RNA Concentration Research Articles

SARS-CoV-2 surveillance in US wastewater: Leading indicators and data variability analysis in 2023–2024

Wastewater-Based Epidemiology (WBE) has become a powerful tool for assessing disease occurrence in communities. This study investigates the coronavirus disease 2019 (COVID-19) epidemic in the United States during 2023–2024 using wastewater data from 189 wastewater treatment plants in 40 states and the District of Columbia. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and pepper-mild mottle virus normalized SARS-CoV-2 RNA concentration data were compared with COVID-19 hospitalization admission data at both national and state levels. We further investigate temporal features in wastewater viral RNA abundance, with peak timing and cross-correlation lag analyses indicating that wastewater SARS-CoV-2 RNA concentrations precede hospitalization admissions by 2 to 12 days. Lastly, we demonstrate that wastewater treatment plant size has a significant effect on the variability of measured SARS-CoV-2 RNA concentrations. This study highlights the effectiveness of WBE as a non-invasive, timely and resource-efficient disease monitoring strategy, especially in the context of declining COVID-19 clinical reporting.

An epidemiologic surveillance study based on wastewater and respiratory specimens reveals influenza a virus prevalence and mutations in Taiyuan, China during 2023-2024.

Influenza is a major cause of morbidity and mortality. Influenza A virus (IAV) is one of the most important pathogens causing influenza and often causes global pandemics due to its tendency to mutate. We aim to use epidemiology based on wastewater and respiratory specimens to understand the occurrence of influenza A virus infections in Taiyuan City. A retrospective epidemiology surveillance was carried out at the First Hospital of Shanxi Medical University (FHSMU) and five wastewater treatment plants (WTPs) in Taiyuan city from 2023 to 2024. Reverse transcription real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect influenza A viruses in wastewater and respiratory specimens. High-throughput whole genome sequencing was performed on 17 strains obtained in this study, and subsequent analyses included characterization, phylogenetic construction, amino acid mutation analysis, and antigenic structural variability assessment. 520 wastewater samples and 1,203 throat swab samples were collected. We detected RNA concentration from pH1N1 and H3N2 viruses in wastewater and got 17 genome sequences (5 of pH1N1 and 12 of H3N2) in respiratory specimens. Whole-genome sequencing showed co-prevalence of pH1N1 viruses in the branches of 6B.1A.5a.2a.1 and H3N2 viruses in the branches of 3C.2a1b.2a.2a.3a.a in Taiyuan from 2023 to 2024. Moreover, a HA mutation (N138D), predicted to be of high phenotypic consequence, was found in 8 Taiyuan H3N2 sequences. This study highlights the predominant presence of pH1N1 and H3N2 strains in Taiyuan. The analysis also identified amino acid site variations in the HA antigenic epitopes in H3N2 strains, which may contribute to immune escape.

Influencing Factors Analysis of Infectious SARS-CoV-2 Aerosols Sampling

Assessing the potential infectivity of airborne viruses is critical for evaluating the risk of their transmission through air. This study investigated the factors influencing the collection of infectious SARS-CoV-2 aerosols using three common aerosol samplers: the impactor sampler AGI-30, the SKC Biosampler, and a cyclone sampler WA-400III. It was found that the sampling process over time significantly impacted the infectivity of SARS-CoV-2 captured in the sampler, and the infectivity loss of different SARS-CoV-2 variants in the process varied. Additionally, adding newborn calf serum in the collection suspension could effectively preserve viral infectivity in the sampler. Further tests conducted under various ventilation occasions indicated that ventilation can reduce the virus concentration in the environment and rapidly clear viral particles after aerosol generation. Although the flow rate of WA-400III is higher, it only could collect higher concentrations of viral RNA instead of live viruses than AGI-30 or SKC Biosampler when aerosol was generated constantly in the environment. After aerosol generation stopped, the WA-400III collected more infectious viruses and viral RNA. This study highlights the impact of the sampling process on captured virus should be assessed and protective agent is needed to preserve viral viability. And it is crucial to select appropriate sampler based on environmental characteristics and research objectives for obtaining optimal results. The AGI-30 and SKC Biosampler are preferable for collecting infectious viruses in environments with high aerosol concentrations, while the cyclone sampler WA-400III is more effective for collecting viral nucleic acids and enriching virus samples in confined spaces with lower viral loads.

Acid‐Responsive Polymer Additives Increase RNA Transfection from Lipid Nanoparticles

AbstractLipid nanoparticles (LNPs) are the most clinically advanced RNA delivery technology, but their efficiency is limited by low RNA release after endosome disruption. To improve RNA release, an acid‐responsive polymer is synthesized with which to formulate LNPs for RNA encapsulation and release. Specifically, three acid‐responsive poly(lactic acid)‐block‐poly(carboxybetaine) zwitterionic derivatives are designed and synthesized that are cationic and complexed with RNA at pH 7.4, but are neutral following cleavage at endosomal pH, thereby having lower affinity to RNA. The polymers are formulated into each of the clinically approved Onpattro, Moderna, or Pfizer LNP formulations to produce hybrid polymer‐lipid nanoparticles (PLNPs). With the PLNPs, the IC50 values of multiple small interfering RNAs (siRNAs) decreased up to 5.4‐fold compared to parent LNPs in several cell lines. Moreover, messenger RNA (mRNA) transfection increased up to two fold. The acid‐responsive polymers in PLNPs accounted for the enhanced RNA transfection as this phenomenon is lost with acid‐inert polymers. Confocal microscopy confirmed that cytosolic RNA concentration increased using the acid‐responsive polymers; conversely, uptake and endosomal escape are identical to existing LNPs. This confirmed that enhanced RNA transfection is due to increased RNA dissociation from its carrier. The novel polymer represents a versatile strategy to increase RNA transfection from LNPs.

Salivary Extracellular Vesicles Separation: Analysis of Ultracentrifugation-Based Protocols.

The clinical potential of extracellular vesicles (EVs) is widely acknowledged, yet the standardization and reproducibility of its separation remain challenging. This study compares three protocols: ultracentrifugation (UC), UC with purification step (UC + PS), and a combined protocol using polymer-based precipitation and UC (PBP + UC). Salivary samples were collected from healthy donors. EVs were separated (UC, UC + PS, and PBP + UC) and characterized using transmission electron microscopy, nanoparticle tracking analysis, EV purity, RNA concentration, and Western blotting. miRNA expression was evaluated by quantitative RT-PCR. Statistical analyses comparing groups were performed using ANOVA. All methods successfully separated CD9+ and CD63+ EVs from saliva. The UC + PS and PBP + UC protocols yielded the highest concentrations of EVs, enriched in < 200 nm vesicles. EV purity and RNA recovery were comparable among all methods. Expression of miR-16, miR-27a, and miR-99a was successfully detected using all methods. The UC + PS and PBP + UC protocols demonstrate comparable efficiency in separating salivary EVs. However, the combined PBP + UC protocol, with its simplified processing capability, offers a significant advantage, particularly in the initial phase of EV separation. This finding suggests its potential application in clinical settings where time-sensitive simple processing is critical. Further validation is needed to confirm its effectiveness for transcriptomic and proteomic analyses.

Highly sensitive and specific electrochemical biosensor for direct detection of hepatitis C virus RNA in clinical samples using DNA strand displacement

Hepatitis C virus (HCV) is a common blood-borne infection that can lead to long-term illnesses such as hepatocellular cancer and liver cirrhosis. Early diagnosis is crucial for effective management, as no vaccine is available for preventing HCV infection. However, the high cost and complexity of current molecular diagnostic tools hinder efforts to achieve early diagnosis and prevent transmission, particularly in resource-limited settings. We developed a novel electrochemical biosensor for point-of-care testing (POCT) of HCV RNA. The sensor utilizes a strand displacement method, where the target RNA displaces a gold nanoparticle-labeled reporter probe (AuRP) from a pre-hybridized duplex with a magnetic nanoparticle (MNP)-labeled capture probe. The amount of displaced AuRP, detected using differential pulse anodic stripping voltammetry (DPASV), is directly proportional to the target RNA concentration. The biosensor exhibited excellent analytical performance, with a detection limit of 4 fM for synthetic targets and 43 ng/µL for RT-PCR products. Importantly, it successfully detected HCV RNA directly in clinical plasma samples without the need for RNA extraction or amplification. The sensor was used to analyze 30 RNA samples from HCV-positive patients, 20 cDNA samples from viral RNA, 30 HCV-positive plasma samples, and 22 HCV-negative plasma samples. The sensor results show good concordance with the RT-PCR results, demonstrating the sensor’s potential for detecting HCV in clinical samples.

Real-Time Visualization of HIV-1 RNA Detection Using Loop-Mediated Isothermal Amplification-Enabled Particle Diffusometry.

Isothermal nucleic acid amplification tests, NAATs, such as reverse transcription-loop-mediated isothermal amplification (RT-LAMP), offer promising capabilities to perform real-time semiquantitative detection of viral pathogens. These tests provide rapid results, utilize simple instrumentation for single-temperature reactions, support efficient user workflows, and are suitable for field use. Herein, we present a novel and robust method for real-time monitoring of HIV-1 RNA RT-LAMP utilizing a novel implementation of particle diffusometry (PD), a diffusivity quantification technique using fluorescent particles, to quantify viral concentration in nuclease-free water. We monitor changes in particle diffusion dynamics of 400 nm fluorescently labeled particles throughout the RT-LAMP of HIV-1 RNA in nuclease-free water, enabling measurement within 20 min and detection of concentrations as low as 25 virus particles per μL. Moreover, in a single-blind study, we demonstrate semiquantitative detection by accurately determining the initial concentration of an unknown HIV-1 RNA within a 10% absolute error margin. These results highlight the potential of real-time PD readout for quantifying HIV-1 RNA via RT-LAMP, offering promise for viral load monitoring of HIV and other chronic infections.

Sewer transport conditions and their role in the decay of endogenous SARS-CoV-2 and pepper mild mottle virus from source to collection

This study presents a comprehensive analysis of the decay patterns of endogenous SARS-CoV-2 and Pepper mild mottle virus (PMMoV) within wastewaters spiked with stool from infected patients expressing COVID-19 symptoms, and hence explores the decay of endogenous SARS-CoV-2 and PMMoV targets in wastewaters from source to collection of the sample. Stool samples from infected patients were used as endogenous viral material to more accurately mirror real-world decay processes compared to more traditionally used lab-propagated spike-ins. As such, this study includes data on early decay stages of endogenous viral targets in wastewaters that are typically overlooked when performing decay studies on wastewaters harvested from wastewater treatment plants that contain already-degraded endogenous material. The two distinct sewer transport conditions of dynamic suspended sewer transport and bed and near-bed sewer transport were simulated in this study at temperatures of 4 °C, 12 °C and 20 °C to elucidate decay under these two dominant transport conditions within wastewater infrastructure. The dynamic suspended sewer transport was simulated over 35 h, representing typical flow conditions, whereas bed and near-bed transport extended to 60 days to reflect the prolonged settling of solids in sewer systems during reduced flow periods. In dynamic suspended sewer transport, no decay was observed for SARS-CoV-2, PMMoV, or total RNA over the 35-h period, and temperature ranging from 4 °C to 20 °C had no noticeable effect. Conversely, experiments simulating bed and near-bed transport conditions revealed significant decreases in SARS-CoV-2 and total RNA concentrations by day 2, and PMMoV concentrations by day 3. Only PMMoV exhibited a clear trend of increasing decay constant with higher temperatures, suggesting that while temperature influences decay dynamics, its impact may be less significant than previously assumed, particularly for endogenous RNA that is bound to dissolved organic matter in wastewater. First order decay models were inadequate for accurately fitting decay curves of SARS-CoV-2, PMMoV, and total RNA in bed and near-bed transport conditions. F-tests confirmed the superior fit of the two-phase decay model compared to first order decay models across temperatures of 4 °C–20 °C. Finally, and most importantly, total RNA normalization emerged as an appropriate approach for correcting the time decay of SARS-CoV-2 exposed to bed and near-bed transport conditions. These findings highlight the importance of considering decay from the point of entry in the sewers, sewer transport conditions, and normalization strategies when assessing and modelling the impact of viral decay rates in wastewater systems. This study also emphasizes the need for ongoing research into the diverse and multifaceted factors that influence these decay rates, which is crucial for accurate public health monitoring and response strategies.

Evaluating survey techniques in wastewater-based epidemiology for accurate COVID-19 incidence estimation

Wastewater-based epidemiology (WBE) requires high-quality survey methods to determine the incidence of infections in wastewater catchment areas. In this study, the wastewater survey methods necessary for comprehending the incidence of infection by WBE are clarified. This clarification is based on the correlation with the number of confirmed coronavirus disease 2019 (COVID-19) cases, considering factors such as handling non-detect data, calculation method for representative values, analytical sensitivity, analytical reproducibility, sampling frequency, and survey duration. Data collected from 15 samples per week for two and a half years using a highly accurate analysis method were regarded as gold standard data, and the correlation between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater and confirmed COVID-19 cases was analyzed by Monte Carlo simulation under the hypothetical situation where the quality of the wastewater survey method was reduced. Regarding data handling, it was appropriate to replace non-detect data with estimates based on distribution, and to use geometric means to calculate representative values. For the analysis of SARS-CoV-2 RNA in samples, using a highly sensitive and reproducible method (non-detect rates of <40 %; ≤0.4 standard deviation) and surveying at least three samples, preferably five samples, per week were considered desirable. Furthermore, conducting the survey over a period of time that included at least 50 weeks was necessary. A WBE that meets these survey criteria is sufficient for the determination of the COVID-19 infection incidence in the catchment. Furthermore, WBE can offer additional insights into infection rates in the catchment, such as the estimated 48 % decrease in confirmed COVID-19 cases visiting a clinic following a COVID-19 legal reclassification in Japan.

Evaluation of potential novel biomarkers for feline hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common cardiomyopathy in cats. The diagnosis can be difficult, requiring advanced echocardiographic skills. Additionally, circulating biomarkers (N-terminal pro-B type natriuretic peptide and cardiac troponin I) have several limitations when used for HCM screening. In previous work, we identified interleukin 18 (IL-18), insulin-like growth factor binding protein 2 (IGFBP-2), brain-type glycogen phosphorylase B (PYGB), and WNT Family Member 5 A (WNT5A) as myocardial genes that show significant differential expression between cats with HCM and healthy cats. The products of these genes are released into the circulation, and we hypothesized that IL-18, IGFBP-2, PYGB, and WNT5A serum RNA and protein concentrations differ between healthy cats, cats with subclinical HCM, and those with HCM and congestive heart failure (HCM + CHF).Reverse transcriptase quantitative polymerase chain reaction (RTqPCR) and enzyme-linked immunosorbent assay (ELISA) were applied to evaluate gene and protein expression, respectively, in the serum of eight healthy controls, eight cats with subclinical HCM, and six cats with HCM + CHF. Serum IGFBP-2 RNA concentrations were significantly different among groups and were highest in cats with subclinical HCM. Compared to healthy controls, serum IL-18 and WNT5A gene expression were significantly higher in cats with HCM + CHF, and WNT5A was higher in cats with subclinical HCM. No differences were observed for PYGB.These results indicate that further investigation via large scale clinical studies for IGFBP-2, WNT5A, and IL-18 may be valuable in diagnosing and staging feline HCM.

Establishment of national standards of SARS-CoV-2 variants in Taiwan

ObjectivesIn response to the pandemic, the Taiwan Food and Drug Administration (TFDA) established an initial SARS-CoV-2 RNA national standard based on the original Wuhan strain. However, with the depletion of the first national standard and continued mutation of the virus, the establishment of new national standards was imminent. MethodsHence, new candidate national standards were established by heat-inactivation for 30 min for six representative strains of SARS-CoV-2, comprising the original strain and five variants with anticipated concentrations of 7.70 Log10 international units (IU)/mL each. To enhance the credibility of these national standards, the TFDA extended invitations to both domestic and international institutions to participate in a collaborative study. A total of eight participants contributed eleven datasets, incorporating two methods and targeting four distinct genes. ResultsBased on these collective findings, the quantified viral RNA concentrations for each SARS-CoV-2 national standard strain are 7.69, 7.70, 7.69, 7.44, 7.52, and 7.29 Log10 IU/mL with Wuhan, alpha, beta, gamma, delta, and omicron strain, respectively. ConclusionsThese newly established national standards will continue to be made available to the industry, serving as a fundamental reference for the development and quality control of nucleic acid in vitro diagnostic (IVD) reagents in Taiwan.

Gene expression of psID gene in Pseudomonas aeruginosa isolated from clinical causes

One hundred clinical samples were collected from Baghdad hospitals including Teaching Laboratories Hospital, Medical City, Baghdad Teaching Hospital and Ghazi Hariri Hospital including burn infections, wound infections, urinary tract infections, otitis media from May 2022 to September 2022, The samples were identefied by microscopic, phenotypic and culturale on MacConkey agar and by biochemical tests including oxidase, catalase, citrate utilization test, triple sugar-iron test (TSI), gelatinase test, motility test and ornithine decarboxylation test. After diagnosis, we received 19 isolates of Pseudomonas aeruginosa . To detection biofilm genes used PslD gene, we all (19) isolates of Pseudomonas aeruginosa have these genes in percentage 100% with a molecular weight of 295 base pair. The gene expression study was done to evaluate the gene expression of the PslD biofilm gene using three concentrations of cefotaxim antibiotic (2, 4 and 8) μg/ml. Only two concentrations 2 and 8 μg/ml of cefotaxim had gene expression results of psID gene, 19.32 at RNA concentration when treated with 2 μg/ml of cefotaxim, 22.56 at RNA concentration when treated with 8 μg/ml of cefotaxim and undetermined at RNA concentration when treated with 4 μl/ml of cefotaxim.

Surveillance of norovirus, SARS-CoV-2, and bocavirus in air samples collected from a tertiary care hospital in Thailand

This study aims to determine the presence of norovirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and bocavirus in air samples from a tertiary care hospital in Bangkok, Thailand. Air samples were collected in water using the BioSampler and concentrated using speedVac centrifugation. Based on RT-qPCR, norovirus RNA and SARS-CoV-2 RNA were detected in 13/60 (21.7%) and 3/60 (5.0%) of samples, respectively. One air sample had a weak positivity for both norovirus and SARS-CoV-2 RNAs. Detection rate of norovirus genogroup (G) II (13.3%) was higher than norovirus GI (6.7%). One air sample (1.7%) tested positive for GI and GII. The norovirus GI RNA concentration was 6.0 × 102 genome copies/m3. The norovirus GII RNA concentrations ranged from 3.4 × 101 to 5.0 × 103 genome copies/m3. Based on RT-nested PCR, norovirus GII was detected in two (3.3%) samples. All samples tested negative for GI RNA and bocavirus DNA. By phylogenetic analysis, GII.17, which is closely related to the outbreak Kawasaki308/JPN/2015 strain, was found in the RT-nested PCR-positive samples. This study highlights the potential of aerosols for norovirus and SARS-CoV-2 transmission and probably cause gastrointestinal and respiratory illnesses, respectively.

Effect of highly-active antiretroviral therapy on neuroendocrine regulation of immunogenesis in HIV-infected children

Comparative assay has been made against the parameters of the immune and endocrine systems in 84 HIV-infected children born from HIV-infected mothers. One group of analyzed children (36 patients) did not receive highly-active antiretroviral therapy. Another group (48 patients) received different variants of highly-active antiretroviral therapy. Children aged from 1 to 182 months were examined. Venous blood samples taken from young patients were used to determine leukocyte blood composition considering the relative (%) and absolute number of blood cell counts. Hormone concentration was determined concurrently. CD-molecule expression by mononuclear cells was registered using flow cytofluorimeter. Plasma viral load in HIV-infected children was quantitatively detected with RT-PCR. Statistically significant lowering in the levels of free thyroxin, cortisol and progesterone was observed in children against a background of highly-active antiretroviral therapy as compared to those without HAART use. Correlation assay between the hormone level and the immunological parameters in children not receiving the antiretroviral preparations revealed marked positive correlations among the somatotropic hormone level and CD3+, CD4+ and CD8+ absolute numbers. Similar positive correlation with absolute T-subset number was found against free T4. The progesterone level also positively correlated with relative CD3+ and CD8+ numbers and showed negative correlation with absolute CD4+ amount. There is another positive correlation with relative T-subset number against the dehydroepiandrosterone level in the group of children without antiretroviral preparation therapy. As for HIV-infected group of children, against a background of highly-active antiretroviral therapy, the results of correlation assay between the hormone concentrations and cell parameters were found to significantly vary. There were observed positive correlations between the levels of cortisol and CD3+ (%), cortisol and CD8+ (%), estradiol and CD4/CD8, progesterone and absolute CD8+ number. As with children not receiving the antiretroviral preparations marked positive relation was revealed between the concentration of free thyroxin and absolute values of CD4+. Negative correlations were recorded between the estradiol level and the relative CD3+ numbers. Against a background of applying the antiretroviral preparations the correlation assay conducted between the viral RNA concentration (lg of copy number of mRNA/ml) and analyzed endocrinological parameters was found to have marked positive correlation with HIV concentration demonstrated by estradiol and testosterone. During the antiretroviral therapy, however, the negative correlation between the thyrotropin level and lg concentration of viral RNA was observed. Analytical results of correlation among the viral RNA concentration (lg copy number of mRNA/ml) and analyzed immunological parameters in this group of children evidence for specific ‘normalization’ due to highly-active antiretroviral therapy as the only positive correlation with virus concentration was detected for CD4+T subsets. Therefore, the alteration in endocrine system state in children born from HIV-infected mothers could be of great significance while monitoring the systemic regulation of the immunogenesis.

A new approach for extracellular RNA recovery from Rhodovulum sulfidophilum

The development of RNA-based drugs is highly pursued due to the possibility of creating viable and effective therapies. However, their translation to clinical practice strongly depends on efficient technologies to produce substantial levels of these biomolecules, with high purity and high quality. RNAs are commonly produced by chemical or enzymatic methods, displaying these limitations. In this sense, recombinant production arises as a promising, cost-effective method, allowing large-scale production of RNA. Rhodovulum sulfidophilum (R. sulfidophilum), a marine purple bacterium, offers the advantage of RNA secretion into the extracellular medium, which contains low levels of RNases and other impurities. Therefore, RNA recovery can be simplified compared to standard extraction protocols involving cell lysis, resulting in a more clarified sample and an improved downstream process. In this work, R. sulfidophilum was transformed with a plasmid DNA encoding pre-miR-29b-1, which is known to be involved in the Alzheimer's disease pathway. After production, the pre-miR-29b-1 was recovered through different extraction methods to verify the most advantageous one. A protocol for extracellular RNA recovery was then established, revealing to be a simpler and less time-consuming method, reducing around 16 h in execution time and the quantity of reagents needed when compared to other established methods. The new strategy brings the additional advantage of eliminating the toxic organic compounds routinely used in intracellular RNA extractions. Overall, the optimized process described here, using isopropanol as the precipitation agent, offers a greener, safer, and faster alternative for recombinant RNA recovery and concentration, with an extracellular RNA recovery of 7 μg/mL and target pre-miRNA-29b-1 recovery of 0.7 μg/L of medium.

Changes in cerebrospinal fluid proteins across the spectrum of untreated and treated chronic HIV-1 infection.

Using the Olink Explore 1536 platform, we measured 1,463 unique proteins in 303 cerebrospinal fluid (CSF) specimens from four clinical centers contributed by uninfected controls and 12 groups of people living with HIV-1 infection representing the spectrum of progressive untreated and treated chronic infection. We present three initial analyses of these measurements: an overview of the CSF protein features of the sample; correlations of the CSF proteins with CSF HIV-1 RNA and neurofilament light chain protein (NfL) concentrations; and comparison of CSF proteins in HIV-associated dementia (HAD) and neurosymptomatic CSF escape (NSE). These reveal a complex but coherent picture of CSF protein changes with highest concentrations of many proteins during CNS injury in the HAD and NSE groups and variable protein changes across the course of systemic HIV-1 progression that included two common patterns, designated as lymphoid and myeloid patterns, related to principal involvement of their underlying inflammatory cell lineages. Antiretroviral therapy reduced CSF protein perturbations, though not always to control levels. The dataset of these CSF protein measurements, along with background clinical information, is posted online. Extended studies of this unique dataset will supplement this report to provide more detailed characterization of the dynamic impact of HIV-1 infection on the CSF proteome across the spectrum of HIV-1 infection, advancing the mechanistic understanding of HIV-1-related CNS pathobiology.

Impact of Chemical Dynamics of Commercial PURE Systems on Malachite Green Aptamer Fluorescence.

The malachite green aptamer (MGapt) is known for its utility in RNA measurement in vivo and in lysate-based cell-free protein systems. However, MGapt fluorescence dynamics do not accurately reflect RNA concentration. Our study finds that MGapt fluorescence is unstable in commercial PURE systems. We discovered that the chemical composition of the cell-free reaction strongly influences MGapt fluorescence, which leads to inaccurate RNA calculations. Specific to the commercial system, we posit that MGapt fluorescence is significantly affected by the system's chemical properties, governed notably by the presence of dithiothreitol (DTT). We propose a model that, on average, accurately predicts MGapt measurement within a 10% margin, leveraging DTT concentration as a critical factor. This model sheds light on the complex dynamics of MGapt in cell-free systems and underscores the importance of considering environmental factors in RNA measurements using aptamers.

Characterizing the Cell-Free Transcriptome in a Humanized Diffuse Large B-Cell Lymphoma Patient-Derived Tumor Xenograft Model for RNA-Based Liquid Biopsy in a Preclinical Setting.

The potential of RNA-based liquid biopsy is increasingly being recognized in diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma. This study explores the cell-free transcriptome in a humanized DLBCL patient-derived tumor xenograft (PDTX) model. Blood plasma samples (n = 171) derived from a DLBCL PDTX model, including 27 humanized (HIS) PDTX, 8 HIS non-PDTX, and 21 non-HIS PDTX non-obese diabetic (NOD)-scid IL2Rgnull (NSG) mice were collected during humanization, xenografting, treatment, and sacrifice. The mice were treated with either rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), CD20-targeted human IFNα2-based AcTaferon combined with CHOP (huCD20-Fc-AFN-CHOP), or phosphate-buffered saline (PBS). RNA was extracted using the miRNeasy serum/plasma kit and sequenced on the NovaSeq 6000 platform. RNA sequencing data of the formalin-fixed paraffin-embedded (FFPE) tissue and blood plasma samples of the original patient were included. Flow cytometry was performed on immune cells isolated from whole blood, spleen, and bone marrow. Bulk deconvolution was performed using the Tabula Sapiens v1 basis matrix. Both R-CHOP and huCD20-Fc-AFN-CHOP were able to control tumor growth in most mice. Xenograft tumor volume was strongly associated with circulating tumor RNA (ctRNA) concentration (p < 0.001, R = 0.89), as well as with the number of detected human genes (p < 0.001, R = 0.79). Abundance analysis identified tumor-specific biomarkers that were dynamically tracked during tumor growth or treatment. An 8-gene signature demonstrated high accuracy for assessing therapy response (AUC 0.92). The tumoral gene detectability in the ctRNA of the PDTX-derived plasma was associated with RNA abundance levels in the patient's tumor tissue and blood plasma (p < 0.001), confirming that tumoral gene abundance contributes to the cell-free RNA (cfRNA) profile. Decomposing the transcriptome, however, revealed high inter- and intra-mouse variability, which was lower in the HIS PDTX mice, indicating an impact of human engraftment on the stability and profile of cfRNA. Immunochemotherapy resulted in B cell depletion, and tumor clearance was reflected by a decrease in the fraction of human CD45+ cells. Lastly, bulk deconvolution provided complementary biological insights into the composition of the tumor and circulating immune system. In conclusion, the blood plasma-derived transcriptome serves as a biomarker source in a preclinical PDTX model, enables the assessment of biological pathways, and enhances the understanding of cfRNA dynamics.

Evaluation of clinically significant miRNAs level by machine learning approaches utilizing total transcriptome data

Analysis of the mechanisms underlying the occurrence and progression of cancer represents a key objective in contemporary clinical bioinformatics and molecular biology. Utilizing omics data, particularly transcriptomes, enables a detailed characterization of expression patterns and post-transcriptional regulation across various RNA types relative to the entire transcriptome. Here, we assembled a dataset comprising transcriptomic data from approximately 16.000 patients encompassing over 160 types of cancer. We employed state-of-the-art gradient boosting algorithms to discern intricate correlations in the expression levels of four clinically significant microRNAs, specifically hsa-mir-21, hsa-let-7a-1, hsa-let-7b, and hsa-let-7i, with the expression levels of the remaining 60.660 unique RNAs. Our analysis revealed a dependence of the expression levels of the studied microRNAs on the concentrations of several small nucleolar RNAs and regulatory long non-coding RNAs. Notably, the roles of these RNAs in the development of specific cancer types had been previously established through experimental evidence. Subsequent evaluation of the created database will facilitate the identification of a broader spectrum of overarching dependencies related to changes in the expression levels of various RNA classes in diverse cancers. In future it will make possible discovery of unique alterations specific to certain types of malignant transformations.

ISOLATION OF THE EXOSOMES FROM hUC-MSCs

The studies on the potential application of exosomes isolated from stem cells are expanding every year. Researchers and medical clinics are interested in them in the regard that exosomes contain different molecules that have therapeutic and regeneration potential. This study aimed to investigate the exosomes of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). The hUC-MSCs were isolated from the human umbilical cord using the standard protocol. Obtained cells were cultivated till 3rd passage. Then, the conditioned medium (CM) was collected, centrifuged and ultracentrifuged using the standard protocol for exosome isolation. The isolated exosomes were identified in 63 x magnification in the inverted microscope and the concentration of DNA and RNA was measured by nanodrope equipment. The obtained hUC-MSCs showed typical morphology of MSCs. We isolated exosomes from the CM of UC-MSCs. The obtained exosomes were round-shaped and had a distinct border. The DNA concentration and RNA concentration in obtained exosomes were 13.4 ng/ml and 17.0 ng/ml, respectively. Our hUC-MSCs-derived exosomes demonstrated the initial characteristics of normal exosomes and also contained DNA and RNA. Further study is needed to determine their functional properties.