Real-time Reverse Transcription-polymerase Research Articles

Examination of SARS-CoV-2 serological test results from multiple commercial and laboratory platforms with an in-house serum panel.

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) is a novel human coronavirus that was identified in 2019. SARS-CoV-2 infection results in an acute, severe respiratory disease called coronavirus disease 2019 (COVID-19). The emergence and rapid spread of SARS-CoV-2 has led to a global public health crisis, which continues to affect populations across the globe. Real time reverse transcription polymerase chain reaction (rRT-PCR) is the reference standard test for COVID-19 diagnosis. Serological tests are valuable tools for serosurveillance programs and establishing correlates of protection from disease. This study evaluated the performance of one in-house enzyme linked immunosorbent assay (ELISA) utilizing the pre-fusion stabilized ectodomain of SARS-CoV-2 spike (S), two commercially available chemiluminescence assays Ortho VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total Reagent Pack and Abbott SARS-CoV-2 IgG assay and one commercially available Surrogate Virus Neutralization Test (sVNT), GenScript USA Inc., cPass SARS-CoV-2 Neutralization Antibody Detection Kit for the detection of SARS-CoV-2 specific antibodies. Using a panel of rRT-PCR confirmed COVID-19 patients' sera and a negative control group as a reference standard, all three immunoassays demonstrated high comparable positivity rates and low discordant rates. All three immunoassays were highly sensitive with estimated sensitivities ranging from 95.4-96.6 %. ROC curve analysis indicated that all three immunoassays had high diagnostic accuracies with area under the curve (AUC) values ranging from 0.9698 to 0.9807. High positive correlation was demonstrated among the conventional microneutralization test (MNT) titers and the sVNT inhibition percent values. Our study indicates that independent evaluations are necessary to optimize the overall utility and the interpretation of the results of serological tests. Overall, we demonstrate that all serological tests evaluated in this study are suitable for the detection of SARS-CoV-2 antibodies.

Influence of genotypes and crop growth stage at time of infection on seed transmission of Maize chlorotic mottle virus

Understanding the effect of seed transmission of Maize chlorotic mottle virus (MCMV) is important in managing the spread of the disease especially secondary transmission caused by vectors. This study was set out to investigate the effect of different genotypes and crop growth stage at the time of infection to rate of MCMV transmission from seeds to seedlings. The experiments were laid out in randomised complete block design with three replications. Detection of MCMV in the grow outs was determined using double antibody sandwich enzyme-linked immunosorbent assay and real time reverse transcription polymerase chain reaction. The results showed low rates of seed transmission from the genotypes in which their was detection of MCMV in their samples (0.08–1%). Similarly, crop growth stage at time of infection also showed low transmission rate of up to 0.1%, with highest transmission rate at the four-leaf stage.

Respiratory syncytial virus in pediatric patients with severe acute respiratory infections in Senegal: findings from the 2022 sentinel surveillance season

In 2022, many regions around the world experienced a severe respiratory syncytial virus (RSV) epidemic with an earlier-than-usual start and increased numbers of paediatric patients in emergency departments. Here we carried out this study to describe the epidemiology and genetic characteristics of RSV infection in patients hospitalized with severe acute respiratory infections in 2022. Samples were tested for RSV by multiplex real time reverse transcription polymerase chain reaction. Subsequently, a subset of RSV positive samples was selected for NGS sequencing. RSV was detected in 16.04%, among which RSV-A was confirmed in 7.5% and RSV-B in 76.7%. RSV infection were more identified in infants aged ≤ 11 months (83.3%) and a shift in the circulation pattern was observed, with highest incidences between September–November. Phylogenetic analyses revealed that all RSV-A strains belonged to GA2.3.5 genotype and all RSV-B strains to GB5.0.5a genotype. Three putative N-glycosylation sites at amino acid positions 103, 135, 237 were predicted among RSV-A strains, while four N-linked glycosylation sites at positions 81, 86, 231 and 294 were identified in RSV-B strains. Globally, our findings reveal an exclusive co-circulation of two genetic lineages of RSV within the pediatric population in Senegal, especially in infants aged ≤ 11 months.

Association of Polymorphism of the Enzyme Catechol-O-Methyltransferase with Fibromialgic Syndrome and its Clinical Repercussions

Objective: Fibromyalgia syndrome (FMS) is a clinical condition that mostly affects women, with chronic diffuse pain, physical disability, mood swings, anxiety, fatigue and insomnia. There are genetic contributions to its pathophysiology. Some single nucleotide polymorphisms (SNPs) can change the function of proteins that participate in pain modulation. Catechol-O-methyltransferase (COMT) is an enzyme responsible for the inactivation of catecholamines in the central nervous system, participating in descending nociceptive inhibitory pathways. This study verified the association of SNPs rs4680, rs6269, rs4633 and rs4818 of the COMT gene with clinical aspects in patients with FMS in Brazil. Methods: Forty-seven volunteers whith FMS were selected, in which the Fibromyalgia Impact Questionnaire, the Beck’s Depression and Anxiety Inventories, the Insomnia Severity Index and the Mini-Mental State Examination were applied. The DNA was extracted by salting out and the SPNs were evaluated by real time reverse transcription polymerase chain (RT-PCR). The association between clinic and SPNs was tested by the Fisher’s exact test. A 95% CI and p value < 0.05 were adopted. Results: The results showed that there was no association between such SNPs and the participants’ clinic regarding the tests used. Conclusions: This study showed that, although the disease has an important impact on patients’ daily lives, increasing the chances of depression, anxiety, insomnia and cognitive losses, it is not associated with the SNPs researched. Further investigations, with larger samples, are needed to assess these and other associations between genetics factors and FMS.

Tou Nong powder obstructs ulcerative colitis through the regulation of NF-κB/NLRP3/Caspase-1/GSDMD inflammasome pyroptotic pathway

Ethnopharmacological relevanceTou Nong Powder (TNP), a classical Chinese medicinal formula originated from the Chinese Ming Dynasty, has been applied to treat skin ulcers in patients with deficient constitutions. According to theory of traditional Chinese medicine, colonic ulcers share similar pathological conditions with skin ulcers, and consequently, TNP has been applied to ulcerative colitis (UC) safely and effectively. Aim of studyTo investigate whether TNP obstructs 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced enteric inflammation through regulation of NLRP3 inflammasome and attenuating enteric pyroptosis. Materials and methodsNetwork pharmacology and UPLC-Q-TOF/MS were operated to identify compounds and pharmacological potential targets. The therapeutic effects of TNP were assessed on TNBS induced colitis via general symptoms (disease activity index, colonic weight and length) and histopathological observation. The NF-κB/NLRP3/Caspase-1/GSDMD signaling pathway regulation was investigated by Western blot and real time reverse transcription polymerase chain reaction (RT-qPCR). ResultsTNP ameliorates the disease activity index, reverses the increase of colonic weight increase, alleviates colonic shortening and colonic histopathological injury. A decrease in tumor necrosis factor α (TNF-α), diamine oxidase (DAO), intercellular adhesion molecule-1 (ICAM-1), and endo-toxin (ET) were investigated in peripheral circulation. Moreover, TNP significantly obstructed the NF-κB/NLRP3/Caspase-1/GSDMD signaling pathway. ConclusionTNP displays a promising therapeutic effect on UC via suppressing NF-κB/NLRP3/Caspase-1/GSDMD signaling pathway and reducing the expression of IL-1β and IL-18.

Infectivity of Maize Chlorotic Mottle Virus from Contaminated Maize Seeds

Seeds have been identified as major sources of introduction and spread of pathogens, with viruses being detected in the seed and also on the seed coat. In this study, the infectivity of Maize chlorotic mottle virus (MCMV) through seeds was investigated. Maize seeds that had tested positive for MCMV previously using double antibody enzyme-linked immunosorbent assay (DAS-ELISA) and real-time reverse transcription polymerase chain reaction (real time RT-PCR) were obtained from various sources. The seeds were soaked in phosphate buffer overnight and the solution used to inoculate maize seedlings. The whole seed was also ground and mixed with the buffer and used for inoculation of seedlings by hand rubbing. Visible MCMV symptoms were observed on less than 2% of the 547 seedlings inoculated with the seed soak and seed extract from contaminated seed 28 days after inoculation and this was confirmed using DAS-ELISA. Use of real time reverse transcription polymerase chain reaction revealed infectivity of MCMV from one of the seed sources used. The mean cycle threshold (Ct) values of samples that showed infectivity ranged from 28.21 to 29.40 cycles. The means were significantly different (P<0.001) from the other samples tested, the healthy and negative controls. When compared to seedlings inoculated with MCMV-infected leaf sap, there was visible development of symptoms associated with MCMV infection, with a severity score of three and Ct values as low as 11.53. The results show evidence of infection of MCMV on maize seedlings caused by virus present in seed extract. Despite rare occurrence of infectivity, the presence of viable virus may cause spread of the virus in the field, leading to development of maize lethal necrosis disease where a cereal potyvirus is present.

The Predictors of Pneumonia in Children with COVID-19

The purpose of this study was to evaluate the relationship between the presence of pneumonia and blood parameters in casesof Coronavirus disease (COVID-19) and to examine their predictive characteristics in terms of pneumonia. We reviewed the filerecords of 151 pediatric patients with a diagnosis of COVID-19 confirmed by the real time-reverse transcription polymerase chainreaction test in nasopharyngeal swabs. The patients were divided into two groups based on direct chest X-ray and computedtomography results in [Group 1 (n:41), with pneumonia findings, and Group 2 (n:110), with no pneumonia findings]. The groups’demographic data, clinical and laboratory findings were compared. Pulmonary involvement was determined in 41 (27.1%) ofthe 151 patients. The [body mass index (BMI) Z-score], red blood cell distribution width (RDW), mean platelet volume (MPV),neutrophil lymphocyte ratio, passive leg raise, and D-dimer levels were significantly higher in patients with pneumonia than thosewithout pneumonia in our study. Based on multivariate logistic regression analysis, BMI Z-score, MPV, and RDW were found tobe independent risk factors of pneumonia in patients. The current study showed higher levels of blood parameters in patients withcoronavirus disease 2019 (COVID -19) presenting with pneumonia than those without pneumonia. We suggest that BMI-Z scoreand MPV value may assist in predicting pulmonary involvement in patients with COVID-19.

Effects of probiotic (Saccharomyces cerevisiae) and ascorbic acid on oxidative gene damage biomarker, heat shock protein 70 and interleukin 10 in broiler chickens exposed to heat stress

Heat stress is a prominent factor responsible for losses economically in poultry meat industry due to adverse effects on the general performance of broiler chickens. In this study, we evaluated the effects of probiotic (Saccharomyces cerevisiae) and ascorbic acid on oxidative gene damage biomarker, heat shock protein 70 (HSP70) and interleukin 10 (IL-10) in broiler chickens exposed to heat stress under natural conditions. Fifty-six broiler chickens served as the subjects, they were divided into 4 groups of 14 as follows: group I (control), group II (probiotic S. cerevisiae at 1 g/kg of feed), group III (ascorbic acid at 200 mg/kg of feed) and group IV (probiotic + ascorbic acid at 1 g/kg and 200 mg/kg of feed, respectively). The treatments were administered via feed for 35 days (D1 to D35). Enzyme-linked immunosorbent assay (ELISA) and one step real time reverse transcription polymerase chain reaction (RT-PCR) was utilised to study the effects of heat stress on the expression levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), HSP70 and IL-10 respectively, in broiler chickens raised during the hot summer season. The level of 8-OHdG gene was significantly lower in the probiotic administered group. The expression level of HSP70 was lowest in the ascorbic acid group while, IL-10 level of expression was highest in the probiotic + ascorbic acid group. The administered antioxidants were efficient in exhibiting anti-stress effects at the level of gene expression. We conclude that probiotic, ascorbic acid and probiotic + ascorbic acid reduced oxidative gene damage, affected the expression of HSP70 and increased the level of IL-10 gene respectively, in broiler chickens exposed to heat stress.

Molecular detection of SARS-CoV-2 infection in three geo-political zones of Nigeria: a cross-sectional study.

sequel to the emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its subsequent spread to all continents of the world, humans have continued to experience severe devastation to their health and economies. To control the spread of this virus, it is important to detect the infection in recently infected and asymptomatic individuals who are capable of infecting others. This study was designed to detect ongoing SARS-CoV-2 Infection among asymptomatic individuals in open markets across three geopolitical zones in Nigeria. nasal and oropharyngeal swab samples were collected from 2,158 study participants between December 20th, 2020 and March 20th, 2021 from large open markets across three geo-political zones (Southwest, Northwest and Southeast) of Nigeria. Virus RNA was extracted from these swab samples and real time reverse transcription polymerase chain reaction (RT-PCR) was carried out for the detection of SARS-CoV-2 specific genes. Data were analysed using descriptive statistics. a total of 163 (7.6%) of the 2,158 participants enrolled for the study tested positive for SARS-CoV-2 by RT-PCR. The rate of infection was significantly higher in the North-western States of the country when compared to the western and Eastern regions (P=0.000). Similarly, the rate of infection was higher among buyers than sellers (P=0.000) and among males when compared with females, though the difference was not significant (p=0.31). this study shows that there is a continuous spread of SARS-CoV-2, especially among active, asymptomatic individuals across many States in the country. There is therefore need to continuously educate citizens on the need to adhere to both the non-pharmaceutical and pharmaceutical preventive measures to protect themselves and ultimately curb the spread of the virus.

A two-step process for in silico screening to assess the performance of qRTPCR kits against variant strains of SARS-CoV-2

BackgroundSince inception of the COVID-19 pandemic, early detection and isolation of positive cases is one of the key strategies to restrict disease transmission. Real time reverse transcription polymerase chain reaction (qRTPCR) has been the mainstay of diagnosis. Most of the qRTPCR kits were designed against the target genes of original strain of SARS-CoV-2. However, with the emergence of variant strains of SARS-CoV-2, sensitivity of the qRTPCR assays has reportedly reduced. In view of this, it is critical to continuously monitor the performance of the qRTPCR kits in the backdrop of variant strains of SARS-CoV-2. Real world monitoring of assay performance is challenging. Therefore, we developed a two-step in-silico screening process for evaluating the performance of various qRTPCR kits used in India.ResultsWe analysed 73 qRT-PCR kits marketed in India, against the two SARS-CoV-2 VoCs. Sequences of both Delta (B.1.617.2) and Omicron (B.1.1.529) VoCs submitted to GISAID within a specific timeframe were downloaded, clustered to identify unique sequences and aligned with primer and probe sequences. Results were analysed following a two-step screening process. Out of 73 kits analysed, seven were unsatisfactory for detection of both Delta and Omicron VoCs, 10 were unsatisfactory for Delta VoC whereas 2 were unsatisfactory for only Omicron VoC.ConclusionOverall, we have developed a useful screening process for evaluating the performance of qRTPCR assays against Delta and Omicron VoCs of SARS-CoV-2 which can be used for detecting SARS-CoV-2 VoCs that may emerge in future and can also be redeployed for other evolving pathogens of public health importance.

"Investigation of the Diagnostic Accuracy of Rapid Influenza Diagnostic Kit Method in Comparison to the Real Time Reverse Transcription Polymerase Chain Reaction"

Bimalesh Kumar Jha1*, Rachana Mehta1 and Krishna Das manandhar2 Author Affiliations 1National Public Health Laboratory, Nepal 2Central Department of Biotechnology, Tribhuvan University, Nepal Received: August 26, 2022 | Published: September 23, 2022 Corresponding author: Bimalesh Kumar Jha, National Public Health Laboratory, Teku, Kathmandu, Nepal DOI: 10.26717/BJSTR.2022.46.007341

STUDY OF TWO YEARS EPIDEMIOLOGICAL TREND OF SWINE FLU (H1N1) CASES BY CROSS SECTIONAL ANALYSIS IN TERTIARY CARE TEACHING HOSPITAL OF WESTERN INDIA

BACKGROUND & OBJECTIVE: To study region-wise monthly incidence, alteration in seasonal trend and geographical distribution of Swine Flu (H1N1) cases in district of western India for two years. After Ethics Committee approval, a cross-sectionalMATERIAL AND METHOD: study was carried out with analysis of two years' demographic prole, geographical distribution and seasonal variation of real time Reverse Transcription–Polymerase Chain Reaction (RT-PCR) - H1N1 positive cases. Annual change in pattern of positive cases was analyzed by independent sample student's t - test (P < 0.05). Out of 3,524 Seasonal Flu cases investigated by Inuenza A (H1N1) RT-PCR, total 1,600RESULT: Seasonal Flu–positive cases were analyzed. On looking at Age-wise distribution of patients, during rst year, maximum number of Seasonal Flu positive cases was from age group of 1 to 10 years, while during next year, maximum number of cases was from age group of 51 to 60 years. During rst year of epidemic, incidence of 836.12 cases per 1,00,000 population per year was detected with peak during winter as compared to 121.49 cases per 1,00,000 population per year with peak during monsoon in second year of epidemic. DuringINTERPRETATION & CONCLUSION: Epidemic of Seasonal Flu, Swine Flu (H1N1) positive cases got reduced annually, and seasonal pattern of H1N1 positive cases changed with peak during monsoon as compared to conventional winter peak. Overall change in seasonal trend, pediatric age distribution suggests need of in depth analysis of development of herd immunity, and risk factors involved for monsoon peak with alarming precaution by epidemic preparedness.

Resistance of breast cancer cells to all-trans retinoic acid is associated with a decrease in the basal level of nuclear receptor <i>RARα</i> expression and induction of cytochrome <i>CYP26A1</i> and <i>CYP26B1</i> expression

Introduction. Retinoic acid (RA) is a key regulator of cell differentiation and a critical player in such systemic processes in the body as embryonic development, immune system cell maturation and functioning, tissue remodeling and several others. This compound displays an antitumor activity due to its ability to stimulate differentiation, induce apoptosis and inhibit proliferation of malignant cells. The rapid acquisition of resistance to RA and its analogues by solid tumor cells is one of the main problems limiting the widespread use of retinoids in the therapy of malignant neoplasms. The mechanisms of RA-resistance are still poorly understood.The study objective – assessment of the relationship between the basal expression level of the nuclear RARα receptor and the RA-induced expression of the cytochromes CYP26A1and CYP26B1 with the resistance of breast cancer cells to the action of all-trans-retinoic acid.Materials and methods. Cell lines were cultured, the sensitivity of breast cancer cells to the action of fully trans-retinoic acid, RNA isolation, reverse transcription reaction and real-time polymerase chain reaction were analyzed).Results. In present study, using an experimental model represented by 9 breast cancer cell lines with different level of sensitivity to RA, we showed that the expression of the RA nuclear receptor RARα, as well as the level of mRNA induction of CYP26A1 and CYP26B1 cytochromes in response to RA treatment correlate with RA-sensitivity.Conclusion. Thus, a decrease of RARα expression as well as the reduced ability to catabolize RA are factors associated with RA-resistance of breast cancer cells.

Differential Expression of Endocrine Regulatory Genes in Apis cerana and Apis mellifera (Hymenoptera: Apidae) at High Temperature

Abstract Drastic changes in temperature can interfere with the normal physiological and biochemical activities of bees. Temperature stress affects the endocrine system of bees and induces a series of stress responses. However, the changes that occur in hormones in bees that are exposed to environmental stress are not well understood. In this study, we analyzed the expression patterns of four genes by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) in Apis mellifera L. and Apis cerana F. (Hymenoptera: Apidae) at different temperatures and different treatment times. The expression of juvenile hormone esterase, vitellogenin, corticotropin-releasing hormone binding protein, and adipokinetic hormone receptor genes was found to be increasingly affected by an increase in temperature and treatment time. Temperature stress affects the endocrine system of bees, and endogenous hormones in bees can respond to environmental stimuli. Our findings provide a basis for determining the mechanisms by which insect endocrine systems adapt to high temperatures.

MRNA and long non-coding RNA expression profiles of rotator cuff tear patients reveal inflammatory features in long head of biceps tendon

BackgroundThis study aimed to identify the differentially expressed mRNAs and lncRNAs in inflammatory long head of biceps tendon (LHBT) of rotator cuff tear (RCT) patients and further explore the function and potential targets of differentially expressed lncRNAs in biceps tendon pathology.MethodsHuman gene expression microarray was made between 3 inflammatory LHBT samples and 3 normal LHBT samples from RCT patients. GO analysis and KEGG pathway analysis were performed to annotate the function of differentially expressed mRNAs. The real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was admitted to verify their expression. LncRNA-mRNA co-expression network, cis-acting element, trans-acting element and transcription factor (TF) regulation analysis were constructed to predict the potential molecular regulatory mechanisms and targets for LHB tendinitis.Results103 differentially expressed lncRNAs and mRNAs, of which 75 were up-regulated and 28 were down-regulated, were detected to be differentially expressed in LHBT. The expressions of 4 most differentially expressed lncRNAs (A2MP1, LOC100996671, COL6A4P, lnc-LRCH1-5) were confirmed by qRT-PCR. GO functional analysis indicated that related lncRNAs and mRNAs were involved in the biological processes of regulation of innate immune response, neutrophil chemotaxis, interleukin-1 cell response and others. KEGG pathway analysis indicated that related lncRNAs and mRNAs were involved in MAPK signaling pathway, NF-kappa B signaling pathway, cAMP signaling pathway and others. TF regulation analysis revealed that COL6A4P2, A2MP1 and LOC100996671 target NFKB2.ConclusionsLlncRNA-COL6A4P2, A2MP1 and LOC100996671 may regulate the inflammation of LHBT in RCT patients through NFKB2/NF-kappa B signaling pathway, and preliminarily revealed the pathological molecular mechanism of tendinitis of LHBT.

Comprehensive analysis of lncRNA-miRNA-mRNA networks during osteogenic differentiation of bone marrow mesenchymal stem cells

BackgroundLong non-coding RNA (lncRNA) plays crucial role in osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs), involving in regulation of competing endogenous RNA (ceRNA) mechanisms and conduction of signaling pathways. However, its mechanisms are poorly understood. This study aimed to investigate lncRNAs, miRNAs and mRNAs expression profiles in rat BMMSCs (rBMMSCs) osteogenic differentiation, screen the potential key lncRNA-miRNA-mRNA networks, explore the putative functions and identify the key molecules, as the basis of studying potential mechanism of rBMMSCs osteogenic differentiation driven by lncRNA, providing molecular targets for the management of bone defect.MethodsHigh-throughput RNA sequencing (RNA-seq) was used to determine lncRNAs, miRNAs, and mRNAs expression profiles at 14-day rBMMSCs osteogenesis. The pivotal lncRNA-miRNA and miRNA-mRNA networks were predicted from sequencing data and bioinformatic analysis, and the results were exported by Cytoscape 3.9.0 software. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used for functional exploration. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to validate lncRNAs, miRNAs and mRNAs.ResultsrBMMSCs were identified, and the osteogenic and adipogenic differentiation ability were detected. A total of 8634 lncRNAs were detected by RNA-seq, and 1524 differential expressed lncRNAs, of which 812 up-regulated and 712 down-regulated in osteo-inductive groups compared with control groups. 30 up-regulated and 61 down-regulated miRNAs, 91 miRNAs were differentially expressed in total. 2453 differentially expressed mRNAs including 1272 up-expressed and 1181 down-expressed were detected. 10 up-regulated lncRNAs were chosen to predict 21 down-regulated miRNAs and 650 up-regulated mRNAs. 49 lncRNA-miRNA and 1515 miRNA–mRNA interactive networks were constructed. GO analysis showed the most important enrichment in cell component and molecular function were “cytoplasm” and “protein binding”, respectively. Biological process related to osteogenic differentiation such as “cell proliferation”, “wound healing”, “cell migration”, “osteoblast differentiation”, “extracellular matrix organization” and “response to hypoxia” were enriched. KEGG analysis showed differentially expressed genes were mainly enriched in “PI3K-Akt signaling pathway”, “Signaling pathway regulating pluripotency of stem cells”, “cGMP-PKG signaling pathway”, “Axon guidance” and “Calcium signaling pathway”. qRT-PCR verified that lncRNA Tug1, lncRNA AABR07011996.1, rno-miR-93-5p, rno-miR-322-5p, Sgk1 and Fzd4 were consistent with the sequencing results, and 4 lncRNA-miRNA-mRNA networks based on validations were constructed, and enrichment pathways were closely related to “PI3K-Akt signaling pathway”, “Signaling pathway regulating pluripotency of stem cells” and “Wnt signaling pathway”.ConclusionslncRNAs, miRNAs and mRNAs expression profiles provide clues for future studies on their roles for BMMSCs osteogenic differentiation. Furthermore, lncRNA–miRNA–mRNA networks give more information on potential new mechanisms and targets for management on bone defect.

Impact of SARS-CoV-2 viral load and duration of symptoms before hospital admission on the mortality of hospitalized COVID-19 patients.

PurposeAssess the impact of viral load estimated by cycle threshold (Ct) of reverse transcription real time-polymerase chain reaction (rRT-PCR) and the days from symptoms onset on mortality in hospitalized patients with COVID19.MethodsRetrospective observational study of 782 patients with a positive rRT-PCR from a nasopharyngeal swab was performed within the first 24 h from admission. Demographic data, clinical manifestations and laboratory parameters were collected. Uni- and multivariate analyses were performed to identify factors associated with mortality at 60 days.ResultsCt was divided into three groups and the mortality rate decreased from 27.3 to 20.7% and 9.8% for Ct values of ≤ 20, 21–25 and > 25, respectively (P = 0.0001). The multivariate analysis identified as predictors of mortality, a Ct value < 20 (OR 3.13, CI 95% 1.38–7.10), between 21–25 (OR 2.47, CI 95% 1.32–4.64) with respect to a Ct value > 25. Days from symptoms onset is a variable associated with mortality as well (DSOA) ≤ 6 (OR 1.86, CI 95% 1.00–3.46), among other factors. Patients requiring hospital admission within 6 DSOA with a Ct value ≤ 25 had the highest mortality rate (28%).ConclusionsThe inclusion of Ct values and DSOA in the characterization of study populations could be a useful tool to evaluate the efficacy of antivirals.

Changes in expression of miRNAs from the DLK1-DIO3 locus are characteristic of relapsing-remitting multiple sclerosis regardless of the disease activity

The role of miRNAs – small, regulatory, noncoding RNAs – in the multiple sclerosis (MS) development is being intensively investigated. Previously, we the first who observed a significant increase in the expression of 26 microRNA genes localized in the DLK1-DIO3 locus in men with relapsing-remitting MS (RRMS) in peripheral blood mononuclear cells (PBMCs), using RNA sequencing.Objective: to evaluate the possible involvement of miRNAs in the regulation of the activity of the pathological process in RRMS by comparing the miRNA genes expression levels from this locus in patients in remission, relapse, and healthy individuals, separately for men and women.Patients and methods. Analysis of miR-431-5p, miR-127-3p, miR-379, miR-376c, miR-381, miR-410 and miR-656-3p microRNA expression was performed by reverse transcription and subsequent real-time polymerase chain reaction in PBMCs of 16 patients in the relapse stage, 20 patients in remission who did not receive immunomodulatory drugs, and 20 healthy individuals.Results and discussion. The expression levels of all studied miRNAs did not differ in patients in remission and relapse stages, in men and women. At the same time, men (in remission and relapse) showed a significant increase in the levels of all miRNAs compared with healthy men; in women, no changes in expression were observed. A high level of correlation of miRNA expression from the DLK1-DIO3 locus was shown in both patients and healthy individuals from the control group, regardless of gender. Coexpression was observed not only for miRNA genes from the same cluster (14q32.2 or 14q32.31), but also for genes from different clusters.Conclusion. The miRNAs genes from the DLK1-DIO3 locus are involved in the pathophysiology of RMS onset, but not in the processes associated with the transition from remission to relapses. The high consistency of miRNA expression, regardless of the localization of their genes within this region, suggests the presense of a common mechanism that regulates their transcription.

Identification of tRNA-Related Fragments and Their Potential Regulatory Effects in Thyroid-Associated Ophthalmopathy.

Recently, the potential role of tRNA-related fragments (tRFs) in ophthalmic diseases has been extensively researched. However, systematic studies on the potential regulatory effects of tRFs in thyroid-associated ophthalmopathy (TAO) are lacking. We used high-throughput sequencing techniques to measure expression levels of mRNAs and tRFs in patients with TAO, and the results were verified by real-time quantitative reverse transcription polymerase chain reaction (q-PCR). Next, the potential biological regulatory effect of differentially expressed tRFs was analyzed, and potential downstream target RNAs of differentially expressed tRFs were predicted to explore the potential role of tRFs as therapeutic targets and biomarkers of TAO. A total of 50 tRFs and 361 mRNAs were dysregulated in the TAO group, and tRF5-GluCTC, PMAIP1, HSD17B2 and ATF3 were verified to be significantly differentially expressed in TAO. Our research reveals that several associated pathways likely play a role in the pathogenesis of TAO. By targeting ATF3, HSD17B2 and PMAIP1, tRF5-GluCTC may play a potential role in regulating the orbital fibroblast adipogenic response and fibrotic hyperplasia in patients with TAO.

Rapid Antigen Test LumiraDxTM vs. Real Time Polymerase Chain Reaction for the Diagnosis of SARS-CoV-2 Infection: A Retrospective Cohort Study.

Background: Real time reverse transcription polymerase chain reaction (real time RT-PCR) testing is the gold standard for the diagnosis of SARS-CoV-2 infections. However, to expand the testing capacity, new SARS-CoV-2 rapid antigen tests (Ag-RDTs) have been implemented. Ag-RDTs are more rapid, but less reliable in terms of sensitivity, and real-life data on their performance in comparison with the real time RT-PCR test are lacking. Methods: We aimed at assessing the diagnostic performance of the third-generation antigenic swab LumiraDx™ compared with real time RT-PCR in a retrospective cohort study at the Infectious Diseases Unit of Padua. All of the patients who were consecutively tested for SARS-CoV-2 in our centre (by both real time RT-PCR and Ag-RTD LumiraDxTM) from 19 January to 30 May 2021, were included. Cycle-threshold (Ct) values of positive real time RT-PCR were recorded as well as the number of days from symptoms’ onset to testing. Results: Among the 282 patients included, 80.9% (N = 228) tested positive to real time RT-PCR, and among these, 174 tested positive also to LumiraDx™. Compared with real time RT-PCR, which is considered as the gold standard for the assessment of the presence/absence of SARS-CoV-2 infection, LumiraDx™ showed an overall sensitivity of 76.3% and specificity of 94.4%. Sensitivity increased to 91% when testing was performed <10 days from symptoms’ onset, and to 95% when considering Ct < 25. Multivariable binomial logistic regression showed that false negative LumiraDx™ results were significantly associated with high Ct values, and with further testing from symptoms’ onset. Conclusions: The results of our study suggested that the LumiraDx™ SARS-CoV-2 antigen assay may be appropriate for the detection of SARS-CoV-2 infection, especially in its early phase when the test largely meets the performance requirements of the European Centre for Disease Prevention and Control (ECDC).