. To evaluate its sensitivity, we conducted experiments using serially diluted cloned plasmids and in vitro transcribed RNA transcripts of the target viruses. We also assessed the specificity of the assay by including aphid-transmitted viruses and other viruses known to infect cucurbits. The diagnostic method successfully detected all five targets simultaneously and allowed for the quantification of up to 100 copies using a mixture of healthy? RNA and in vitro transcribed RNA. Our aim with this study was to develop a highly specific and sensitive one-step multiplex RT-qPCR system for the simultaneous detection of viruses transmitted by whiteflies in cucurbits. This system offers significant advantages for early detection, enabling prompt control measures to mitigate the further spread of viral infections and reduce yield losses. Additionally, we demonstrated the ability to simultaneously detect mixed viruses (CCYV, CYSDV, CuLCrV, and SqVYV) in individual whiteflies and quantify the number of viral copies carried by each whitefly. The multiplex RT-qPCR assay outperforms currently available techniques for detecting many samples at a given time and can be effectively utilized for early monitoring of plant viruses in individual whiteflies and symptomless plants.

Rapid, easy, sensitive, low‐cost and on‐site detection of environmental DNA and RNA using CRISPR‐Cas13

Abstract Environmental DNA (eDNA) monitoring of species distribution has become a critical tool in ecology, conservation biology and fisheries for identifying the presence and distribution of diverse organisms, including important, threatened and invasive species. However, eDNA detection still has room for improvement in sensitivity, requiring time‐consuming real‐time polymerase chain reaction (qPCR) steps and costly machines. In this study, we report a CRISPR‐Cas13‐based method for rapid, easy, sensitive, low‐cost and on‐site detection of eDNA. The assay for the detection of common carp (Cyprinus carpio) and medaka (Oryzias latipes) nucleic acid employs a two‐step process, starting with recombinase polymerase amplification (RPA) and followed by cleavage using Cas13 nuclease, to effectively identify mitochondrial DNA or RNA. Our results showed that the Cas13‐based method has a higher sensitivity than the qPCR‐based method in detecting tiny amounts of eDNA. When combined with reverse transcription of environmental RNA (eRNA), our method increased detection sensitivity by approximately one order of magnitude. Cas13‐based detection could achieve on‐site detection of eDNA using a quick nucleic acid extraction solution and lateral flow strips. Cas13‐based detection of eDNA and eRNA requires minimal training efforts and can be performed in 1 h, without the need for centrifugation and qPCR machines. The portability of the Cas13‐based method, along with its accuracy and relative ease in designing primers and CRISPR RNA (crRNA), underscores its potential to broaden the application of eDNA and eRNA in various fields, including biodiversity conservation.

LncRNA Nostrill promotes interferon-γ-stimulated gene transcription and facilitates intestinal epithelial cell-intrinsic anti-Cryptosporidium defense.

Intestinal epithelial cells possess the requisite molecular machinery to initiate cell-intrinsic defensive responses against intracellular pathogens, including intracellular parasites. Interferons(IFNs) have been identified as cornerstones of epithelial cell-intrinsic defense against such pathogens in the gastrointestinal tract. Long non-coding RNAs (lncRNAs) are RNA transcripts (>200 nt) not translated into protein and represent a critical regulatory component of mucosal defense. We report here that lncRNA Nostrill facilitates IFN-γ-stimulated intestinal epithelial cell-intrinsic defense against infection by Cryptosporidium, an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children. Nostrill promotes transcription of a panel of genes controlled by IFN-γ through facilitating Stat1 chromatin recruitment and thus, enhances expression of several genes associated with cell-intrinsic defense in intestinal epithelial cells in response to IFN-γ stimulation, including Igtp, iNos, and Gadd45g. Induction of Nostrill enhances IFN-γ-stimulated intestinal epithelial defense against Cryptosporidium infection, which is associated with an enhanced autophagy in intestinal epithelial cells. Our findings reveal that Nostrill enhances the transcription of a set of genes regulated by IFN-γ in intestinal epithelial cells. Moreover, induction of Nostrill facilitates the IFN-γ-mediated epithelial cell-intrinsic defense against cryptosporidial infections.

The sensing of circRNA with tetrahedral DNA nanostructure modified microfluidic chip

BackgroundCircular ribonucleic acids (circRNAs) are a type of covalently closed noncoding RNA with disease-relevant expressions, making them promising biomarkers for diagnosis and prognosis. Accurate quantification of circRNA in biological samples is a necessity for their clinical application. So far, methods developed for detecting circRNAs include northern blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), microarray analysis, and RNA sequencing. These methods generally suffer from disadvantages such as large sample consumption, cumbersome process, low selectivity, leading to inaccurate quantification of circRNA. It was thought that the above drawbacks could be eliminated by the construction of a microfluidic sensor. ResultsHerein, for the first time, a microfluidic sensor was constructed for circRNA analysis by using tetrahedral DNA nanostructure (TDN) as the skeleton for recognition probes and target-initiated hybridization chain reaction (HCR) as the signal amplification strategy. In the presence of circRNA, the recognition probe targets the circRNA-specific backsplice junction (BSJ). The captured circRNA then triggers the HCR by reacting with two hairpin species whose ends were labeled with 6-FAM, producing long DNA strands with abundant fluorescent labels. By using circ_0061276 as a model circRNA, this method has proven to be able to detect circRNA of attomolar concentration. It also eliminated the interference of linear RNA counterpart, showing high selectivity towards circRNA. The detection process can be implemented isothermally and does not require expensive complicated instruments. Moreover, this biosensor exhibited good performance in analyzing circRNA targets in total RNA extracted from cancer cells. SignificanceThis represents the first microfluidic system for detection of circRNA. The biosensor showed merits such as ease of use, low-cost, small sample consumption, high sensitivity and specificity, and good reliability in complex biological matrix, providing a facile tool for circRNA analysis and related disease diagnosis in point-of care application scenes.

Telomere-to-telomere genome assembly of the goose Anser cygnoides

Our study presents the assembly of a high-quality Taihu goose genome at the Telomere-to-Telomere (T2T) level. By employing advanced sequencing technologies, including Pacific Biosciences HiFi reads, Oxford Nanopore long reads, Illumina short reads, and chromatin conformation capture (Hi-C), we achieved an exceptional assembly. The T2T assembly encompasses a total length of 1,197,991,206 bp, with contigs N50 reaching 33,928,929 bp and scaffold N50 attaining 81,007,908 bp. It consists of 73 scaffolds, including 38 autosomes and one pair of Z/W sex chromosomes. Importantly, 33 autosomes were assembled without any gap, resulting in a contiguous representation. Furthermore, gene annotation efforts identified 34,898 genes, including 436,162 RNA transcripts, encompassing 806,158 exons, 743,910 introns, 651,148 coding sequences (CDS), and 135,622 untranslated regions (UTR). The T2T-level chromosome-scale goose genome assembly provides a vital foundation for future genetic improvement and understanding the genetic mechanisms underlying important traits in geese.