Amount Of RNA Research Articles

A global view of oat immunogenicity for celiac disease, from genes to cellular response

Celiac disease is an autoimmune disease triggered by the ingestion of gluten proteins and manifested in the small intestine. Celiac patients must keep a strict gluten-free diet. Although pure oats technically fulfil the criteria set for gluten-free products, the safety of oats in general or of distinct cultivars has been questioned. In this study we investigated 23 oat cultivars and lines that are commercially relevant, or have been suggested to induce adverse activity, or both. The oat seeds were investigated systematically for celiac epitope gene expression by RNA sequencing, peptide detection and quantification by mass spectrometry, and patient-derived T-cell activation to increase the knowledge on their risks. The RNA expression data showed three known avenin-specific T-cell epitopes to be expressed in low abundance in all cultivars with some degree of variation. Two of the three epitopes were also detectable in comparable amounts at the protein level in all cultivars, but their amounts were quite low, together averaging 2.6 mg/kg. Avenin preparations did not induce comprehensive IFN-γ secretion in celiac patients' peripheral blood cells. Any particularly ‘toxic’ cultivar and all commercial oat cultivars presented generally very low abundances of celiac epitopes on both gene and protein levels and are considered safe for consumption.

Pseudouridine Detection and Quantification Using Bisulfite Incorporation Hindered Ligation.

Pseudouridine (Ψ) is a widespread RNA modification found in various RNA species, including rRNA, tRNA, snRNA, mRNA, and long noncoding RNA (lncRNA). Understanding the function of Ψ in these RNA types requires a robust method for the detection and quantification of the Ψ level at single-nucleotide resolution. A previously used method utilizes Ψ labeling by N-cyclohexyl-N'-β-(4-methylmorpholinium)ethylcarbodiimide (CMC). The quantification of Ψ is based on the stop ratio after reverse transcription. However, the use of CMC followed by strong alkaline treatment causes severe RNA degradation, often requiring a large amount of RNA. The removal of CMC and recovery of RNA by ethanol precipitation are also time-consuming. Here, we introduce a Bisulfite Incorporation Hindered ligation-based method (BIHIND), which can detect and quantify Ψ sites on rRNA, mRNA, and noncoding RNA. BIHIND can be coupled with quantitative PCR (BIHIND-qPCR) for quantitative detection of Ψ fraction at individual modification sites, as well as with next-generation sequencing (BIHIND-seq) for high-throughput sequencing of Ψ without requiring reverse transcription. We validated the robustness of BIHIND with the elucidation of Ψ dynamics following pseudouridine synthase depletion.

Zilebesiran: The First siRNA Drug Therapy for Hypertension

Blood pressure, which includes ischemic heart disease, stroke, and chronic kidney disease, is the leading preventable cause of death from cardiovascular illnesses on a global scale. Worldwide, arterial hypertension ranks first among cardiovascular diseases (CVDs) and has done so for a long time. One of the first drugs to target hypertension using small interfering RNA (siRNA) technology is zilebesiran. Zilebesiran, an RNA interference therapy drug now in development, binds strongly to the hepatic asialoglycoprotein receptor. A therapeutic target for hypertension, it aims to decrease angiotensinogen production by measuring hepatic angiotensinogen messenger RNA (mRNA) quantities. Zilebesiran is a novel, ground-breaking siRNA therapy for the treatment of hypertension that is now in the second stage of clinical studies. How much of it crosses the placenta and whether it might be utilized to treat preeclampsia should be addressed in future research.

Insecticidal, physiological, and biochemical effects of Schinus molle essential oil on the adults of Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae)

Rhyzopertha dominica (F.) is considered as a major stored product pest. Conventional insecticides induce adverse effects on the environment. Plant derivative products constitute a promising alternative for pest control. The purpose of our research was to identify the chemical profile of essential oil (EO) extracted from Schinus molle L. (Anacardiaceae) and to evaluate the fumigant and the contact toxicity, and the repellent activity of this oil on adults of Rhyzopertha dominica F. (Coleoptera), a major pest in products that are stored around the world. The effects of this oil were examined on biochemical profile (energy reserves and Proteins), selective biomarkers like acetylcholinesterase (AChE), glutathione S-transferases (GST) and glutathione (GSH), and nucleic acids (DNA and RNA). Digestive enzymes were also investigated. The major chemical compounds identified by the GC-MS were α-phellandrene (16.613%), ß-phellandrene (12.104%), elemol (9.028%) and β-eudesmol (5.872%). Then, we evaluated the repellent activity, and also the effects in nutritional reserves and some biomarkers. This EO exhibited an insecticidal and repellent activity as a function of the exposure time and concentrations. Contact (LC50 at 24 h: 19.99 μL/mL) is the most effective mode of application compared to fumigation (LC50 at 24h: 174.30 μL/L air). S. molle EO was also investigated in digestive enzymes’ activities (α-amylase, lipase, chitinase and protease). Our results revealed a decrease in both α-amylase, lipase, protease and chitinase activities in treated adults compared to controls. In addition, we noticed a neurotoxic activity of S. molle EO traduced by a decrease of AChE. An induction of the detoxification system was observed via a decrease in GSH rate and an increase in GST activity. Moreover, the treatment induces an alteration of biochemical profile of R. dominica adults. Lastly, essential oil was examined on nucleic acids. Results showed an increase in DNA and RNA amount in treated adults when compared with control. Due to their effective source of bioactive compounds, the EO from the studied plant could be used as an alternative strategy for the management of stored product pests.

P-127 The RNA m6A landscape of mouse oocytes and preimplantation embryos

Abstract Study question Despite the significance of N6-methyladenosine (m6A) in gene regulation, the requirement for large amounts of RNA has hindered m6A profiling in mammalian early embryos. Summary answer We apply picoMeRIP-seq to map m6A in mouse oocytes and preimplantation embryos. We define the landscape of m6A during the maternal-to-zygotic transition. What is known already N6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic messenger RNA (mRNA), plays key regulatory roles in many biological processes (for example, RNA stability, splicing, transport and translation) and is involved in a variety of physiological processes (for example, cell differentiation and reprogramming, embryonic development and stress responses). Study design, size, duration We recently developed an m6A mapping assay for low RNA input, low-input methyl RNA immunoprecipitation and sequencing (picoMeRIP-seq), which enables mapping of the m6A methylome using a low number of cells. In this article, we applied picoMeRIP-seq to multiple developmental stages of mouse oocytes and early embryos to profile their m6A landscapes. Participants/materials, setting, methods We use mouse as study materials.Using picoMeRIP-seq, we generated transcriptome-wide m6A maps for six stages (GV, MII, zygote, two-cell, eight-cell and blastocyst), with two biological replicates per stage and 49–110 oocytes/embryos per replicate. Main results and the role of chance We define the landscape of m6A during the maternal-to-zygotic transition, including stage-specifically expressed transcription factors essential for cell fate determination. Both the maternally inherited transcripts to be degraded post fertilization and the zygotically activated genes during zygotic genome activation are widely marked by m6A. In contrast to m6A-marked zygotic ally-activated genes, m6A-marked maternally inherited transcripts have a higher tendency to be targeted by microRNAs. Moreover, RNAs derived from retrotransposons, such as MTA that is maternally expressed and MERVL that is transcriptionally activated at the two-cell stage, are largely marked by m6A. Our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryonic development. Limitations, reasons for caution The effect of m6A on the turnover of retrotransposon-derived transcripts abundant in oocytes and early embryos needs to be further addressed in future studies. Wider implications of the findings Our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryonic development. Trial registration number not applicable

Utility Analyses of AVITI Sequencing Chemistry.

DNA sequencing is a critical tool in modern biology. Over the last two decades, it has been revolutionized by the advent of massively parallel sequencing, leading to significant advances in the genome and transcriptome sequencing of various organisms. Nevertheless, challenges with accuracy, lack of competitive options and prohibitive costs associated with high throughput parallel short-read sequencing persist. Here, we conduct a comparative analysis using matched DNA and RNA short-reads assays between Element Biosciences' AVITI and Illumina's NextSeq 550 chemistries. Similar comparisons were evaluated for synthetic long-read sequencing for RNA and targeted single-cell transcripts between the AVITI and Illumina's NovaSeq 6000. For both DNA and RNA short-read applications, the study found that the AVITI produced significantly higher per sequence quality scores. For PCR-free DNA libraries, we observed an average 89.7% lower experimentally determined error rate when using the AVITI chemistry, compared to the NextSeq 550. For short-read RNA quantification, AVITI platform had an average of 32.5% lower error rate than that for NextSeq 550. With regards to synthetic long-read mRNA and targeted synthetic long read single cell mRNA sequencing, both platforms' respective chemistries performed comparably in quantification of genes and isoforms. The AVITI displayed a marginally lower error rate for long reads, with fewer chemistry-specific errors and a higher mutation detection rate. These results point to the potential of the AVITI platform as a competitive candidate in high-throughput short read sequencing analyses when juxtaposed with the Illumina NextSeq 550.

Assessment of performance and comparison of three commercial HDV RNA detection assays: implications for diagnosis and treatment monitoring.

Precise HDV-RNA detection and quantification are pivotal for diagnosis and monitoring of response to newly approved treatment. We evaluate the performance of three HDV RNA detection and quantification assays. Hepatitis Delta RT-PCR system kit, EurobioPlex HDV assay, and RoboGene HDV RNA Quantification kit 2.0 were used for testing 151 HBsAg-positive samples, 90 HDV-RNA negative and 61 HDV-RNA positive. We also evaluated serial dilutions of the WHO international standard for HDV, PEI 7657/12. All HDV-RNA positive samples were genotyped using a next-generation sequencing strategy. Qualitative results indicated a 100% concordance between tests. Quantitative results correlated well, r2 = 0.703 (Vircell-vs-Eurobio), r2 = 0.833 (Vircell-vs-RoboGene), r2 = 0.835 (Robogene-vs-Eurobio). Bias index was 2.083 (Vircell-vs-Eurobio), -1.283 (Vircell-vs-RoboGene), and -3.36 (Robogene-vs-Eurobio). Using the WHO IS, Vircell overestimated the viral load by 0.98 log IU/mL, Eurobio by 1.46 log IU/mL, and RoboGene underestimated it by 0.98 log IU/mL. Fifty-nine samples were successfully genotyped (Genotype 1, n=52; Genotype 5, n=7; Genotype 6, n=1), with similar results for correlation and bias. This study underscores the necessity of using reliable HDV-RNA detection and quantification assays, as evidenced by the high concordance rates in qualitative detection and the observed variability in quantitative results. These findings highlight the importance of consistent assay use in clinical practice to ensure accurate diagnosis and effective treatment monitoring of HDV infection.

Investigation of MIRs in Amphetamine Misusers

Introduction: Drug addiction is characterized by uncontrolled drug use and recurrence of need for medication. Changes in gene expression play an important role in addictive neuroplasticity, but the mechanisms by which addictive drugs regenerate brain motive circuits are unclear. MicroRNAs (miRNAs) are a group of non-coding small RNAs that bind to the translocated regions; primers of their target mRNAs inhibit translation and suppress gene expression. This study investigated the profiles and different expression of MIR 24 and MIR 329 in the patients with amphetamine dysfunction. Methods: This study was performed on 30 patients .of medical centers. RNA extraction was performed according to the kit. The amount of RNA extracted was evaluated by a nanodrop machine. These RNAs were then added to Poly (A) tail and then transformed into cDNA using specific primers and reverse transcriptase. Finally, their expression was quantitatively measured by qRT-PCR method compared to the control group. Results: In the present study, the results indicated that MIR 24 gene expression was significantly increased compared to U6. The MIR 24 gene showed an increase of 9.02 (P-value: 0.049) compared to the control group in blood samples. The MIR 329 gene showed a decrease of 0.007 (P-value: 0.000) compared to the control group in blood samples. Conclusion: The results showed that there was a significant association between miR-24 and miR-329 expression in blood samples of amphetamine users, which may be related to amphetamine abuse disorder. Used as a noninvasive diagnostic marker for the detection of amphetamine abuse disorder.

Rapid and portable quantification of HIV RNA via a smartphone-enabled digital CRISPR device and deep learning

For the 29.8 million people in the world living with HIV/AIDS and receiving antiretroviral therapy, it is crucial to monitor their HIV viral loads. To this end, rapid and portable diagnostic tools that can quantify HIV RNA are critically needed. We report herein a rapid and quantitative digital CRISPR-assisted HIV RNA detection assay that has been implemented within a portable smartphone-based device as a potential solution. Specifically, we first developed a fluorescence-based reverse transcription recombinase polymerase amplification (RT-RPA)-CRISPR assay that can efficiently detect HIV RNA at 42 °C. We then implemented this assay within a commercial stamp-sized digital chip, where RNA molecules were quantified as strongly fluorescent digital reaction wells. The isothermal reaction condition and the strong fluorescence in the digital chip simplified the design of thermal and optical modules, allowing us to engineer a palm-size device measuring 70 × 115 × 80 mm and weighing less than 0.6 kg. We also capitalized the smartphone by developing a custom app to control the device, perform the digital assay, and capture fluorescence images throughout the assay using the smartphone's camera. Moreover, we trained and verified a deep learning-based algorithm for analyzing fluorescence images and identifying positive digital reaction wells with high accuracy. Using our smartphone-enabled digital CRISPR device, we successfully detected as low as 75 copies of HIV RNA in just 15 min, showing its potential toward monitoring of HIV viral loads and aiding the global effort to combat the HIV/AIDS epidemic.

SEPTIC SHOCK: LPS TOLERANCE PROTECTS MITOCHONDRIAL BIOGENESIS AND RESPIRATION.

Mitochondrial dysfunction is a recognized feature of sepsis, characterized by ultrastructural damage, diminished oxidative phosphorylation, and depletion of mitochondrial antioxidant capacity observed in deceased septic patients. LPS tolerance induces a controlled response to sepsis. This study aimed to evaluate the function of tolerant mitochondria after cecal ligation and puncture (CLP)-induced sepsis. Mytochondrial oxygen consumption was determined using polarography. Extraction and quantification of RNA for the expression of Tfam, Nrf-1, and Ppargc-1α, and respiratory complex activity were measured. CLP-tolerant animals presented preserved respiratory rates of S3 and S4 and a ratio of respiratory control (RCR) compared to CLP-nontolerant animals with reduced oxidative phosphorylation and increased uncoupled respiration. Complex I Vmax was reduced in septic animals; however, CLP animals sustained normal Vmax. Mitochondrial biogenesis was preserved in CLP-tolerant animals compared to the CLP-nontolerant group, likely due to increased TFAM expression. LPS tolerance protected septic animals from mitochondrial dysfunction, favoring mitochondrial biogenesis and preserving mitochondrial respiration and respiratory complex I activity.

Strategies to Reduce Promoter-Independent Transcription of DNA Nanostructures and Strand Displacement Complexes.

Bacteriophage RNA polymerases, in particular T7 RNA polymerase (RNAP), are well-characterized and popular enzymes for many RNA applications in biotechnology both in vitro and in cellular settings. These monomeric polymerases are relatively inexpensive and have high transcription rates and processivity to quickly produce large quantities of RNA. T7 RNAP also has high promoter-specificity on double-stranded DNA (dsDNA) such that it only initiates transcription downstream of its 17-base promoter site on dsDNA templates. However, there are many promoter-independent T7 RNAP transcription reactions involving transcription initiation in regions of single-stranded DNA (ssDNA) that have been reported and characterized. These promoter-independent transcription reactions are important to consider when using T7 RNAP transcriptional systems for DNA nanotechnology and DNA computing applications, in which ssDNA domains often stabilize, organize, and functionalize DNA nanostructures and facilitate strand displacement reactions. Here we review the existing literature on promoter-independent transcription by bacteriophage RNA polymerases with a specific focus on T7 RNAP, and provide examples of how promoter-independent reactions can disrupt the functionality of DNA strand displacement circuit components and alter the stability and functionality of DNA-based materials. We then highlight design strategies for DNA nanotechnology applications that can mitigate the effects of promoter-independent T7 RNAP transcription. The design strategies we present should have an immediate impact by increasing the rate of success of using T7 RNAP for applications in DNA nanotechnology and DNA computing.

Assessment of long-term stored specimens in the Siriraj Hospital colorectal cancer biobank for RNA sequencing and profiling

Abstract Objectives Biobanks play an important role in advancing cancer research, yet concerns persist regarding the molecular integrity of long-term stored samples. This study assessed fresh frozen (FF) tissues and formalin-fixed paraffin-embedded (FFPE) tissues from the Siriraj Hospital colorectal cancer (CRC) biobank collected during two distinct periods (2011–2012 and 2020–2021). Methods In 2022, FF and FFPE primary cancer tissues from 75 CRC patients were evaluated. RNA sequencing (RNA-Seq) analyzed comprehensive gene expression profiles in FF tissues preserved at −80 °C, while nCounter profiling elucidated cancer-specific RNA transcripts in FFPE tissues stored at ambient temperature. Comparative analyses were conducted between specimens from 2011 to 2012 and 2020–2021. Results The FF tissues stored for approximately 10.5 years were well-suited for RNA-Seq compared to the intact tissues preserved for 1.5 years. Despite consistencies in RNA quantity, RNA integrity, amount of sequencing reads, and CRC gene signature, gene enrichment analysis revealed the decreased ribosome biogenesis, spliceosome and antifolate resistance pathways in the 2011–2012 group. Moreover, the FFPE tissues also showed no alteration in RNA quantity between the two periods, and the nCounter profiling demonstrated comparable CRC-specific gene counts in spite of the significant reduction of raw counts in the 2011–2012 group. Conclusions We report that FF tissues from CRC patients, stored for 10 years, are viable for whole transcriptome RNA-Seq, despite altered pathways such as ribosome biogenesis, spliceosome, and antifolate resistance. Moreover, 10-year-stored FFPE CRC tissues remain suitable for specific RNA profiling using the nCounter pan-cancer panel, despite a significant reduction in raw counts. These findings underscore the enduring contribution of biobanks to molecular research, highlighting their value a decade post-collection.

Defining low-HER2 cancers using RNA quantification by next generation sequencing.

e13044 Background: The recent success of treatment with HER2-directed antibody-drug conjugate T-DXd (trastuzumab-deruxtecan) in treating low-HER2 expression breast cancer raises the possibility of similar success in other tumors that express similarly low levels of HER2. HER2 amplification has been reported in various types of tumors at relatively low frequency. However, “low-HER2” expression in various types of tumors remains undefined. In this study, we utilized next generation sequencing to explore the levels of ERBB2 (HER2), ERBB3, ERBB4, PIK3CA, and ERS1 mRNA in various types of cancers and attempted to set limits reflecting distinct biology of tumors based of level of ERBB2 expression. Methods: RNA was extracted from FFPE samples from patients with cancers of breast (N=203), ovary (N=95), endometrium (N=75), and esophagus/stomach (N=46). RNA was sequenced via a 1408 gene panel using a standard hybrid capture approach. Results: Based on previous data correlating gene amplification with gene expression (DOI: 10.1002/imed.1051), we defined ERBB2 expression as high, low, and very-low when ERBB2 expression measured >1500 TPM (transcripts per million), 800-1500 TPM, and <800 TPM, respectively. ERBB2 high, low, and very-low were detected in 20%, 32%, and 48% of breast cancer; 5%, 29%, and 65% of endometrial cancer; 9%, 26%, and 65% of ovarian cancer; 12%, 16%, and 72% of esophageal/stomach cancer; and 8%, 19%, and 73% of lung cancer. With minor exceptions, cases classified as ERBB2 high and low from the five tumor types showed no correlation with levels of PIK3CA, ESR1, or ERBB4 mRNA. In contrast, cases with relatively very-low ERBB2 showed significant correlation with PIK3CA, ESR1, and ERBB4 mRNA. Endometrial cancer was an exception and showed no correlation with PIK3CA mRNA in very-low ERBB2 cases. Activation of PIK3CA has been reported to be associated with resistance to anti-HER2 therapy. These findings suggest that cases showing high or low ERBB2 are independent of PIK3CA and likely less influenced by the negative effects of PIK3CA on response to HER2 inhibitors. Cases classified as ERBB2 high or low showed significantly higher (P<0.0001) PIK3CA mutations as compared with ERBB2 very-low cases (27% vs 14%). ERBB2 mutations were relatively rare, detected in only 7%, 8%, 14%, 1%, and 4% of breast, endometrial, esophageal/stomach, ovarian, and lung cancers, respectively. However, significantly (P=0.001) higher mutation rate is detected in ERBB2-high and -low as compared with very-low ERBB2 cases (8% vs 6%). Conclusions: ERBB2/HER2 RNA quantification in cancers can define a biologically distinct group of patients. Patients defined as very-low ERBB2 as classified in this approach appear to be biologically distinct and more likely to be resistant to anti-HER2 therapy. Clinical trials using this approach in selecting patients for treatment with antibody-drug conjugates are justified and needed.

Integrative genomic analysis of the lung tissue microenvironment in SARS-CoV-2 and NL63 patients

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 virus has affected over 700 million people, and caused over 7 million deaths throughout the world as of April 2024, and continues to affect people through seasonal waves. While over 675 million people have recovered from this disease globally, the lingering effects of the disease are still under study. Long term effects of SARS-CoV-2 infection, known as 'long COVID,' include a wide range of symptoms including fatigue, chest pain, cellular damage, along with a strong innate immune response characterized by inflammatory cytokine production. Three years after the pandemic, data about long covid studies are finally emerging. More clinical studies and clinical trials are needed to understand and determine the factors that predispose individuals to these long-term side effects.In this methodology paper, our goal was to apply data driven approaches in order to explore the multidimensional landscape of infected lung tissue microenvironment to better understand complex interactions between viral infection, immune response and the lung microbiome of patients with (a) SARS-CoV-2 virus and (b) NL63 coronavirus. The samples were analyzed with several machine learning tools allowing simultaneous detection and quantification of viral RNA amount at genome and gene level; human gene expression and fractions of major types of immune cells, as well as metagenomic analysis of bacterial and viral abundance. To contrast and compare specific viral response to SARS-COV-2, we analyzed deep sequencing data from additional cohort of patients infected with NL63 strain of corona virus.Our correlation analysis of three types of RNA-seq based measurements in patients i.e. fraction of viral RNA (at genome and gene level), Human RNA (transcripts and gene level) and bacterial RNA (metagenomic analysis), showed significant correlation between viral load as well as level of specific viral gene expression with the fractions of immune cells present in lung lavage as well as with abundance of major fractions of lung microbiome in COVID-19 patients.Our methodology-based proof-of-concept study has provided novel insights into complex regulatory signaling interactions and correlative patterns between the viral infection, inhibition of innate and adaptive immune response as well as microbiome landscape of the lung tissue. These initial findings could provide better understanding of the diverse dynamics of immune response and the side effects of the SARS-CoV-2 infection and demonstrates the possibilities of the various types of analyses that could be performed from this type of data.

Selection and Multiplexing of Reverse Transcription-Quantitative PCR Tests Targeting Relevant Honeybee Viral Pathogens.

Verifying the inclusivity of molecular detection methods gives indications about the reliability of viral infection diagnosis because of the tendency of viral pathogens to undergo sequence variation. This study was aimed at selecting inclusive probes based on reverse transcription-quantitative PCR (RT-qPCR) assays for the diagnosis of the most widespread and detrimental viruses infecting honeybees, namely the acute bee paralysis virus (ABPV), the black queen cell virus (BQCV), the chronic paralysis bee virus (CBPV), the deformed wing virus variants A (DWVA) and B (DWVB), and the sacbrood virus (SBV). Therefore, previously described detection methods were re-evaluated in silico for their specificity and inclusivity. Based on this evaluation, selected methods were modified, or new ones were designed and tested in duplex RT-qPCR reactions. The limits of detection (LODs), effect of multiplexing on sensitivity and the viral RNA quantification potential in bees and hive debris were assessed. This study made available diagnostic assays able to detect an increased number of virus variants compared with previously described tests and two viral pathogens in a single PCR reaction.

Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation

Extracellular vesicles (EVs) are lipid-membrane enclosed structures that are associated with several diseases, including those of genitourinary tract. Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000g to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time.

A molecular standard for circulating HBV RNA detection and quantification assays in patients with chronic hepatitis B

BackgroundCirculating HBV RNAs have been proposed as a biomarker that reflects the transcriptional activity of cccDNA and may help to evaluate HBV treatments activity. Different research assays have been proposed and, although 2 PCR-based research use only (RUO) investigational assays (IA) have been developed, the lack of standardized protocols represents an important limitation. Here we have designed and generated a stable clonal cell line producing an RNA-based standard for the calibration of PCR-based circulating HBV RNA assays. MethodsHBV RNA producing Huh7-derived stable cell lines were generated by transfecting pTriEX plasmids containing 1.1 length HBV DNA genomes carrying mutations in the catalytic site (YMAA mutation) and the TP-domain (Y63F) of the polymerase, and the ε-loop of the pgRNA (mutation A1G). ResultsThe clonal cell line (Huh7-3D29), carrying a double YMAA and Y63F mutation, displayed, and maintained over several passages in culture, a high RNA secretion phenotype with negligible residual secreted HBV DNA. Density gradient centrifugation showed that most of the secreted HBV RNA from Huh7-3D29 cells was detected in naked capsid and virions-like particles and only a minority in small extracellular vescicles (sEV). Nanopore sequencing of 5’RACE products shows that the majority of the Huh7-3D29 secreted HBV RNAs start at the 5' end of pgRNA and pgRNA derived spliced RNAs. Finally, Huh7-3D29 cells showed a high and up-scalable secreted RNA yield allowing 1,300 standard curves in 9 days from 1 flask. ConclusionWe generated a clonal cell line that produces high amounts of HBV RNAs with very low quantities of contaminating HBV DNAs, representing a stable source of RNA standard for HBV RNA assays calibration. Impact and ImplicationsSeveral investigational assays and 2 research use only (RUO) assays have been developed to detect and quantify circulating HBV RNAs, an emerging biomarker of cccDNA transcriptional activity and target engagement by new HBV treatments. The lack of a unique molecular standard for circulating HBV RNAs quantification represents an important limitation. Here we describe the generation of a stable clonal cell line producing and secreting an RNA-based standard containing all the HBV RNA species found in HBV patients’ sera (e.g., pgRNA, HBx transcripts). This new RNA standard will serve for the calibration of all PCR-based assays for circulating HBV RNA quantification to evaluate in a non-invasive manner the size of the transcriptionally active cccDNA pool and the activity of novel strategies aiming at curing HBV infection.

An Emission-Based Probe for the Detection and Quantification of DNA and RNA.

Sequence-independent detection of low concentrations of nucleic acids is important for applications in forensics and diagnostics. An emission-based probe for detecting and quantifying DNA and RNA utilizing a water-soluble dicationic tetraphenylethene (TPE) derivativewas developed. The recognition is based on the electrostatic and other non-covalent interactions between the phosphate backbone of nucleic acids and the cationic probe, which cause the restriction of rotation of the aryl units of the probe, ensuing in the enhancement of the fluorescence signal. The binding was validated by different spectroscopic techniques and also by electrophoretic mobility shift assay. The probable mode of binding with the nucleic acids was studied by blind-docking studies that correlated well with the experimental results.

Development of a SARS-CoV-2 viral dynamic model for patients with COVID-19 based on the amount of viral RNA and viral titer.

The target-cell limited model, which is one of the mathematical modeling approaches providing a quantitative understanding of viral dynamics, has been applied to describe viral RNA profiles of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in previous studies. However, these models have been developed mainly using patient data from the early phase of the pandemic. Furthermore, no reports focused on the profiles of the viral titer. In this study, the dynamics of both viral RNA and viral titer were characterized using data reflecting the current clinical situation in which the Omicron variant has become epidemic and vaccines for SARS-CoV-2 have become available. Consecutive data for 5212 viral RNA levels and 5216 viral titers were obtained from 720 patients with coronavirus disease 2019 (COVID-19) in a phase II/III study for ensitrelvir. Our model assumed that productively infected cells would produce only infectious viruses, which could be transformed into non-infectious viruses, and has been used to describe the dynamics of both viral RNA levels and viral titer. The time from infection to symptom onset (tinf) of unvaccinated patients was estimated to be 3.0 days, which was shorter than that of the vaccinated patients. The immune-related parameter as a power function for the vaccinated patients was 1.1 times stronger than that for the unvaccinated patients. Our model allows the prediction of the viral dynamics in patients with COVID-19 from the time of infection to symptom onset. Vaccination status was identified as a factor influencing tinf and the immune function.

RNA degradation in human mitochondria: the journey is not finished.

Mitochondria are vital organelles present in almost all eukaryotic cells. Although most of the mitochondrial proteins are nuclear-encoded, mitochondria contain their own genome, whose proper expression is necessary for mitochondrial function. Transcription of the human mitochondrial genome results in the synthesis of long polycistronic transcripts that are subsequently processed by endonucleases to release individual RNA molecules, including precursors of sense protein-encoding mRNA (mt-mRNA) and a vast amount of antisense noncoding RNAs. Because of mitochondrial DNA (mtDNA) organization, the regulation of individual gene expression at the transcriptional level is limited. Although transcription of most protein-coding mitochondrial genes occurs with the same frequency, steady-state levels of mature transcripts are different. Therefore, post-transcriptional processes are important for regulating mt-mRNA levels. The mitochondrial degradosome is a complex composed of the RNA helicase SUV3 (also known as SUPV3L1) and polynucleotide phosphorylase (PNPase, PNPT1). It is the best-characterized RNA-degrading machinery in human mitochondria, which is primarily responsible for the decay of mitochondrial antisense RNA. The mechanism of mitochondrial sense RNA decay is less understood. This review aims to provide a general picture of mitochondrial genome expression, with a particular focus on mitochondrial RNA (mtRNA) degradation.