Total RNA Research Articles

Optimal isolation of extracellular vesicles from pleural fluid and profiling of their microRNA cargo

AbstractPleural effusion occurs in both benign and malignant pleural disease. In malignant pleural effusions, the diagnostic accuracy and sensitivity of pleural fluid cytology is less than perfect, particularly for the diagnosis of malignant pleural mesothelioma, but also in some cases for the diagnosis of metastatic pleural malignancy with primary cancer in the lung, breast or other sites. Extracellular vesicles (EVs) carry an enriched cargo of microRNAs (miRNAs) which are selectively packaged and differentially expressed in pleural disease states. To investigate the diagnostic potential of miRNA cargo in pleural fluid extracellular vesicles (PFEVs), we evaluated methods for isolating the extracellular vesicle (EV) fraction including combinations of ultracentrifugation, size‐exclusion chromatography (SEC) and ultrafiltration (10 kDa filter unit). PFEVs were characterized by total and EV–associated protein, nanoparticle tracking analysis and visualisation by transmission electron microscopy. miRNA expression was analyzed by Nanostring nCounter® in separate EV fractions isolated from pleural fluid with or without additional RNA purification by ultrafiltration (3 kDa filter unit). Optimal PFEV yield, purity and miRNA expression were observed when PFEV were isolated from a larger volume of pleural fluid processed through combined ultracentrifugation and SEC techniques. Purification of total RNA by ultrafiltration further enhanced the detectability of PFEV miRNAs. This study demonstrates the feasibility of isolating PFEVs, and the potential to examine PFEV miRNA cargo using Nanostring technology to discover disease biomarkers.

Alterations in Lung Carcinoma Cells and Cell-Derived Extracellular Vesicles after Human Adenovirus Type 5 Administration

Human adenoviruses (HAdVs) affect the respiratory system of healthy individuals and people with pre-existing health conditions. These highly infectious viruses can alter a myriad of cellular and pathophysiological processes. HAdVs can modulate their infection ability and pathogenicity by regulating extracellular vesicles (EVs) formation and function. EVs are nanovesicles that facilitate intracellular communication, cell signaling/trafficking, and immune regulation. We propose that HAdVs can exploit EV formation, secretion, and release various pathways to promote infection and transmission between neighboring cells. In the present study, we explored the impact of HAdV serotype 5 (HAdV-5) on the A549-epithelial carcinoma cells and EVs derived from A549 cells. A549 cells were cultured in an exosome-depleted medium and administered different concentrations of HAdV-5 at varying time points. The effect of HAdV-5 infection on A549 cells and A549-derived EVs was determined by 4’,6-diamidino2-phenylindole (DAPI) stain, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, electron microscopy, particle size, zeta potential analysis, immunosorbent analysis, total RNA levels, and total protein content. Our findings illustrated that HAdV-5 reduces A549-cell viability and promotes variations within the protein content of A549 cells. Our data explicitly demonstrated that HAdV-5 significantly altered EV numbers, biogenesis, and composition. These findings suggest that HAdV-5 modulates EVs’ biological properties and physiological functions in human lung carcinoma cells.

Plasma Exosomal miRNAs as Response Biomarkers of Immunotherapy in Extensive-Stage Small-Cell Lung Cancer

Immunotherapy combined with chemotherapy has become the first-line standard treatment for patients with extensive-stage small-cell lung cancer (ES-SCLC). The reliable biomarkers stratifying true responders of immunotherapy effectively are unknown, and it is urgent to identify novel biomarkers in clinical. Exosomal miRNAs are considered to play a role in intercellular communication among immune cells and interaction between immune cells and tumor cells. The purpose of this study was to explore the possibility of using plasma-derived exosomal miRNAs as potential biomarkers for identifying responses to immunotherapy in ES-SCLC. From March 2020 to September 2021, 24 patients with ES-SCLC who received PD-L1 inhibitors were enrolled. Tumor assessments were conducted after every two treatment cycles according to RECIST 1.1. Plasma samples of these patients were collected before administering PD-L1 inhibitors as the baseline, and after every four cycles until the occurrence of disease progression. Plasma exosomes were isolated by ultracentrifugation, then total RNA was extracted. The miRNA profile was analyzed with small RNA next-generation sequencing followed by differential expression analysis. Of the 24 patients, 15 underwent immunotherapy maintenance after completing four cycles of PD-L1 inhibitor plus chemotherapy. In order to identify biomarkers for a better response to immunotherapy, all five responders (patients achieving PR) and four non-responders (patients achieving PD) at tumor assessment within eight cycles of the maintenance phase were included for differential expression analysis. Surprisingly, hsa-miR-320c, hsa-miR-320d, and hsa-miR-320e showed a trend of increased expression in the non-responders compared with the responders at baseline and were significantly downregulated in the post-treatment plasma exosomes compared with pre-treatment samples of the responders. Exosomal miRNA profiles are discordant between responders and non-responders of anti-PD-L1 treatment. Hsa-miR-320c, hsa-miR-320d, and hsa-miR-320e were identified as potential biomarkers for predicting the efficacy of immunotherapy in patients with ES-SCLC.

New JAK3-INSL3 Fusion Transcript-An Oncogenic Event in Cutaneous T-Cell Lymphoma.

Constitutively activated tyrosine kinase JAK3 is implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCL). The mechanisms of constitutive JAK3 activation are unknown although a JAK3 mutation was reported in a small portion of CTCL patients. In this study, we assessed the oncogenic roles of a newly identified JAK3-INSL3 fusion transcript in CTCL. Total RNA from malignant T-cells in 33 patients with Sézary syndrome (SS), a leukemic form of CTCL, was examined for the new JAK3-INSL3 fusion transcript by RT-PCR followed by Sanger sequencing. The expression levels were assessed by qPCR and correlated with patient survivals. Knockdown and/or knockout assays were conducted in two CTCL cell lines (MJ cells and HH cells) by RNA interference and/or CRISPR/Cas9 gene editing. SS patients expressed heterogeneous levels of a new JAK3-INSL3 fusion transcript. Patients with high-level expression of JAK3-INSL3 showed poorer 5-year survival (n = 19, 42.1%) than patients with low-level expression (n = 14, 78.6%). CTCL cells transduced with specific shRNAs or sgRNAs had decreased new JAK3-INSL3 fusion transcript expression, reduced cell proliferation, and decreased colony formation. In NSG xenograft mice, smaller tumor sizes were observed in MJ cells transduced with specific shRNAs than cells transduced with controls. Our results suggest that the newly identified JAK3-INSL3 fusion transcript confers an oncogenic event in CTCL.

Prognostic Value of Hypoxia Gene Expression Signature in Early-Stage Non-Small Cell Lung Cancer Patients Treated with Stereotactic Body Radiation Therapy

Stereotactic body radiation therapy (SBRT) is an effective alternative to surgery in the treatment of early-stage non-small cell lung cancer (NSCLC). However, disease recurrence occurs in approximately 20-30% of patients. Hypoxia is a well-known factor promoting solid tumor progression, radiation resistance, and immune evasion. In this study, we utilize a previously published hypoxia gene expression signature (Buffa) and measure its association with clinical outcomes in patients with NSCLC treated with SBRT. Our study focused on patients with localized, node-negative NSCLC treated with SBRT, and we identified 92 patients treated at our institution between 2008 and 2018 with available gene expression data. Total RNA from formalin-fixed paraffin-embedded archival biopsy specimens (pre-therapy) was isolated and mRNA expression was analyzed using an assay for human samples. For each gene in the Buffa signature, the top 50% of mRNA abundance values were given a score of +1, and the bottom 50% were given a score of -1 to generate a tumor hypoxia score. High scores suggest a hypoxic tumor and low scores imply normoxia. Kaplan-Meier curves were used to assess overall survival, disease-free survival, local recurrence, regional nodal recurrence, and distant recurrence. Cox proportional hazards were performed to account for confounding due to age, T stage, ECOG performance status, biologically effective dose, patient sex, and histology (squamous vs non-squamous). The patient's gene expression data were dichotomized based on their median hypoxia score (Table 1). Median follow-up was 23.9 months (95% CI 20.6 - 26.6). A high hypoxia score was associated with squamous histology (p = 0.003). On univariate analysis, a high hypoxia score was significantly associated with local recurrence (hazard ratio [HR] = 5.63; 95% CI 1.03 - 30.78; p = 0.046) and distant recurrence (HR = 1.85; 95% CI 1.14 - 3.02; p = 0.013). There was no significant difference in overall survival (HR = 1.93; 95% CI 0.98 - 3.82; p = 0.058), disease-free survival (HR = 2.13; 95% CI 0.93 - 4.89; p = 0.074), or regional nodal recurrence (HR = 1.45; 95% CI 0.41 - 5.14; p = 0.57). On multivariate analysis, a high hypoxia score was associated with worse overall survival (HR = 2.73; 95% CI 1.23 - 6.07; p = 0.014), local recurrence (HR = 12.85; 95% CI 1.08 - 152.49; p = 0.043) and distant recurrence (HR = 1.91; 95% CI 1.13 - 3.22; p = 0.016). Our findings demonstrate that a hypoxia gene signature is significantly associated with worse survival and increased local and distant recurrence after SBRT for localized NSCLC. This hypoxia signature may be useful in prognostication, selecting patients for surgery versus radiation, or selecting patients for treatment intensification with radiation or other systemic agents, and should be prospectively validated.

Effects of ruminal lipopolysaccharide exposure on primary bovine ruminal epithelial cells

The objective of this study was to investigate the immunopotential of ruminal lipopolysaccharides (LPS) on cultured primary bovine rumen epithelial cells (REC). Primary bovine REC were isolated from 6 yearling steers and grown in culture for 3 experiments. Experiment 1 aimed to determine the immunopotential of ruminal LPS, experiment 2 aimed to assess tolerance to chronic LPS exposure, and experiment 3 aimed to evaluate antagonistic interactions between ruminal and Escherichia coli LPS. In experiments 1 and 2, REC were exposed to nonpyrogenic water, 20 μg/mL E. coli LPS (EC20), 10 μg/mL ruminal LPS, 20 μg/mL ruminal LPS, and 40 μg/mL ruminal LPS, either continuously or intermittently. For the continuous exposure, REC underwent a 6 h exposure, whereas for the intermittent exposure, the procedure was: (1) a 12 h continuous exposure to treatments followed by LPS removal for 24 h and then another 12 h of exposure (RPT), and (2) a 12 h continuous exposure to treatments followed by LPS removal and a recovery period of 36 h (RCV). In experiment 3, REC were exposed to nonpyrogenic water, 1 μg/mL E. coli LPS, 1 μg/mL ruminal LPS to 1 μg/mL E. coli LPS, 10 μg/mL ruminal LPS to 1 μg/mL E. coli LPS, and 50 μg/mL ruminal LPS to 1 μg/mL E. coli LPS. Each experiment was done as a complete randomized block design with 6 REC donors. The REC-donor was used as blocking factor. Each treatment had 2 technical replicates, and treatment responses for all data were analyzed with the MIXED procedure of SAS. For all experiments, total RNA was extracted from REC and real-time quantitative PCR was performed to determine the relative expression of genes for toll-like receptors (TLR2 and TLR4), proinflammatory cytokines (TNF, IL1B, and IL6), chemokines (CXCL2 and CXCL8), growth factor-like cytokines (CSF2 and TGFB1), and a lipid mediator (PTGS2). In experiment 1, the targeted genes were upregulated by EC20, whereas all ruminal LPS treatments resulted in a lower transcript abundance. Regarding RPT, and RCV condition, in experiment 2, the expression of targeted genes was not affected or was at a lower abundance to EC20 when compared with ruminal LPS treatments. Lastly, in experiment 3, all targeted genes resulted in lower or similar transcript abundance on all ruminal LPS ratios. Overall, our results indicate that ruminal LPS have a limited capacity to activate the TLR4/NF-kB pathway and to induce the expression of inflammatory genes.

Differential Oxidative Stress Responses in Type II Airway Epithelial Cells Impact Premature Senescence and Lung Fibrosis Susceptibility

Radiation-Induced Pulmonary Fibrosis (RIPF) is a late toxicity characterized by premature senescence in Type II airway epithelial cells (AECII) and accumulation of alternatively activated (M2) macrophages. Differential susceptibility to RIPF is observed across mouse strains. Based on our prior study of the effects of macrophage variation on RIPF, we hypothesized that intrinsic differences in AECII oxidative stress response across mouse strains also impact susceptibility to RIPF. Ten-week-old female mice from C57L, C57BL6 and C3H/HeN strains were exposed to thoracic irradiation (5x6 Gy, n>5 per group). Fifteen weeks after radiation, lung tissue was collected and examined with Masson-Trichrome staining (histologic changes) and β-galactosidase activity assay (senescence). AECII prepared from mice of each strain were exposed to irradiation. To assess differential gene expression, total RNA was extracted and assessed with a multiplex analysis platform and quantitative PCR. Senescence was assessed by β-galactosidase activity assay in primary AECII after irradiation or after co-culture with M2 macrophages polarized with IL13. Susceptibility to radiation-induced lung injury, survival, and premature AECII senescence vary by mouse strain: C57L (fibrosis-prone), C57BL6J (-intermediate) and C3H/HeN (-resistant). Enriched AECII from each strain exhibited differential expression of genes related to inflammatory responses including SASP production after irradiation. Minimal increased expression of Il1r1 was observed in irradiated and unirradiated AECII from C3H/HeN, however Il1rn levels were markedly elevated in response to irradiation. The expression of Thioredoxin (Txn) and Thioredoxin reductase 1 (Txnrd1) in AECII from C3H/HeN was significantly higher than those observed in other strains. In Vivo, C3H/HeN mouse lungs exhibited the least premature senescence in AECII after irradiation. Premature senescence in AECII irradiated In Vitro or co-cultured with superoxide anion-producing M2 macrophages was substantially less in AECII from C3H/HeN compared to other strains. A comparison of primary AECII from three different mouse strains identified intrinsic differences in expression of major inflammatory signaling (IL1R and IL1RN) and redox homeostasis status (TXN and TXNDR1) molecules. This study is the first to demonstrate that intrinsic differences in AECII impacts susceptibility to premature senescence and lung fibrosis after irradiation.

Artemia enrichment as modulator of gene expression patterns of Enteroctopus megalocyathus paralarvae at pre-settlement stage

Enteroctopus megalocyathus, the Patagonian red octopus, is the only octopus species for which a complete production cycle in captivity has been accomplished in Chile. The rearing of E. megalocyathus paralarvae from hatching to the settlement of juveniles lasts 90 days and it represents the bottleneck in the culture of this species. The paralarval stages from 1 to 60 days after hatching (DAH) are fed on enriched Artemia while, when pre-settlement behavior appears, from 60 DAH onwards, the feeding protocol is a mixed diet of enriched Artemia and juvenile marine crabs. Marine crustaceans represent a suitable diet to improve E. megalocyathus paralarvae survival, complementing the enriched Artemia to feed the pre-settling paralarvae. To determine the effect of Artemia enrichment on the response of E. megalocyathus at the pre-settling paralarval stage, paralarvae were fed two mixed diets containing juvenile marine crabs (Petrolisthes spp.) and differing only in Artemia enrichment: enriched (complete diet, CD), or unenriched (limited diet, LD). After 30 days of feeding trial, samples consisting of whole paralarvae were collected for transcriptomic analysis using high-quality total RNA to generate six mRNA libraries (two treatments x three biological replicates). A total of 270 genes were differentially expressed between paralarvae fed LD and those fed CD. When classifying the cellular processes in which the genes of LD and CD paralarvae differed significantly, it was observed that the downregulated/upregulated gene ratio was 2/1 in genes related to membrane organization and neuron differentiation. The genes of paralarvae fed a LD for which upregulation was equal to or >20 times compared with those fed a CD were related with increased rearrangement of tissues, molecular transport, energy production, improvement of sensory organs, cellular processes, transcription, and protein catabolic processes. The downregulated genes in paralarvae fed a LD were involved with reduced DNA repair and mRNA replication and elongation, molecular transport, cellular protein modification, cellular component organization, inflammatory cytokines, and biosynthetic processes. It was concluded that removing enrichment from Artemia, even under co-feeding with marine crustaceans, results in notable changes in the transcription that, while not generating a significant detriment to growth in 30 days of experimentation, could affect subsequent growth phases.

Age-Related Gene and Protein Expression in Mouse Mandibular Condyle Analyzed by Cap Analysis of Gene Expression and Immunohistochemistry

Introduction: Aging, an inevitable physiological process, leads to morphological and histological degenerative changes in the mandibular condylar cartilage (MCC); however, the molecular mechanism has not yet been elucidated, and little information is available on age-related factors. Therefore, this study was designed to identify age-related factors by investigating the age-related differentially expressed genes (DEGs) and localization of their translated protein expression in the mandibular condyle. Methods: Mandibular condyles were collected from 10- and 50-week-old mice. Total RNA was extracted from the samples and then analyzed using cap analysis of gene expression (CAGE) to identify age-related DEGs. Gene ontology (GO) enrichment analysis was performed to determine which biological processes were most affected by aging in terms of gene expression using Metascape. The mandibular condyle samples were processed for histology to investigate morphological changes caused by aging and for immunohistochemistry to localize the protein expression encoded by age-related genes identified with CAGE. Semi-quantitative immunohistochemistry was performed to assess age-related extracellular matrix (ECM) protein levels in the MCC. The histological sections were also used for Alcian blue histochemistry to detect glycosaminoglycans (GAGs). Results: GO enrichment analysis revealed that the genes related to “extracellular matrix organization,” including Acan, Col1a1, Col1a2, Col2a1, Mmp3, Mmp9, and Mmp13, were most differentially expressed in the aged mandibular condyle. Among these seven genes, Mmp3 was upregulated, and the others were downregulated with aging. Histological examination showed the age-related morphological and histological changes in the MCC. Immunohistochemical investigation showed the localization of matrix metalloproteinases (MMPs)-3, -9, and -13 and their substrate proteins, aggrecan, type I collagen, and type II collagen, in the mandibular condyle at 10 and 50 weeks, indicating different localizations between the young and the aged. In the aged MCC, semi-quantitative immunohistochemistry showed a significant decrease in the aggrecan protein level, and Alcian blue histochemistry showed a decrease in GAGs. Conclusion: MMP-3, MMP-9, and MMP-13 contribute to the remodeling of the ECM of the MCC and subchondral bone during aging by degrading ECM proteins at specific times and sites under the regulation of their production and secretion.

Documentation of Viruses Infecting Lablab (Lablab purpureus L.) Beans in Tamil Nadu, India

Field surveys were conducted in lablab-bean growing areas of Tamil Nadu to document the occurrence and distribution of virus diseases. Samples collected from lablab bean field showed symptoms like bright yellow mosaic patches and puckering on leaves. The total DNA and RNA were isolated from the symptomatic samples separately. The DNAs were subjected to polymerase chain reaction using Begomovirus universal primers and the positive samples amplified 1200 bp amplicon. Similarly, the isolated RNAs were subjected to reverse transcription PCR using Potyvirus universal primers and which amplified 350 bp in preliminary confirmation of viruses. The amplified products were sequenced and the DNA sequences were compared with the sequences available in the NCBI database and the phylogenetic tree was constructed using Mega 11 software. The occurrence of viruses viz., Bean common mosaic virus (BCMV) and Dolichos yellow mosaic virus (DoYMV) were documented. The results indicated a higher incidence of Begomovirus, DoYMV with a mean incidence of 86 per cent followed by the Potyvirus, BCMV with an incidence of around 70 per cent.

B-242 Fast and Cost-effective RNA Extraction in Onco-hematology Laboratories

Abstract Background Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of a BCR-ABL1 rearrangement; its detection and quantification by real-time quantitative PCR (RQ-PCR) plays a central role in CML diagnosis, therapy monitoring, and sequencing to identify resistance mutations in case of therapy failure. To evaluate the molecular response of CML, BCR-ABL1 quantification must be calculated on the international scale (IS%) for which RQ-PCR has the necessary sensitivity (up to 10−5). Currently, BCR-ABL1 quantifications are ubiquitous in onco-hematology laboratories (OHLs; ∼70% of tests performed in our OHL), whereby quality, cost, and turn-around-time are of critical importance. Today, the majority of OHLs in Brazil rely on laborious manual RNA-extraction methods (e.g., Trizol) that usually require 8 mL of peripheral blood (PB) to monitor CML. Here, we report a strategy that requires only 2 mL of PB in combination with the automated magnetic DNA/RNA-extraction kit Extracta-MPTA (Loccus do Brasil) for CML diagnosis and monitoring. Methods: BCR:ABL1 was quantified and its IS% determined using our developed kit, which was calibrated using ERM-AD263 and UK-NEQAS (external quality assessment). Total RNA was extracted from 40 PB samples, after erythrocyte lysis using two different methods: 1) Leukocytes from 8 mL of PB were extracted manually using Trizol; 2) leukocytes from 2 mL of PB were automatically extracted using Extracta-MPTA (Loccus do Brasil). BCR-ABL1 quantification, BCR-ABL1 IS%, and BCR quantification (internal control) were compared statistically (paired t-test). The reproducibility was evaluated by analyzing 20 replicates; the low limit of the leucocyte number to BCR:ABL1 quantification using Extracta-MPTA were also evaluated. Median and moving means of 135 unpaired samples were used to monitor BCR-ABL1 IS% between Trizol, Extracta-MPTA, while EQA from UK-NEQAS was also evaluated using both methods. In parallel, RNA was extracted by both methods from 20 samples for the diagnosis of acute lymphoblastic leukemia (ALL) (BCR-ABL p190 transcript) and acute promyelocytic leukemia (APL) (PML-RARA transcript). Results A comparison of BRC-ABL1, BCR, and %IS in 40 paired samples showed that the results obtained from Trizol or Extracta-MPTA are comparable (paired t-test: P = 0.4222, P = 0.9263, and P = 0.071, respectively). The reproducibility was evaluated by BCR quantification (24%). A comparison of the medians from 135 unpaired samples extracted using Trizol and Extracta-MPTA revealed that BCR-ABL1 IS% distribution among our patients is stable over time (P = 0.7704; Mann Whitney: r2 = 0.0012). EQA was extracted using both methods and the results were identical. A comparison between Trizol and EXTRACTA_MPTA for ALL and APL was 100% identical (Fischer; P = 1.000). Conclusion Both RNA extraction methods are suitable for OHLs for PML-RARA and BCR-ABL (p190 and p210) detection and CML diagnosis/monitoring by BCR-ABL1 detection/quantification. By using Extracta-MPTA the amount of PB used to obtain RNA can be reduced by 75%, test results can be obtained within 4 h, and direct reagent costs are reduced by 25% with a minimum of hands-on time.

A-294 Duplicate Testing of Samples Suspected of SARS-CoV-2 Infection Could Prevent Misdiagnosis of Covid-19

Abstract Background There is no doubt that the whole world has been shaken by the SARS-CoV-2 pandemic, which has affected both global health and the global economy. At the same time the importance of molecular methods for the accurate diagnosis of respiratory infections has been reinforced together with the need of testing methodologies that minimize the risk of misleading results. During the last three years of the pandemic, SARS-CoV-2 exhibited a tremendous adaptation capability through numerous mutations, leading to the appearance of the well-known Variants of Concern (VOCs) responsible of increased transmission rates. Therefore, the development of molecular methods capable of detecting all variants has become a challenge. To overcome this, potential genetic targets with low mutation rates have been identified, including ORF1ab, N gene, S gene and E gene. While Real-time PCR based detection assays of viral genomes are very accurate, testing naso/oropharyngeal samples using singlet analysis does not guarantee the error free detection of true positive results for SARS-CoV-2 infections. Furthermore, it has been documented that the probability of a false-negative testing results is influenced by a number of variables . Therefore, testing protocol adaptations, such as replicate testing, has been shown to efficiently reduce the probability of missing SARS-CoV-2 positive cases. The pilot study presented here investigates the outcome of testing 10 014 nasopharyngeal samples collected from patients with a suspected SARS-CoV-2 infection using two separate detection kits. In this study the error rate of single vs duplicate testing is discussed. Methods 10 014 nasopharyngeal swabs, collected from three different U.K. hospital trusts and Kent based care homes, were tested at the North Kent Pathology Service (NKPS). Each swab collected was placed in sterile transport media and stored at 4°C prior to viral genome isolation using the HigherPurity™ Viral RNA extraction kit. All collected specimens were processed within 24 h from collection. Total RNA isolated was immediately analyzed using two commercial CE-IVD kits: Vitassay qPCR SARS-CoV-2 and Primerdesign™ LTD, Coronavirus (COVID-19) Genesig Real-Time PCR assay. All samples were tested in duplicate. Results Following the testing and subsequent analysis, 491 samples (4.9%) were detected positive for SARS-CoV-2 using both assay kits. Of these, 302 samples (61.5%) were reported as duplicate positive (on both replicate 1 and 2) whereas 189 samples (38.5%) were detected positive on either replicate 1 or 2. In particular, 85 (17.3%) were detected positive on replicate 1 and 104 (21.2%) were detected positive on replicate 2. Conclusions Approximately 19% of tested individuals were detected as SARS-CoV-2 positive based on a positive Real-time PCR result in one of the two reactions. This percentage represents the potential SARS-CoV-2 infections that can be missed when a single reaction Real-time PCR testing is employed for infection detection in the wider population, and equates to 940 individuals out of 100 000 potentially being falsely confirmed as SARS-CoV-2 negative The authors believe that in the presence of fast transmitting variants this is a significant number of false negative tests and should be taken into consideration when testing protocols are developed and disease modelling is employed.

First report of Nerine yellow stripe virus infecting crinium lily (Crinium sp.) in Texas.

Crinum sp. (family Amaryllidaceae) is an ornamental flower bulb that is commonly called crinum lily, cape lily, cemetery plant, spider lily, and swamp lily. In April 2023, two plants of Crinum sp. var. Maiden's Blush with yellow stripe symptoms (Fig. S1) were submitted to the Texas Plant Virus Diagnostic Laboratory, Weslaco, TX for virus diagnosis. Due to the resemblance of the observed symptoms to those described for potyviruses infecting ornamental flower bulbs (Pearson et al. 2009), total RNA extracts were made from each sample using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, USA), according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from 2 µg total RNA per sample with Oligo(dT) primers using the PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio, USA) as recommended by the manufacturer. A 2µL aliquot of each cDNA template was initially subjected to PCR using the generic primer pair CIFor/CIRev (Ha et al., 2008) that targets a fragment of the cylindrical inclusion (CI) body of potyviruses. The expected ~700 bp DNA band was amplified from both samples using the Taq DNA polymerase, dNTPack kit (Sigma-Aldrich). The amplicons were cloned and sequenced (three recombinant clones per sample) as described by Hernandez et al. (2021) and the BLASTX analyses of the consensus sequence (GenBank acc. no. OR137018) returned significant hits only to nerine yellow stripe virus (NeYSV; Potyvirus, Potyviridae) at 100% query coverage. To further confirm the results, another pair of universal primers (Jordan et al. 2011) was used to amplify the expected ∼1,600 bp product specific to the partial nuclear inclusion body (NIb), coat protein (CP) cistron, and 3' untranslated region of potyviruses from the same samples. The amplicons were similarly cloned, and a consensus sequence obtained (OR137019). In pairwise comparisons, the partial CI sequence of NeYSV from Texas (NeYSV-TX; OR137018) shared 83% nucleotide (nt)/93% amino acids (aa) identities with the corresponding sequences of NeYSV isolate 63 (MT396083) from the United Kingdom. The partial (649 nt) NIb sequences of NeYSV-TX (OR137019) and the complete CP (OR137019) of NeYSV-TX shared 77-94%/88-94% and 83-99%/89-98% nt/aa identities with the corresponding sequences of global NeYSV isolates that were retrieved from GenBank. Phylogenetic analysis revealed a closer relationship between NeYSV-TX and the isolates Stenomesson (EU042758) and DC (MG012805) from the Netherlands and USA, respectively based on the partial NIb and CP cistrons (Fig. S2), suggesting that NeYSV-TX may have been introduced from foreign and/or domestic sources. NeYSV has been documented previously from the United Kingdom, the Netherlands, Australia, New Zealand, and India; its first report from the United States was a decade ago from Amaryllis belladonna in California (Guaragna et al. 2013). To the best of our knowledge, this is the first report of NeYSV in Texas, thus expanding the geographical range of the virus in the USA. Anecdotal information from the sample submitter implicated infected crinum lily bulbs as the likely source of NeYSV introduction into the property, with subsequent vegetative propagation of plants resulting in 100% incidence of symptomatic lilies (n>100) over time. Thus, the results underscore the importance of ensuring that only virus-free vegetative plant materials are distributed and propagated by florists to curtail virus spread.

Altered Peripheral Blood Gene Expression in Childhood Cancer Survivors With Anthracycline-Induced Cardiomyopathy - A COG-ALTE03N1 Report.

Background Anthracycline-induced cardiomyopathy is a leading cause of premature death in childhood cancer survivors, presenting a need to understand the underlying pathogenesis. We sought to examine differential blood-based mRNA expression profiles in anthracycline-exposed childhood cancer survivors with and without cardiomyopathy. Methods and Results We designed a matched case-control study (Children's Oncology Group-ALTE03N1) with mRNA sequencing on total RNA from peripheral blood in 40 anthracycline-exposed survivors with cardiomyopathy (cases) and 64 matched survivors without (controls). DESeq2 identified differentially expressed genes. Ingenuity Pathway Analyses (IPA) and Gene Set Enrichment Analyses determined the potential roles of altered genes in biological pathways. Functional validation was performed by gene knockout in human-induced pluripotent stem cell-derived cardiomyocytes using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) technology. Median age at primary cancer diagnosis for cases and controls was 8.2 and 9.7 years, respectively. Thirty-six differentially expressed genes with fold change ≥±2 were identified; 35 were upregulated. IPA identified "hepatic fibrosis" and "iron homeostasis" pathways to be significantly modulated by differentially expressed genes, including toxicology functions of myocardial infarction, cardiac damage, and cardiac dilation. Leading edge analysis from Gene Set Enrichment Analyses identified lactate dehydrogenase A (LDHA) and cluster of differentiation 36 (CD36) genes to be significantly upregulated in cases. Interleukin 1 receptor type 1, 2 (IL1R1, IL1R2), and matrix metalloproteinase 8, 9 (MMP8, MMP9) appeared in multiple canonical pathways. LDHA-knockout human-induced pluripotent stem cell-derived cardiomyocytes showed increased sensitivity to doxorubicin. Conclusions We identified differential mRNA expression profiles in peripheral blood of anthracycline-exposed childhood cancer survivors with and without cardiomyopathy. Upregulation of LDHA and CD36 genes suggests metabolic perturbations in a failing heart. Dysregulation of proinflammatory cytokine receptors IL1R1 and IL1R2 and matrix metalloproteinases, MMP8 and MMP9 indicates structural remodeling that accompanies the clinical manifestation of symptomatic cardiotoxicity.

GPER deletion triggers inhibitory effects in triple negative breast cancer (TNBC) cells through the JNK/c-Jun/p53/Noxa transduction pathway

The G protein-coupled estrogen receptor (GPER) mediates estrogen action in different pathophysiological conditions, including cancer. GPER expression and signaling have been found to join in the progression of triple-negative breast cancer (TNBC), even though controversial data have been reported. In present study, we aimed at providing new mechanistic and biological discoveries knocking out (KO) GPER expression by CRISPR/Cas9 technology in MDA-MB-231 TNBC cells. GPER KO whole transcriptome respect to wild type (WT) MDA-MB-231 cells was determined through total RNA sequencing (RNA-Seq) and gene ontology (GO) enrichment analysis. We ascertained that anti-proliferative and pro-apoptotic gene signatures characterize GPER KO MDA-MB-231 cells. Thereafter, we determined that these cells exhibit a reduced proliferative, clonogenic and self-renewal potential along with an increased mitochondria-dependent apoptosis phenotype. In addition, we recognized that decreased cAMP levels trigger the JNK/c-Jun/p53/Noxa axis, which in turn orchestrates the pro-apoptotic effects observed in GPER KO cells. In accordance with these data, survival analyses in TNBC patients of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset indicated that high Noxa expression correlates with improved outcomes in TNBC patients. Furthermore, we demonstrated that GPER KO in TNBC cells impairs the expression and secretion of the well-acknowledged GPER target gene named CTGF, thus resulting in the inhibition of migratory effects in cancer-associated fibroblasts (CAFs). Overall, the present study provides novel mechanistic and biological insights on GPER KO in TNBC cells suggesting that GPER may be considered as a valuable target in comprehensive therapeutic approaches halting TNBC progression.

A Single-Tube Colorimetric Loop-Mediated Isothermal Amplification for Rapid Detection of SARS-CoV-2 RNA.

Since SARS-CoV-2 is a highly transmissible virus, a rapid and accurate diagnostic method is necessary to prevent virus spread. We aimed to develop and evaluate a new rapid colorimetric reverse transcription loop--mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection in a single closed tube. Nasopharyngeal and throat swabs collected from at-risk individuals testing for SARS-CoV-2 were used to assess the sensitivity and specificity of a new RT-LAMP assay against a commercial qRT-PCR assay. Total RNA extracts were submitted to the RT-LAMP reaction under optimal conditions and amplified at 65 °C for 30 min using three sets of specific primers targeting the nucleocapsid gene. The reaction was detected using two different indicator dyes, hydroxynaphthol blue (HNB) and cresol red. A total of 82 samples were used for detection with HNB and 94 samples with cresol red, and results were compared with the qRT-PCR assay. The sensitivity of the RT-LAMP-based HNB assay was 92.1% and the specificity was 93.2%. The sensitivity of the RT-LAMP-based cresol red assay was 80.3%, and the specificity was 97%. This colorimetric feature makes this assay highly accessible, low-cost, and user-friendly, which can be deployed for massive scale-up and rapid diagnosis of SARS-CoV-2 infection, particularly in low-resource settings.

Discovery and Comprehensive Characterization of Novel Circular RNAs of the Apoptosis-Related BOK Gene in Human Ovarian and Prostate Cancer Cells, Using Nanopore Sequencing.

CircRNAs have become a novel scientific research hotspot, and an increasing number of studies have shed light on their involvement in malignant progression. Prompted by the apparent scientific gap in circRNAs from apoptosis-related genes, such as BOK, we focused on the identification of novel BOK circRNAs in human ovarian and prostate cancer cells. Total RNA was extracted from ovarian and prostate cancer cell lines and reversely transcribed using random hexamer primers. A series of PCR assays utilizing gene-specific divergent primers were carried out. Next, third-generation sequencing based on nanopore technology followed by extensive bioinformatics analysis led to the discovery of 23 novel circRNAs. These novel circRNAs consist of both exonic and intronic regions of the BOK gene. Interestingly, the exons that form the back-splice junction were truncated in most circRNAs, and multiple back-splice sites were found for each BOK exon. Moreover, several BOK circRNAs are predicted to sponge microRNAs with a key role in reproductive cancers, while the presence of putative open reading frames indicates their translational potential. Overall, this study suggests that distinct alternative splicing events lead to the production of novel BOK circRNAs, which could come into play in the molecular landscape and clinical investigation of ovarian and prostate cancer.

Screening of genes encoding proteins that interact with Nrf2: Probing a cDNA library from Mytilus coruscus using a yeast two-hybrid system

The Nuclear factor Erythroid 2-related factor 2 (Nrf2) is the most important endogenous antioxidant factor in organisms, and it has been demonstrated that it exerts extensive control over the immune response by interacting with crucial innate immunity components directly or indirectly. Although Nrf2 has been widely confirmed to be involved in stress resistance in mammals and some fish, its contribution to mollusks oxidative stress resistance has not frequently been documented. In this investigation, total RNA was taken from the digestive gland of M. coruscus, and a cDNA library was constructed and screened using the GATEWAY recombination technology. The Nrf2 cDNA sequence of M. coruscus was cloned into the pGBKT7 vector to prepare the bait plasmid. Using yeast two-hybrid system, after auxotrophic medium screening, sequencing, and bioinformatics analysis, 13 binding proteins that interacted with Nrf2 were finally identified. They were QM-like protein, 40S ribosomal protein S4 (RPS4), ribosomal protein S2 (RPS2), ribosomal protein L12 (RPL12), EF1-alpha mRNA for elongation factor 1 alpha (eEF1-alpha), ferritin, alpha-amylase, trypsin, vdg3, period clock protein, cyclophilin A isoform 1 (CYP A), serine protease CFSP2, histone variant H2A.Z (H2A.Z). For a better understanding the physiological function of Nrf2 in animals and as a potential target for future research on protein roles in Nrf2 interactions, it is crucial to clarify these protein interactions.

First report of beet western yellows virus in radish in Korea.

Radish (Raphanus sativus L.) is an important root vegetable widely consumed in kimchi in Korea. In October 2021, radish leaves with virus-like symptoms of mosaic and yellowing were collected in three fields around Naju, Korea (Fig. S1). A pooled sample (n = 24) was screened for causal viruses by high-throughput sequencing (HTS), with detection confirmed by reverse transcription (RT) PCR. Total RNA was extracted from symptomatic leaves using the Plant RNA Prep kit (Biocube System, Korea), and a cDNA library was constructed and sequenced on an Illumina NovaSeq 6000 system (Macrogen, Korea). De novo transcriptome assembly yielded 63,708 contigs, which were analyzed against the viral reference genome database in GenBank by BLASTn and BLASTx searches. Two large contigs were clearly of viral origin. BLASTn analysis showed that a 9,842-bp contig (4,481,600 mapped reads, mean read coverage 68,758.6×) had 99% identity (99% coverage) with isolate CCLB of turnip mosaic virus (TuMV) from radish in China (KR153038). A second contig of 5,711 bp (7,185 mapped reads, mean read coverage 189.9×) had 97% identity (99% coverage) with isolate SDJN16 of beet western yellows virus (BWYV) from Capsicum annuum in China (MK307779). To confirm the presence of these viruses, total RNA extracted from 24 leaf samples was subjected to RT-PCR using primers specific for TuMV (N60_5'-ACATTGAAAAGCGTAACCA-3' and C30_5'-TCCCATAAGCGAGAATACTAACGA-3', amplicon 356 bp) and BWYV (95F_5'-CGAATCTTGAACACAGCAGAG-3' and 784R_5'-TGTGGG ATCTTGAAGGATAGG-3', amplicon 690 bp) for virus detection. Of the 24 samples, 22 were positive for TuMV and 7 were co-infected with BWYV. Single infection of BWYV was not detected. Infection with TuMV, the predominant virus in radish in Korea, was previously reported (Choi and Choi, 1992; Chung et al., 2015). To determine the complete genomic sequence of the BWYV isolate (BWYV-NJ22) from radish, RT-PCR was conducted using eight overlapping primer pairs designed according to the alignment of previously reported BWYV sequences (Table S2). Terminal sequences of the viral genome were analyzed by 5' and 3' rapid amplification of cDNA ends (RACE; Thermo Fisher Scientific Corp.). The assembled complete genome sequence of BWYV-NJ22 was 5,694 nt long and was deposited in GenBank (accession no. OQ625515). The Sanger sequences shared 96% nt identity with the HTS-derived sequence. BLASTn analysis showed that BWYV-NJ22 had high nucleotide identity (98%) at the complete genome level with a BWYV isolate (OL449448) from C. annuum in Korea. BWYV (genus Polerovirus, family Solemoviridae), is an aphid-borne virus with a host range that includes > 150 plant species and is one of the most important viruses causing yellowing and stunting of vegetable crops (Brunt et al., 1996; Duffus 1973). In Korea, BWYV was first reported to infect paprika, followed by pepper, motherwort, and figwort (Jeon et al., 2021; Kwon et al., 2016; 2018; Park et al., 2018). During fall and winter 2021, 675 radish plants with virus-like symptoms of mosaic, yellowing, and chlorosis were collected from 129 farms in major cultivation areas in Korea and analyzed by RT-PCR using the BWYV detection primers. The incidence of BWYV in radish plants was 4.7%, and all infections were mixed infections with TuMV. To our knowledge, this is the first report of BWYV infecting radish in Korea. The symptoms of single BWYV infection are unclear, as radish is a new host plant of BWYV in Korea. Further research on the pathogenicity and impact of this virus in radish is therefore needed.

Novel circular RNAs of the apoptosis-related BAX and BCL2L12 genes identified in a chronic lymphocytic leukemia cell line using nanopore sequencing.

Circular RNAs (circRNAs), a novel RNA type generated by back-splicing, are key regulators of gene expression, with deregulated expression and established involvement in leukemia. The products of BCL2 and its homologs, including BAX and BCL2L12, are implicated in chronic lymphocytic leukemia (CLL). However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their role in CLL. We sought to further elucidate the contribution of BAX and BCL2L12 in CLL by unraveling the identity, localization, and potential role of their circRNAs. Therefore, total RNA from the EHEB cell line and peripheral blood mononuclear cells (PBMCs) of CLL patients and non-leukemic blood donors was extracted and reverse-transcribed using random hexamers. Next, nested PCRs with divergent primers were performed and the purified PCR products were subjected to 3rd generation nanopore sequencing. Nested PCRs were also applied to first-strand cDNAs synthesized from total RNA extracts of PBMCs from CLL patients and non-leukemic blood donors. Lastly, a single-molecule resolution fluorescent in situ hybridization (smFISH) method called circFISH was used to visualize the circRNA distribution in EHEB cells. We discovered several novel circRNAs produced by BAX and BCL2L12, which were characterized by great exon structure diversity. In addition, intriguing findings regarding their formation emerged. Interestingly, visualization of the most abundant circRNAs showed distinct intracellular localization. Moreover, a complex BAX and BCL2L12 circRNA expression pattern was revealed in CLL patients and non-leukemic blood donors. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B-cell CLL.