Detection Of mRNA Research Articles

Susceptibility of Pimephales promelas and Carassius auratus to a strain of koi herpesvirus isolated from wild Cyprinus carpio in North America

Cyprinid herpesvirus-3 (CyHV-3, syn. koi herpesvirus) is an important pathogen worldwide and a common cause of mass mortality events of wild common carp (Cyprinus carpio) in North America, however, reference strains and genomes obtained from wild carp are not available. Additionally, it is unclear if fishes in North America are susceptible to CyHV-3 infection due to incomplete susceptibility testing. Here we present the first North American type strain and whole-genome sequence of CyHV-3 isolated from wild carp collected from a lake with a history and recent incidence of carp mortality. Additionally, the strain was used in an in-vivo infection model to test the susceptibility of a common native minnow (Pimephales promelas) and goldfish (Carrasius auratus) which is invasive in North America. Detection of CyHV-3 DNA was confirmed in the tissues of a single fathead minnow but the same tissues were negative for CyHV-3 mRNA and samples from exposed fathead minnows were negative on cell culture. There was no detection of CyHV-3 DNA or mRNA in goldfish throughout the experiment. CyHV-3 DNA in carp tissues was reproducibly accompanied by the detection of CyHV-3 mRNA and isolation on cell culture. Additionally, environmental CyHV-3 DNA was detected on all tank filters during the study. These findings suggest that fathead minnows and goldfish are not susceptible to CyHV-3 infection and that detection of CyHV-3 DNA alone in host susceptibility trials should be interpreted with caution.

Systematic comparison of high-throughput single-cell RNA-seq methods for immune cell profiling

BackgroundElucidation of immune populations with single-cell RNA-seq has greatly benefited the field of immunology by deepening the characterization of immune heterogeneity and leading to the discovery of new subtypes. However, single-cell methods inherently suffer from limitations in the recovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropout events. This issue is often compounded by limited sample availability and limited prior knowledge of heterogeneity, which can confound data interpretation.ResultsHere, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. We prepared 21 libraries under identical conditions of a defined mixture of two human and two murine lymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluated methods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expression signatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5′ v1 and 3′ v3 methods. We demonstrate that these methods have fewer dropout events, which facilitates the identification of differentially-expressed genes and improves the concordance of single-cell profiles to immune bulk RNA-seq signatures.ConclusionOverall, our characterization of immune cell mixtures provides useful metrics, which can guide selection of a high-throughput single-cell RNA-seq method for profiling more complex immune-cell heterogeneity usually found in vivo.

Quantitative analysis of RNAscope staining for c-fos expression in mouse brain tissue as a measure of Neuronal Activation

The expression of c-fos mRNA is an indirect marker of neuronal activity. RNAscope ACD Bio RNAscope (now Biotechne) is a proprietary in-situ mRNA detection technology using branched DNA amplification and z paired probes to deliver a robust and specific assay designed primarily for use on formalin fixed paraffin sections [1]. In the present study we adapted this technology to be used in frozen sections to allow quantitative analysis of c-fos gene expression in different mouse brain regions during neuropharmacology studies. The method was applied by Cosi et al. 2021 [2] and the image analysis is described here in details.•The patented RNAscope (ACD Bio) flourescent in-situ hybridisation technology designed primarily for use on formalin fixed paraffin sections was adapted to be used on frozen section from mouse brain.•We carefully controlled sample preparation and handling to maximise mRNA preservation and used the fluorescent properties of the fast Red substrate combined with fluorescent whole slide scanning and image analysis.•A customized algorithm was set up for image analysis•The method developed permitted the quantitative analysis of c-fos expression in specific brain regions from whole sections.

Versatile Multiplex Endogenous RNA Detection with Simultaneous Signal Normalization Using Mesoporous Silica Nanoquenchers.

Detection of endogenous tumor-related RNA is vital for cancer diagnostics. Despite advancements made, live-cell RNA detection still faces numerous problems, such as low signal output and cell-to-cell variations arising from differences in probe uptake. To address these issues, we designed a versatile and highly sensitive mRNA/miRNA nanosensor featuring, for the first time, signal amplification and in-built signal normalization. Using dye-loaded mesoporous silica nanoquenchers (qMSNs) capped with target-corresponding antisense oligos (ASOs), direct fluorescence "Turn-ON" with signal amplification was achieved upon target binding. By readily varying the capping ASOs as well as cargo dyes, a suite of RNA nanosensors for multiplex target detection could be easily prepared. Further modification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA-responsive molecular beacons (MBs) onto our nanosensor enabled dual detection of target RNA and GAPDH mRNA, allowing for target signal normalization using GAPDH as a reference. We demonstrated that this newly developed nanosensor could successfully differentiate between noncancer and cancer cells, as well as accurately monitor the relative expression levels of multiple tumor-related RNAs simultaneously in different cancer cell lines, with a high degree of specificity and sensitivity, functioning as a noninvasive "qPCR mimic" imaging tool in live cells.

Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples.

Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans.

Highly sensitive fluorescence biosensing of BCR-ABL1 fusion gene based on exponential transcription-triggered hemin catalysis

Simple, sensitive and specific detection of the transcription level of BCR-ABL1 mRNA possesses vital clinical significance in diagnosis and treatment of chronic myeloid leukemia (CML). In this study, an innovative fluorescence biosensing methodology has been developed for sensitive and specific detection of BCR-ABL1 mRNA by integrating high-efficiency of exponential transcription and superior catalytic performance of DNA-grafted hemin. Exponential transcription was triggered by BCR-ABL1 mRNA to produce plenty of RNA products. They can specifically hybridize with circular dual-labeled hemin (DLH) probe to dissociate the intramolecular hemin dimmers into highly active hemin monomers for catalyzing fluorescence substrate tyramine. This exponential transcription-triggered hemin catalysis (ET-HC) strategy showed highly sensitive and specific for BCR-ABL1 detection with a limit of detection at 0.5 aM and a good linear range from 2 aM to 200 fM. This method was successfully applied to directly detect as low as 0.001% e13a2 transcript isoforms from complex genomic RNA extraction. Compared with clinical routine, the overall process is a thermostatic reaction and eliminates additional reverse transcription operation. Therefore, the developed ET-HC strategy might provide a promising alternative tool for precise diagnosis and personalized treatment of CML.

Detection of parvovirus mRNAs as markers for viral activity in endomyocardial biopsy-based diagnosis of patients with unexplained heart failure.

Erythroparvovirus (B19V) genomes have been detected in various organs of infected individuals including endothelial cells of the heart muscle. However, the role of B19V as a causative pathogen of myocardial damage is still unknown. The majority of reports focus on the presence of viral DNA ignoring proof of viral RNAs as important markers for viral activity. During this study, we established (RT-) qPCR to characterize expression of B19V RNAs (NS1 and VP1/2) in endomyocardial biopsies (EMBs) of 576 patients with unexplained heart failure. 403/576 (70%) EMBs were positive for B19V DNA. B19V mRNAs NS1 and/or VP1/2, indicating viral activity, could be detected in 38.5% of B19V DNA positive samples using the newly established B19V RT-PCRs. 22.1% of samples were characterized by only NS1 mRNA detection while 6.0% revealed only VP1/2 mRNA expression. Detection of both intermediates was successful in 10.4% of samples. Applying the molecular testing, our study revealed that a high proportion (38.5%) of B19V DNA positive EMBs was characterized by viral transcriptional activity. Further prospective studies will evaluate relevance of viral transcription intermediates as a diagnostic marker to differentiate between latent B19V infection and clinically relevant transcriptionally active B19V-infection of the heart muscle.

Role of Human Papillomavirus Infection in Head and Neck Cancer in Italy: The HPV-AHEAD Study.

Simple SummaryThis is the largest and most comprehensive assessment of the role of human papillomavirus (HPV) in head and neck cancer (HNC) in Italy, which is a region currently considered bearing a low burden of HPV-driven HNC. p16INK4a, HPV-DNA, and HPV RNA biomarkers were used to assess the HPV status in head and neck cancer in a retrospective cohort of approximately 700 patients. In our study, HPV prevalence in oropharyngeal cancers was much higher than in oral and laryngeal cancers, and HPV positivity conferred better prognosis only in oropharyngeal cancers. Importantly, we have observed an increase of the prevalence of HPV positivity in oropharyngeal cancers in the most recent calendar periods, suggesting that this disease is increasing in Italy, as has happened before in other developed regions.Literature on the role of human papillomavirus (HPV) in head and neck cancer (HNC) in Italy is limited, especially for non-oropharyngeal tumours. Within the context of the HPV-AHEAD study, we aimed to assess the prognostic value of different tests or test algorithms judging HPV carcinogenicity in HNC and factors related to HPV positivity at the European Institute of Oncology. We conducted a retrospective cohort study (2000–2010) on a total of 696 primary HNC patients. Formalin-fixed, paraffin-embedded cancer tissues were studied. All HPV-DNA-positive and a random sample of HPV-DNA-negative cases were subjected to HPV-E6*I mRNA detection and p16INK4a staining. Multivariate models were used to assess for factors associated with HPV positivity and proportional hazards for survival and recurrence. The percentage of HPV-driven cases (considering HPV-E6*I mRNA positivity) was 1.8, 2.2, and 40.4% for oral cavity (OC), laryngeal (LC), and oropharyngeal (OPC) cases, respectively. The estimates were similar for HPV-DNA/p16INK4a double positivity. Being a non-smoker or former smoker or diagnosed at more recent calendar periods were associated with HPV-E6*I mRNA positivity only in OPC. Being younger was associated with HPV-E6*I mRNA positivity in LC. HPV-driven OPC, but not HPV-driven OC and LC, showed better 5 year overall and disease-free survival. Our data show that HPV prevalence in OPC was much higher than in OC and LC and observed to increase in most recent years. Moreover, HPV positivity conferred better prognosis only in OPC. Novel insights on the role of HPV in HNC in Italy are provided, with possible implications in the clinical management of these patients.

A simple and sensitive direct mRNA multiplexed detection strategy for amoA-targeted monitoring of ammonia-oxidizing activity in water environment

Nitrogenous pollutants are the main causes of water eutrophication, which greatly interfere with the balance of the aquatic ecosystem. The biological nitrogen removal using bacteria is the leading technology to control nitrogenous pollutants. The α-subunit (amoA) of ammonia monooxygenase (AMO) of aerobic ammonia-oxidizing bacteria (AOB) is an ammonia-depleting biomarker in nitrogen removal processes. Currently, RT-PCR and metatranscriptome are the general methods for analyzing amoA gene expression, however they can introduce amplification bias and are time consuming. Herein we report a new direct mRNA multiplexed detection strategy based on xMAP array, for analyzing gene expression without reverse transcription and amplification steps. After analyzing all amoA sequences in database, we design a universal probe for AOB with coverage of 79.8% whose high coverage is further confirmed by metatranscriptome. Moreover, we classify AOB into 10 clusters, and then design the specific probes of each cluster. This technique enables single-nucleotide, wide linear dynamic range (5–50,000 amol) and high sensitivity (5 amol with detection volume of 1 µL) detection with using locked-nucleic-acid probes. Particularly, the sensitivity is by far among the highest level for multiplex mRNA detection without amplification. We found that mRNA fragmentation is not necessary in our direct detection, simplifying our strategy. We have successfully detected amoA mRNA semi-quantitatively and determined the predominant cluster in sludge from wastewater treatment plants, which was consistent with RT-PCR and metatranscriptome results. Meanwhile, the ammonia-oxidizing activities of sludge verified the amoA mRNA detection results. In conclusion, we have developed a mRNA direct, simple, specific and sensitive multiplexed detection strategy, which shows great potential for application in gene expression analysis, environmental microbial detection and activity diagnosis of wastewater treatment plants.

In vivo imaging of tagged mRNA in plant tissues using the bacterial transcriptional antiterminator BglG.

RNA transport and localization represent important post-transcriptional mechanisms to determine the subcellular localization of protein synthesis. Plants have the capacity to transport messenger (m)RNA molecules beyond the cell boundaries through plasmodesmata and over long distances in the phloem. RNA viruses exploit these transport pathways to disseminate their infections and represent important model systems to investigate RNA transport in plants. Here, we present an in vivo plant RNA-labeling system based on the Escherichia coli RNA-binding protein BglG. Using the detection of RNA in mobile RNA particles formed by viral movement protein (MP) as a model, we demonstrate the efficiency and specificity of mRNA detection by the BglG system as compared with MS2 and λN systems. Our observations show that MP mRNA is specifically associated with MP in mobile MP particles but hardly with MP localized at plasmodesmata. MP mRNA is clearly absent from MP accumulating along microtubules. We show that the in vivo BglG labeling of the MP particles depends on the presence of the BglG-binding stem-loop aptamers within the MP mRNA and that the aptamers enhance the coprecipitation of BglG by MP, thus demonstrating the presence of an MP:MP mRNA complex. The BglG system also allowed us to monitor the cell-to-cell transport of the MP mRNA, thus linking the observation of mobile MP mRNA granules with intercellular MP mRNA transport. Given its specificity demonstrated here, the BglG system may be widely applicable for studying mRNA transport and localization in plants.

Utility and Limitations of Albumin mRNA In Situ Hybridization Detection in the Diagnosis of Hepatobiliary Lesions and Metastatic Carcinoma to the Liver.

Albumin messenger RNA (mRNA) in situ hybridization is a sensitive and specific biomarker for hepatocellular carcinoma (HCC). Intrahepatic cholangiocarcinoma (ICC) shows variable sensitivity, whereas extrahepatic cholangiocarcinoma (ECC) and metastatic carcinoma are generally negative. We studied the clinical utility and limitations of albumin mRNA detection in a cohort of HCCs, ICCs, ECCs, bile duct adenomas, bile duct hamartomas, and metastatic carcinomas to the liver; and investigated the variability in sensitivity observed for this biomarker in ICCs. We identified 122 cases of hepatobiliary lesions and metastatic carcinomas. Albumin mRNA detection was performed using RNAscope run on formalin-fixed, paraffin-embedded tissue sections. ICCs were categorized according to the classification proposed by Hayashi and colleagues into the small duct, large duct, and indeterminate subtypes. Albumin mRNA was detected in all 17 HCCs and focally in 6/8 (75%) of bile duct adenomas. All 28 nonhepatic carcinomas, 13 bile duct hamartomas, and 9 ECCs were negative. Albumin mRNA was found in 38/47 (80.9%) of ICC with 35/37 (94.6%) in the small duct subtype, 2/3 (66.7%) in the indeterminate subtype, and 1/7 (14.3%) of the large duct subtype (P<0.003). Albumin mRNA detection is a sensitive and specific biomarker for HCCs. It is highly sensitive and moderately specific in the diagnosis of ICC with small gland morphology, but not ICCs with large duct morphology and in metastatic carcinoma. The variability in the sensitivity of albumin mRNA expression in ICCs may depend on the subtypes of ICC.

Identification of Key Genes and Potential New Biomarkers for Ovarian Aging: A Study Based on RNA-Sequencing Data.

Ovarian aging leads to reproductive and endocrine dysfunction, causing the disorder of multiple organs in the body and even declined quality of offspring’s health. However, few studies have investigated the changes in gene expression profile in the ovarian aging process. Here, we applied integrated bioinformatics to screen, identify, and validate the critical pathogenic genes involved in ovarian aging and uncover potential molecular mechanisms. The expression profiles of GSE84078 were downloaded from the Gene Expression Omnibus (GEO) database, which included the data from ovarian samples of 10 normal C57BL/6 mice, including old (21–22 months old, ovarian failure period) and young (5–6 months old, reproductive bloom period) ovaries. First, we filtered 931 differentially expressed genes (DEGs), including 876 upregulated and 55 downregulated genes through comparison between ovarian expression data from old and young mice. Functional enrichment analysis showed that biological functions of DEGs were primarily immune response regulation, cell–cell adhesion, and phagosome pathway. The most closely related genes among DEGs (Tyrobp, Rac2, Cd14, Zap70, Lcp2, Itgb2, H2-Ab1, and Fcer1g) were identified by constructing a protein–protein interaction (PPI) network and consequently verified using mRNA and protein quantitative detection. Finally, the immune cell infiltration in the ovarian aging process was also evaluated by applying CIBERSORT, and a correlation analysis between hub genes and immune cell type was also performed. The results suggested that plasma cells and naïve CD4+ T cells may participate in ovarian aging. The hub genes were positively correlated with memory B cells, plasma cells, M1 macrophages, Th17 cells, and immature dendritic cells. In conclusion, this study indicates that screening for DEGs and pathways in ovarian aging using bioinformatic analysis could provide potential clues for researchers to unveil the molecular mechanism underlying ovarian aging. These results could be of clinical significance and provide effective molecular targets for the treatment of ovarian aging.

Impact of neurotrophic factors combination therapy on retinitis pigmentosa

ObjectiveWe aimed to determine the location of neurotrophic receptors tropomyosin receptor kinase (Trk)B, TrkC, and ciliary neurotrophic factor receptor (CNTFR)α in the retina of retinal degeneration (rd) mice and to explore the dynamic changes of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X-protein (Bax), and microtubule-associated protein light chain 3 (LC3) expression and ultrastructure in the retina of rd mice intravitreally injected with neurotrophic factors.MethodsRd mice aged 2 and 3 weeks post-natally (PN) received intravitreal injections of neurotrophic factors. Two weeks later, their retinas were harvested for the detection of Bax, Bcl-2, and LC3 mRNA and protein expression.ResultsTrkB and TrkC expression levels were lower at 3 weeks PN compared with 0, 1, and 2 weeks PN, but CNTFRα expression was still detected in certain layers. The three receptors were expressed in different retinal layers at the same timepoint. Bax expression was downregulated in, rhBDNF + rhCNTF, rhBDNF + rhNT-3, groups 2 weeks after intravitreal injection; Bcl-2 expression was upregulated in the rhBDNF + rhCNTF + rhNT-3 group at PN-4w; and LC3 expression was upregulated in rhBDNF + rhCNTF + rhNT-3 groups.ConclusionsThe combined use of neurotrophic factors had a more significant effect on Bax, Bcl-2, and LC3 expression than the same factors used alone.

Parvovirus B19 NS1 and VP1/2 mRNAs expression indicates viral activity in endomyocardial biopsy-based diagnosis of patients with unexplained heart failure

Abstract Background Human erythroparvovirus-1 (B19V) is the most frequently found virus in endomyocardial biopsies (EMBs). Recent studies have detected B19V genomes in various organs of symptomatic and asymptomatic individuals including endothelial cells of the heart muscle. Accordingly, B19V as a causative pathogen of myocardial damage has been questioned. To date, the majority of studies focused on the presence of viral DNA in the myocardium, but lack the analysis of viral RNAs as markers for viral activity. In contrast to latent infection, the expression of viral mRNAs is of clinical importance as we could demonstrate in previous studies. Only recently, novel antiviral treatment strategies have become available. A sensitive and specific method to identify and monitor patients that will profit from antiviral treatment is a prerequisite for successful therapy. During this study, we established a new PCR-based diagnostic method to characterize the expression of B19V RNAs coding for the non-structural (NS1) and capsid protein (VP1/2) in endomyocardial biopsies of patients with unexplained heart failure and demonstrated its feasibility in a clinical setting of molecular diagnostics. Methods To quantify B19V RNA expression, we developed real-time (reverse transcription) polymerase chain reactions (qPCRs) targeting the NS1 and VP1/2 regions of B19V genome. 576 randomly selected EMBs of patients with unexplained heart failure were tested for B19V genomes and transcription intermediates using qPCR and nested PCR. Results 403 of 576 (69.9%) EMBs were positive for B19V genomes (B19V DNA). Viral loads ranged from 15 to 51,118 genome equivalents/μg isolated DNA. Of the 403 B19V DNA-positive samples, B19V mRNAs NS1 and/or VP1/2, indicating viral activity, could be detected in 155/403 (38.4%) EMBs using the newly established B19V qPCRs. 89/403 samples were characterized by only NS1 mRNA detection while 24/403 revealed only VP1/2 mRNA expression. Detection of both intermediates was successful in 42/403 samples. Patients characterized by active B19V infection presented with a significantly reduced left ventricular ejection fraction (30.7±13.1%) in contrast to virus free patients (35.5±15.9%; p=0.025) or latently infected patients (34.7±15.7; p=0.043). Conclusion Our newly established molecular testing will improve detection and quantification of B19V. As we could demonstrate here, it is essential to screen for both viral transcription intermediates, the NS1 and VP1/2 viral mRNAs, in order to precisely differentiate between latent infection or a clinically relevant B19V-infection with transcriptional activity of the heart muscle. The results of our study revealed that a remarkable high proportion of 26.9% (155/576) of patients with unexplained heart failure is affected by transcriptional active B19V infection of the myocardium. Prospective studies must be conducted to evaluate our findings and furthermore, effective antiviral treatment strategies need to be adapted. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): IKDT GmbHn

Undernutrition reduces kisspeptin and neurokinin B expression in castrated male sheep.

SummaryUndernutrition impairs reproductive success through suppression of gonadotropin-releasing hormone (GnRH), and subsequently luteinizing hormone (LH), secretion. Given that kisspeptin and neurokinin B (NKB) neurons in the arcuate nucleus (ARC) of the hypothalamus are thought to play key stimulatory roles in the generation of GnRH/LH pulses, we hypothesized that feed restriction would reduce the ARC mRNA abundance and protein expression of kisspeptin and NKB in young, male sheep. Fourteen wethers (castrated male sheep five months of age) were either fed to maintain (FM; n = 6) pre-study body weight or feed-restricted (FR; n = 8) to lose 20% of pre-study body weight over 13 weeks. Throughout the study, weekly blood samples were collected and assessed for LH concentration using RIA. At Week 13 of the experiment, animals were killed, heads were perfused with 4% paraformaldehyde, and brain tissue containing the hypothalamus was collected, sectioned, and processed for detection of mRNA (RNAscope) and protein (immunohistochemistry) for kisspeptin and NKB. Mean LH was significantly lower and LH inter-pulse interval was significantly higher in FR wethers compared to FM wethers at the end of the experiment (Week 13). RNAscope analysis revealed significantly fewer cells expressing mRNA for kisspeptin and NKB in FR wethers compared to FM controls, and immunohistochemical analysis revealed significantly fewer immunopositive kisspeptin and NKB cells in FR wethers compared to FM wethers. Taken together, this data supports the idea that long-term feed restriction regulates GnRH/LH secretion through central suppression of kisspeptin and NKB in male sheep.Lay summaryWhile undernutrition is known to impair reproduction at the level of the brain, the components responsible for this in the brain remain to be fully understood. Using male sheep we examined the effect of undernutrition on two stimulatory molecules in the brain critical for reproduction: kisspeptin and neurokinin B. Feed restriction for several weeks resulted in decreased luteinizing hormone in the blood indicating reproductive function was suppressed. In addition, undernutrition also reduced both kisspeptin and neurokinin B levels within a region of the brain involved in reproduction, the hypothalamus. Given that they have stimulatory roles in reproduction, we believe that undernutrition acts in the brain to reduce kisspeptin and neurokinin B levels leading to the reduction in luteinizing hormone secretion. In summary, long-term undernutrition inhibits reproductive function in sheep through suppression of kisspeptin and neurokinin B within the brain.

Sensitive detection and quantification of SARS-CoV-2 by multiplex droplet digital RT-PCR

The purpose of this study is to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive quantification of SARS-CoV-2 RNA with respect to human-derived RNA and could be used for screening and monitoring of Covid-19 patients. A one-step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19-negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality and to enable quantification of SARS-CoV-2 RNA. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. It was found that assay sensitivity of the RT-ddPCR was not affected by the total NA background while assay sensitivity of the gold standard RT-PCR assay is drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared with water. The present study describes a robust and sensitive one-step ddRT-PCR multiplex assay for reliable quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.

Research progress on analysis of human papillomavirus by microchip capillary electrophoresis

Human papillomavirus (HPV), is a common spherical DNA virus that can lead to six types of cancers later in life, which has recently garnered human's attention. Microchip capillary electrophoresis (MCE) has provided simple, fast, portable, and sensitive HPV typing assay assisted by a variety of signal amplification technologies. This review presents the latest research progress of MCE in routine HPV typing assays, including both of the MCE techniques and MCE combined with the nucleic acid amplification techniques for HPV assay. The introduction on the former part concerns the MCE system, the MCE chips design and electrophoretic separation methods. The typical MCE system includes high voltage power supply, microfluidic chip of separation, sample injection, electrolyte cell, detection unit and so on. Four different chips are reviewed, containing straight separation channel, T-channel, serpentine channel and dual channel, because these microchips are the most used in the last decade. Furthermore, the high integration and high throughput on a single chip are often integrated the sample preparation unit on a chip. The advantages and disadvantages of different designed microchips are introduced at the same time. The separation methods of chip electrophoresis are briefly introduced. With the development and application of MCE for HPV detection, the separation time is greatly shortened from a few hours to several minutes. The review on the second part gives the comments on various kinds of nucleic acid amplification technologies coupled with MCE for HPV assay. Firstly, the comparative analysis is given on the polymerase chain reaction (PCR) combined with MCE, loop-mediated isothermal amplification (LAMP), PCR combined with restriction fragment length polymorphism (RFLP) for HPV DNA detection, and nucleic acid sequence based amplification (NASBA) for the detection of HPV mRNA, nested PCR and so on. Secondly, the reviews on the other methods beside MCE are also summarized, including the PCR coupled with Fourier transform-infrared spectroscopy (FT-IR spectroscopy), nanotechnology, DNA probes combined with electrochemical methods, reductive Cu(Ⅰ) particles catalyzed Zn-doped MoS2 quantum dots and T7 exonuclease with electrochemiluminescence, LAMP with CRISPR/Cas12a based lateral. In these non-MCE methods, the electrochemical sensing, e. g., impedimetric detection, pulse voltammetry method and flow biosensor, is an ideal method due to its low background signal and excellent time control ability. Finally, although MCE technologies have been developed and the developed instruments are applied in recent years, there are still some challenges in MCE techniques, methods and applications. The first challenge faced in the application of MCE technique in HPV typing assay is that the MCE device can not be well utilized for the detection of HPV with high resolution and high sensitivity, because MCE can not do signal amplification of HPV nucleic acid. The second challenge is that even though some researchers have successfully integrated PCR and MCE on one chip, the technique still faces difficulty for wide application and there is still no really integrated PCR-MCE chip for HPV detection. The third one is the MCE technique is lack for the manufacture of miniaturized and automatic instrument. At the end of review, the authors' insights are given on the development of automatic, fast, high stable and reliable detection in the HPV typing via the portable MCE device.

Angiotensin-converting enzyme 2, the SARS-CoV-2 cellular receptor, is widely expressed in human myometrium and uterine leiomyoma.

Objective:Angiotensin-converting-enzyme 2 (ACE2), the cell surface receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is found in a variety of reproductive tissues. The present study evaluated whether uterine fibroids and normal myometrium express ACE2 and, if so, at which tissue compartments.Methods:We included 13 premenopausal women (age range 33–50 years, median 40 years) with uterine fibroids undergoing elective hysterectomy or myomectomy. Samples of leiomyoma (n = 12) and normal myometrial tissue (n = 8) were analyzed by immunohistochemistry for protein localization or by real time PCR for mRNA detection.Results:In normal myometrium, ACE2 immunoreactivity was localized in smooth muscle fibers, arteriolar walls, and endothelial cells. In uterine leiomyoma, ACE2 staining was more intense in smooth muscle cells than in the extracellular matrix, and was also present in vascular endothelium. ACE2 mRNA was detected in myometrium as well as in fibroid samples.Conclusion:Human myometrium and uterine leiomyoma express ACE2 mRNA and have abundant distribution of ACE2 protein in their smooth muscle cells and microvasculature.

Protective effects of valsartan administration on doxorubicin‑induced myocardial injury in rats and the role of oxidative stress and NOX2/NOX4 signaling.

Clinical application of doxorubicin (DOX) is hampered by its potential cardiotoxicity, however angiotensin receptor blockers could attenuate DOX-induced cardiomyopathy. The present study tested the hypothesis that simultaneous administration of valsartan (Val) with DOX could prevent DOX-induced myocardial injury by modulating myocardial NAD(P)H oxidase (NOX) expression in rats. Eight-week-old male Sprague-Dawley rats were randomly divided into control (CON), DOX, and DOX+Val groups. After 10 weeks, surviving rats underwent echocardiography examination, myocardial mRNA and protein expression detection of NOX1, NOX2 and NOX4. H9C2 cells were used to perform in vitro experiments, reactive oxygen species (ROS) production and apoptosis were observed under the conditions of down- or upregulation of NOX2 and NOX4 in DOX- and DOX+Val-treated H9C2 cells. Cardiac function was significantly improved, pathological lesion and collagen volume fraction were significantly reduced in the DOX+Val group compared with the DOX group (all P<0.05). Myocardial protein and mRNA expression of NOX2 and NOX4 was significantly downregulated in DOX+Val group compared with in the DOX group (all P<0.05). In vitro, ROS production and apoptosis in DOX-treated H9C2 cells was significantly reduced by NOX2-small interfering (si)RNA and NOX4-siRNA, and significantly increased by overexpressing NOX2 and NOX4. To conclude, Val applied simultaneously with DOX could prevent DOX-induced myocardial injury and reduce oxidative stress by downregulating the myocardial expression of NOX2 and NOX4 in rats.

Simultaneous detection of multiple mRNAs and proteins in bovine IVD cells and tissue with single cell resolution

Interactions of cells with their neighbors and influences by the surrounding extracellular matrix (ECM) is reflected in a cells transcriptome and proteome. In tissues comprised of heterogeneous cell populations or cells depending on ECM signalling cues such as those of the intervertebral disc (IVD), this information is obscured or lost when cells are pooled for the commonly used transcript analysis by quantitative PCR or RNA sequencing. Instead, these cells require means to analyse RNA transcript and protein distribution at a single cell or subcellular level to identify different cell types and functions, without removing them from their surrounding signalling cues. We developed a simple, sequential protocol combining RNA is situ hybridisation (RISH) and immunohistochemistry (IHC) for the simultaneous analysis of multiple transcripts alongside proteins. This allows one to characterize heterogeneous cell populations at the single cell level in the natural cell environment and signalling context, both in vivo and in vitro. This protocol is demonstrated on cells of the bovine IVD, for transcripts and proteins involved in mechanotransduction, stemness and cell proliferation. A simple, sequential protocol combining RISH and IHC is presented that allows for simultaneous information on RNA transcripts and proteins to characterize cells within a heterogeneous cell population and complex signalling environments such as those of the IVD.