Real-time Quantitative Reverse Transcriptase-polymerase Research Articles

Influence of storage solution, temperature, assay time and concentration on RT-qPCR nucleic acid detection for SARS-CoV-2 detection of SARS-CoV-2 by the RT-qPCR

Real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is an important method for the early diagnosis of coronavirus disease 2019 (COVID-19). This study investigated the effects of storage solution, temperature and detection time on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection by RT-qPCR. Various concentrations of SARS-CoV-2 were added to inactive and non-inactive storage solution and the viral suspensions were stored at various temperatures (room temperature, 4, −20 and −80 °C). Then, at five different detection time points, the Ct values were determined by RT-qPCR. Active and inactive storage solutions and storage temperature have a great impact on the detection of N gene of SARS-CoV-2 at different concentration corridors but have little impact on the ORF gene. The storage time has a greater impact on the N gene and ORF gene at high concentrations but has no effect on the two genes at low concentrations. In conclusion, storage temperature, storage time and storage status (inactivated, non-inactivated) have no effect on the nucleic acid detection of SARS-CoV-2 at the same concentration. For different concentrations of SARS-CoV-2, the detection of N gene is mainly affected.

Selection, identification and evaluation of optimal reference genes in Chinese sturgeon (Acipenser sinensis) under polypropylene microplastics stress

Polypropylene microplastics (PP-MPs) are emerging environmental contaminants that have the potential to cause adverse effects on aquatic organisms. Reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) is a valuable tool for assessing the gene expression profiles under PP-MPs stress. To obtain an accurate gene expression profile of tissue inflammation and apoptosis that reflects the molecular mechanisms underlying the impact of PP-MPs on Chinese sturgeon, identifying reliable reference genes is crucial for RT-qPCR analysis. In this study, we constructed an experiment model of Chinese sturgeon exposed to PP-MPs, assessed the pathological injury, metabolic profile responses and oxidative stress in liver, evaluated the reliability of 8 reliable reference genes by 4 commonly used algorithms including GeNorm, NormFinder, BeatKeeper, Delta Ct, and then analyzed the performance of inflammatory response genes in liver, spleen and kidney with the best reference gene. HE staining revealed that the cytoplasm full small vacuoles and nucleus diameter increased were occurred in the liver cell of PP-MPs in treatment groups. Additionally, oxidative and biochemical parameters were significantly changes in the liver of treatment groups. For the reference genes in PP-MPs exposure experiments, this study screening the optimal reference genes including: EF1α and GAPDH for liver and spleen, and GAPDH and RPS18 for kidney. Besides, 2 inflammatory response genes (NLRP3, TNF-α) were chosen to assess the optimal reference genes using the least stable reference gene (TUB) as a control, verified the practicality of the select reference genes in different tissues. We also found that the low concentration of PP-MPs could induce the liver tissue damage and inflammatory response in Chinese sturgeon. Our study initially evaluated the impact of short-time exposure with PP-MPs in Chinese sturgeon and provided 3 sets of validated optimal reference genes in Chinese sturgeon exposure to PP-MPs.

Utilization of temperature-mediated activation of thyroid hormone-induced molecular memory to evaluate early signaling events in the olfactory epithelium of Rana [Lithobates] catesbeiana tadpoles

The amphibian olfactory system is highly distinct between aquatic tadpole and terrestrial frog life stages and therefore must remodel extensively during thyroid hormone (TH)-dependent metamorphosis. Developmentally appropriate functioning of the olfactory epithelium is critical for survival. Previous studies in other Rana [Lithobates] catesbeiana premetamorphic tadpole tissues showed that initiation of TH-induced metamorphosis can be uncoupled from execution of TH-dependent programs by holding tadpoles in the cold rather than at warmer permissive temperatures. TH-exposed tadpoles at the nonpermissive (5 °C) temperature do not undergo metamorphosis but retain a “molecular memory” of TH exposure that is activated upon shift to a permissive warm temperature. Herein, premetamorphic tadpoles were held at permissive (24 °C) or nonpermissive (5 °C) temperatures and injected with 10 pmoles/g body weight 3,5,3′-triiodothyronine (T3) or solvent control. Olfactory epithelium was collected at 48 h post-injection. RNA-sequencing (RNA-Seq) and reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analyses generated differentially expressed transcript profiles of 4328 and 54 contigs for permissive and nonpermissive temperatures, respectively. Translation, rRNA, spliceosome, and proteolytic processes gene ontologies were enriched by T3 treatment at 24 °C while negative regulation of cell proliferation was enriched by T3 at 5 °C. Of note, as found in other tissues, TH-induced basic leucine zipper-containing protein-encoding transcript, thibz, was significantly induced by T3 at both temperatures, suggesting a role in the establishment of molecular memory in the olfactory epithelium. The current study provides critical insights by deconstructing early TH-induced induction of postembryonic processes that may be targets for disruption by environmental contaminants.

Ethanol extract of Paridis rhizoma attenuates carrageenan-induced paw swelling in rats by inhibiting the production of inflammatory factors

ContextInflammation has been identified as a key factor contributing to the development of numerous diseases. Several anti-inflammatory drugs have been developed to treat inflammation-related diseases. However, some of such drugs are associated with varying degrees of side effects. Therefore, it is imperative to develop new anti-inflammatory drugs with reducing side effects for the treatment of inflammation-related diseases. Natural anti-inflammatory drugs have emerged as an important area of research in recent years. The study was to determine the anti-inflammatory mechanism of Paridis rhizoma extract (PRE) in rat models of acute inflammation induced by carrageenan and RAW264.7 cells models induced by lipopolysaccharide (LPS).Materials and methodsPRE was investigated using the carrageenan-induced paw oedema model on rats in vivo. Histopathology examined the extent of inflammatory infiltration and tissue damage. The effect of PRE on the levels of specific cytokines was determined using enzyme-linked immunosorbent assay (ELISA). The Cell Counting Kit (CCK)-8 assay evaluated the cytotoxic effects of PRE on Raw264.7 cells. The mRNA expression levels of cytokines were quantified using quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Western blot measured TNF-α, IL6, TLR4, p-P65, p-IKB, HO1, SOD1 and SOD2. Fluorescence measured the cellular levels of reactive oxygen species (ROS).ResultsPRE treatment reduced interstitial edema and structural damage in a dose-dependent manner in vivo. PRE inhibited inflammatory responses in vivo and in vitro, as evidenced by the decreased expression of inflammatory factors, production of ROS, and increased expression of SOD1, SOD2, and HO1. Moreover, PRE inhibited the activity of the nuclear factor kappa B (NF-kB) pathway.ConclusionThe anti-inflammatory activity and potential mechanism of PRE were demonstrated according to the results. PRE reduced LPS-induced inflammation in RAW264.7 cells by inhibiting the NF-KB signaling pathway and ROS production in vitro. PRE alleviated interstitial edema and structural damage in the carrageenan-induced paw edema model on rats in vivo. This study provided an idea for future development of PR-based anti-inflammatory drugs.

512. Interim Results of a Prospective Cohort Study to Monitor the Emergence of Resistance in Immunocompromised Non-Hospitalized Patients With COVID-19 Who Were Treated with Sotrovimab in Great Britain: LUNAR Study

Abstract Background Immunocompromised (IC) patients are at risk of adverse COVID-19 outcomes. The risk of treatment emergent resistance may be high in this population. This study investigated clinical and virological outcomes in sotrovimab-treated IC patients in Great Britain while the Omicron variant was predominant. Methods IC, non-hospitalized patients aged ≥ 18 years who were infected with SARS-CoV-2 and received early treatment with sotrovimab 500 mg IV for COVID-19 as per standard of care were included in this multicenter, single arm, observational prospective cohort study. Nasal/oropharyngeal samples were collected at baseline, Day (D) 7, 14, and 28 (+/-2 days) for viral load and sequencing analyses. Clinical (hospitalization, respiratory support, intensive care unit [ICU] admission and death) and safety outcomes were assessed through D28. This interim analysis included patients enrolled from 1 July 2022–31 January 2023. Results Among 195 patients (median age: 58 years), 56% were female, 86% were white, and 98.5% had ≥ 1 COVID-19 vaccine dose prior to enrollment. All patients received sotrovimab within 8 (median: 2) days of diagnosis. Absolute median viral load declined from 7.46 log10 copies/mL at baseline to 0.55 log10 copies/mL at D28 (Table 1). Of 189 patients with spike consensus sequencing data, all harbored the Omicron variant, with 32 sublineages identified. Omicron BA.4, BQ.1, BE.9, BA.5.1.18, and BN.1 were most common in this data cut. We also plan to present interim data on treatment emergent substitutions. No patients were hospitalized due to COVID-19. Six (1.3%) patients had all-cause hospitalizations; none were admitted to ICU. One patient (0.5%; infected with Omicron CH.1.1) with progressive neuromuscular disease needed high flow oxygen/non-invasive mechanical intervention and died on D18 (death deemed not COVID-19 related by investigator) (Table 2). Three mild sotrovimab-related adverse events were reported.Table 1:Summary of absolute viral load (log10 copies/mL) through Day 28, as measured by qRT-PCR from nasal/oropharyngeal swabsNote: Baseline log10 viral load was defined as the non-missing assessment taken at Day 0, excluding the negative viral load results. The post-baseline viral load values with Ct value > 38 were imputed as half the lower limit of detection (i.e. 453 copies/mL*0.5=226.5). Negative viral loads were defined as Ct ≥ 45 and were imputed as 3.57 copies/mL. These imputed values were used to derive log10 viral loads. Ct, cycle threshold; IV, intravenous; qRT-PCR, quantitative real-time reverse transcriptase polymerase chain reactionTable 2:Summary of clinical outcomes through Day 28Notes: N = Number of participants in Completer population (defined as all participants who were enrolled, exposed to study intervention [sotrovimab], and who had been followed for 30 days or discontinued from the study early); n = Number of participants with data available. ECMO, extracorporeal membrane oxygenation; ICU, intensive care unit; IV, intravenous Conclusion Sotrovimab-treated patients had reduced viral load by D7 which further decreased through D28, despite being IC and infected with Omicron subvariants (reduced in vitro neutralization has been reported for some of the subvariants). Few severe clinical outcomes were reported (all unrelated to COVID-19). Disclosures Judith Breuer, MD, GSK: Funding to conduct study|MRC/UKRI: Grant/Research Support|National Institutes of Health: Grant/Research Support|NIHR: Senior investigator|UKNIHR: Grant/Research Support|Vir Biotechnology, Inc: Funding to conduct study|Wellcome Trust: Grant/Research Support Myriam Drysdale, PhD, GSK: Employee|GSK: Stocks/Bonds Jill Walker, PhD, GSK: Employee|GSK: Stocks/Bonds Jennifer Han, MD, GSK: Employment|GSK: Stocks/Bonds Magdalena Gorczycka, MSc, GSK: Employee|GSK: Stocks/Bonds Alicia Aylott, MSc, GSK: Employee|GSK: Stocks/Bonds Melissa K. Van Dyke, PhD, GSK: Employee|GSK: Stocks/Bonds Helen Birch, PhD, GSK: Employee|GSK: Stocks/Bonds Elizabeth McKie, PhD, GSK: Employee|GSK: Stocks/Bonds David M. Lowe, FRCP, PhD, Biotest: Speaker fees|Blood Cancer UK: Grant/Research Support|Bristol Myers Squibb: Grant/Research Support|British Society of Antimicrobial Chemotherapy: Grant/Research Support|Gilead: Speaker fees (educational video)|GSK: Funding to conduct study|Langland: Speaker fees|LifeArc: Grant/Research Support|Merck: Personal fees for a round-table discussion|National Institute of Health Research: Grant/Research Support|Octapharma: Travel, accommodation and conference fees|UK Medical Research Council: Grant/Research Support|Vir Biotechnology, Inc: Funding to conduct study

Gastrodin Regulates PI3K/AKT-Sirt3 Signaling Pathway and Proinflammatory Mediators in Activated Microglia.

Activated microglia and their mediated inflammatory responses play an important role in the pathogenesis of hypoxic-ischemic brain damage (HIBD). Therefore, regulating microglia activation is considered a potential therapeutic strategy. The neuroprotective effects of gastrodin were evaluated in HIBD model mice, and in oxygen glucose deprivation (OGD)-treated and lipopolysaccharide (LPS)activated BV-2 microglia cells. The potential molecular mechanism was investigated using western blotting, immunofluorescence labeling, quantitative realtime reverse transcriptase polymerase chain reaction, and flow cytometry. Herein, we found that PI3K/AKT signaling can regulate Sirt3 in activated microglia, but not reciprocally. And gastrodin exerts anti-inflammatory and antiapoptotic effects through the PI3K/AKT-Sirt3 signaling pathway. In addition, gastrodin could promote FOXO3a phosphorylation, and inhibit ROS production in LPSactivated BV-2 microglia. Moreover, the level P-FOXO3a decreased significantly in Sirt3-siRNA group. However, there was no significant change after gastrodin and siRNA combination treatment. Notably, gastrodin might also affect the production of ROS in activated microglia by regulating the level of P-FOXO3a via Sirt3. Together, this study highlighted the neuroprotective role of PI3K/AKT-Sirt3 axis in HIBD, and the anti-inflammatory, anti-apoptotic, and anti-oxidative stress effects of gastrodin on HIBD.

Activation of UBEC2 by transcription factor MYBL2 affects DNA damage and promotes gastric cancer progression and cisplatin resistance.

Ubiquitin-conjugating enzyme E2 C (UBE2C) plays a carcinogenic role in gastric cancer (GC); yet, its role in cisplatin (DDP) resistance in GC is enigmatic. This study sought to probe into the impact of UBE2C on DDP resistance in GC and its concrete molecular mechanism in GC progression. Bioinformatics analysis was used to analyze differentially expressed mRNAs and predict upstream regulatory molecules in GC. Real-time quantitative reverse transcriptase polymerase chain reaction and western blot were used to detect the expression of UBE2C and MYB proto-oncogene like 2 (MYBL2). Dual luciferase and chromatin immunoprecipitation (ChIP) assays were used to verify the binding relationship. Cell counting kit-8 was used to detect cell viability and calculate IC50 values. Flow cytometry was used to detect the cell cycle. Comet assay was used to detect DNA damage. Western blot was used to detect the expression of DNA loss-related proteins (γ-H2AX, ATM/p-ATM). The knockdown of highly expressed UBE2C in GC cell lines could reduce cell viability, induce G2/M arrest, induce apoptosis, and promote DNA damage and DDP sensitivity. Bioinformatics analysis predicted that the substantially upregulated MYBL2 was an upstream transcription factor in UBE2C. The binding relationship between the UBE2C promoter region and MYBL2 was verified by dual luciferase and ChIP. Overexpression of UBE2C in the rescue experiment was found to reverse the inhibited GC progression and promoted DDP sensitivity brought by the knockdown of MYBL2. In conclusion, the MYBL2/UBE2C regulatory axis may be a potential way to overcome DDP resistance in GC.

Autophagy Markers Induced by Influenza Virus and MUC1 Expressions in Cancer-Derived Cell Lines

Influenza virus-induced autophagy is often accompanied by apoptosis and results in cell death in virus-infected cells. It is well known that autophagy is modulated by the mTOR/PI3K/Akt pathway, which plays an important role in the response to the presence of energy sources and external stimuli. This pathway is modulated by mucin 1 (MUC1), which has extracellular and intracellular components and plays an important role in metastasis and chemotherapeutic resistance. In this study, it was aimed to investigate the expression of MUC1 after the inoculation of influenza viruses into the cancer-derived cell cultures and, accordingly, the changes in autophagy markers such as mTOR and LC3B. In this study, MCF-7, HeLa and A-549 cell lines were used which have adenocarcinoma origin. To control the growth of influenza virus in these cells, the MDCK cell line was also inoculated. Centrifuge-enhanced shell-vial cell culture method was used in all experiments. Influenza A (H1N1) pdm09 strain was inoculated into these cell lines then the expressions of viral nucleic acid and cycle threshold (Ct) of MUC1, mTOR, LC3B associated genes were investigated by quantitative real-time reverse transcriptase polymerase chain reaction (qRTPCR) method in the samples taken from the supernatants of all cells at the end of the 48-hour incubation period. To investigate whether these markers were present in cells, after all cells were permeabilized with paraformaldehyde, cell-coated infected coverslips were stained with fluorescent labeled monoclonal antibodies developed against MUC1, mTOR, LC3B and influenza virus antigens. In the examination of fluorescence microscopy, all of the cell cultures (MCF-7, He-La and A-549) infected with influenza virus yielded positive results in terms of LC3B, mTOR and MUC1 monoclonal antibody staining, whereas all of the non-infected cells were found negative. Cycle threshold values of MUC1, LC3B and mTOR associated genes were found to be lower in A-549 cell line inoculated with influenza virus. Although protein expression was demonstrated in MCF-7 and He-La cell lines, similar changes were not detected in the 1/Ct values of genes in the autophagy pathway. The Ct value of the MUC1 gene was found to be higher only in the MCF-7 cell line after inoculation. In conclusion, it was observed that the specific expression pattern for influenza virus-induced autophagy was formed only in the A-549 cell line among the adenocarcinoma cells. It was thought that this relationship could constitute a dataset in further research on lung adenocarcinoma. However, in future studies, the determination of the expression of these genes at the protein level by using further tests will provide better comparison of the results.

Serum level of MicroRNA 15a and interleukin 6 as biomarkers for sepsis in critically Ill Patients

This study intended to evaluate the diagnostic efficacy of serum interleukin-6 (IL-6) and serum expression level of microRNA 15a (miRNA 15a) in sepsis patients admitted to the medical intensive care unit (ICU), and to correlate the results with the outcome of patients. This observational case-control study, was done from January 2022 to June 2022, included 75 adult ICU patients ≥18 years old, divided into 2 groups. Group 1: included 38 adults, critically ill sepsis patients and Group 2 (control group), included 37 adults, non-sepsis patients admitted to the ICU. Venous blood was withdrawn from cases and controls under aseptic conditions for assessment of serum IL-6 level by ELISA and serum expression level of miRNA 15a by quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR). Patient demographic data in addition to their characteristics including site of infection, blood culture results, length of stay and outcome of patients were included in the study. miRNA 15a was significantly higher in the sepsis group with mean of 3.99±1.61 compared to the controls (1.03±0.03, p< 0.001) while serum IL-6 levels were higher in the sepsis patients compared to the controls, however the difference did not reach statistical significance (p= 0.92). No difference was found in IL-6 and miRNA 15a levels between surviving and non-surviving sepsis patients, shocked and non-shocked sepsis patients. In conclusion, both IL-6 and miRNA 15a were up-regulated in ICU patients with sepsis. Both biomarkers did not show a significant difference regarding the outcome of patients. miRNA 15a could be considered a highly specific and sensitive marker in differentiating sepsis cases from controls, so it could be used as a diagnostic rather than a prognostic biomarker for sepsis.

Reliability of RT-qPCR Pooling Method for COVID-19 Detection in Various Cycle Threshold Values

Background: Reverse Transcriptase Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) is a standard method to detect SARS-CoV-2, the cause of COVID-19 disease, albeit expensive for some laboratory settings. The pooling test is widely used for large-scale screening to speed up the turn-around time and reduce the cost of the RT-qPCR. However, the pooling test involves mixing a certain number of specimens which theoretically increases the possibility of false-negative results. This study aimed to evaluate the accuracy of the pooling test compared with the non-pooling test in different Ct values as a surrogate for viral load.Methods: RT-qPCR was performed in three groups of samples: non-pooling (individual samples), pooling of 5 samples and 11 samples, with various ranges of Ct value in the respective group: x<25 (n=4); 25<x<30 (n=5), x<30 (n=16), and negative sample (n=5). Agreement and kappa values were calculated. Four of twenty-five individual samples resulted in false-negative after pooling.Results: By taking all samples without applying the cut-off value to the calculation, the agreement in pooling of 5 samples was 0.86 (Kappa 0.31) and of 11 samples was 0.64 (Kappa 0.96). When the cut-off value of Ct<37 was applied, percent agreement and kappa were 1.00, respectively, for both pooling methods.Conclusions: Pooling up to 11 samples shows high concordance with RT-qPCR with individual samples with Ct<37. Interpreting pooling results in a very low viral load (Ct≥37) must be considered due to the increased possibility of inconclusive results.

Ellagic acid inhibits the formation of hypertrophic scars by suppressing TGF-β/Smad signaling pathway activity.

Hypertrophic scar (HS) is a benign fibroproliferative skin disease, which lacks the ideal treatment and drugs. Ellagic acid (EA) is a natural polyphenol that prevents fibroblasts from proliferating and migrating. This study aimed to determine the role of EA in HS formation and its possible mechanism by in vitro experiments. HS fibroblasts (HSFs) and normal fibroblasts (NFs) were separated from HS tissue and normal skin tissue, respectively. HSFs were treated with 10 and 50 μM EA to assess their effect on HS formation. In particular, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and scratch assay were used to detect the viability and migration ability of HSFs. Quantitative reverse transcriptase real-time polymerase chain reaction was used to measure the mRNA expression level of basic fibroblast growth factor (bFGF), extracellular matrix (ECM)-related gene collagen-I (COL-I), and fibronectin 1 (FN1) in HSFs. Finally, Western blot was utilized to measure the expression level of TGF-β/Smad signaling pathway-related proteins in HSFs. The viability of HSFs was significantly increased compared with NFs. 10 and 50 μM EA treatment markedly inhibition the cell viability and migration of HSFs. EA treatment upregulated the bFGF expression level and downregulated the COL-I and FN1 expression level in HSFs. In addition, p-Smad2, p-Smad3, and transforming growth factor (TGF)-β1 expression levels as well as p-Smad2/Smad2 and p-Smad3/Smad3 ratios remarkably decreased in HSFs after EA treatment. EA inhibited the formation of HSs by suppressing the viability and migration of HSFs and ECM deposition as well as by preventing the activation of TGF-β/Smad signaling.

Optimization of mRNA extraction from human nasal mucosa biopsies for gene expression profile analysis by qRT-PCR.

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is the gold-standard method for analyzing modifications in gene expression in cells and tissues. However, large quantities of high-quality RNA samples are needed for analyzing the expression of multiple genes from one human tissue sample. Here, we provide an optimized protocol for extracting large amounts of RNA from human nasal mucosal biopsies. The quality and quantity of samples were sufficient for qRT-PCR analyses of the expressions of various genes, in duplicate. In contrast to other protocols, we optimized RNA isolation to increase the amount from nasal biopsy samples for analyses of multiple genes. In most previous publications, expressions of only one or a few genes, including housekeeping genes, were analyzed because the amount of biological material was small. We were able to improve our protocol with respect to the yield and quality of RNA. This is likely to produce better results from molecular analyses of very small biopsy samples of human nasal mucosa.

Antifungal drug susceptibility profiles and molecular mechanisms of azole resistance in Candida blood stream isolates

PurposeThe purpose of this study was to determine the activity of fluconazole, voriconazole, posaconazole, amphotericin B, micafungin and caspofungin against Candida blood stream isolates and to investigate the molecular mechanisms of azole resistance in fluconazole resistant isolates. MethodsThe in vitro susceptibilities of Candida isolates to fluconazole, voriconazole, posaconazole, amphotericin B, micafungin and caspofungin were tested by E-test ERG11, CDR1, CDR2 and MDR1 genes expression of fluconazole resistant, fluconazole (S-DD) and fluconazole intermediate resistant isolates were investigated by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). ResultsC. albicans (41%) was the most frequently isolated fungal species from blood stream infections followed by C. parapsilosis (22%). All C. albicans isolates except one and all C. tropicalis isolates were found to be susceptible to fluconazole. Overall, 21% of C. glabrata isolates were resistant to fluconazole. None of the Candida isolates were found to be resistant to caspofungin except 2 ​C. glabrata isolates. Overexpression of ERG11 and CDR1 genes were detected in all fluconazole S-DD and fluconazole resistant C. glabrata isolates, while CDR2 overexpression was observed in 91% fluconazole S-DD and 67 of % fluconazole resistant C.glabrata isolates. The overexpression of MDR1, ERG11 and CDR1 genes were found 100%, 80% and 80%, respectively, in fluconazole resistant C. parapsilosis isolates. ConclusionMost of the Candida isolates were susceptible to posaconazole and caspofungin. Our data also highlighted that overexpression of efflux pump genes major cause of azole resistance.

The Impact of Prolonged and Intermittent Fasting on PGC-1α, Oct-4, and CK-19 Liver Gene Expression.

Liver stemness refers to the high regenerative capacity of the organ. This intrinsic regeneration capacity allows the restoration of post-resection liver function in up to 50% of liver donors. Liver cirrhosis is one of the terminal liver diseases with a defect in the intrinsic regeneration capacity. Several attempts to restore intrinsic regeneration capacity by conducting in vivo studies on stem cells in various organs have shown the positive impact of fasting on stemness. An increased capacity for stem cell proliferation and regeneration was reported due to fasting. Prolonged fasting (PF) has been reported to maintain the long-term proliferative ability of hematopoietic stem cells. However, clinical trials on intermittent fasting (IF) have not conclusively given positive results for fasting individuals. This research aims to investigate the effect of fasting on liver stemness by comparing the expression of octamer-binding transcription factor 4 (Oct-4), cytokeratin 19 (CK-19), and peroxisome proliferator-activated receptor γ co-activator α (PGC-1α) in liver cells of fasted rabbits with rabbits fed ad libitum. This study compares two types of fasting, which are intermittent (16 hours) and prolonged (40 hours) fasting, for liver stemness and intrinsic regenerative capacity. A total of 18 rabbits were conditioned into 3 different groups. The first group was subjected to an ad libitum diet, the second to intermittent fasting (16-hour fasting), and the third to prolonged fasting (40-hour fasting). Afterward, the RNA was extracted from the liver tissues of each rabbit and analyzed via real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Relative expression was calculated using the Livak method. Compared to the ad libitum diet, a greater increase was reported in PGC-1α, upregulated Oct4, and steady CK-19 gene expressions in the livers of intermittent fasting rabbits. Prolonged fasting increased PGC1α, reduced liver stemness, and a statistically insignificant decrease in intrinsic liver regenerative capacity. Intermittent fasting indicates preferable molecular alterations in liver stemness and intrinsic regenerative capacity compared to prolonged fasting.

Cinnamaldehyde Induces the Expression of MicroRNA-1285-5p and MicroRNA-140-5p in Chondrocytes to Ameliorate the Apoptosis and Inflammatory Response.

Cinnamaldehyde (CA) is an active ingredient of Wenyang Tongluo capsule. This study was performed to investigate the function of CA on human chondrocytes. Different doses of CA were used to treat C28/I2 cells, which were stimulated by interleukin-1β (IL-1β), and then the viability and apoptosis of the cells were examined by cell counting kit-8 and flow cytometry. Interleukin-6 (IL-6), interleukin-20 (IL-20), and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay. Quantitative real-time reverse transcriptase polymerase chain reaction was performed to measure miR-1285-5p, miR-140-5p, IL-20, and high-mobility group box 1 (HMGB1) messenger RNA (mRNA) expression. Western blot assay was performed to detect IL-20, HMGB1, IKBα, phospho-IKBα, IKKα/β, and phospho-IKKα/β expression. Moreover, the relationships between miR-1285-5p and IL-20, as well as miR-140-5p and HMGB1, were validated by dual-luciferase reporter assay. CA promoted the viability and inhibited the apoptosis of C28/I2 cells stimulated by IL-1β and repressed IL-6, IL-20, and TNF-α levels. CA increased miR-1285-5p and miR-140-5p expression levels. MiR-1285-5p and miR-140-5p promoted the viability and inhibited the apoptosis and inflammation of C28/I2 cells. IL-20 was a target gene of miR-1285-5p, and HMGB1 was a target gene of miR-140-5p. Overexpression of IL-20 or HMGB1 could reverse the effect of CA on C28/I2 cells treated with IL-1β. In addition, HMGB1 increased phospho-IKBα and phospho-IKKα/β expression in IL-1β- and CA-treated C28/I2 cells. CA protects chondrocytes via regulating miR-1285-5p/IL-20 axis and miR-140-5p/HMGB1/nuclear factor kappa B pathway.

Identification of reliable reference genes for gene expression studies in mouse models under microplastics stress

Microplastics (MPs) and nanoplastics (NPs) have been deemed to be newly emerged contaminants interfering with various physiological processes closely related with gene expression alteration. Reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) serves as a powerful tool to assess gene expression, however highly dependent on a reliable reference gene. Therefore, it is necessary to identify stable reference genes for gene expression study under MP or NP stress. We constructed a mouse model postexposure to polypropylene microplastics (PP-MPs) to assess PP-MPs bioaccumulation in kidney, evaluate the kidney pathological changes, and then explore potential reference genes via RT-qPCR. Although the hematoxylin-eosin staining showed no obvious damage in kidney tissues, we observed significant PP-MPs accumulation in kidney using Raman spectra analysis supported by spectral multivariate analysis. The expression of 19 candidate reference genes were examined, including the commonly used ones of β-actin, glyceraldehyde 3-phosphate dehydrogenase (Gapdh), Cytochrome c oxidase subunit 4I1 (Cox4i), Histocompatibility 13 (H13) and ribosomal protein. Their expression stability and reliability were assessed by the combination of four algorithms including geNorm, NormFinder, BestKeeper and Delta Cq. The geNorm analysis revealed that the top three genes with the lowest variability were Cox4il, Rps9 and Gapdh, whereas NormFinder results ranked Rps3, Cox4il and Rps18 as the top three ones. Rpl15, Cox4i1 and Rps3 were the most reliable reference genes in BestKeeper results, and Delta Cq proposed Rps3 and Cox4il as the stable genes. The overall ranking indicated by GMR value gave the five most stable reference genes (Cox4i1, Rps3, Rps9, Rps18 and Gapdh). Three genes associated with different biochemical processes (Atp5f1, Crebbp and Dele1) were chosen to verify the characterized reference genes using the least stable gene as a control, exhibiting different expression profiles and implying the essentiality to select the reliable reference genes. Our results documented the expression fluctuations of acknowledged reference gene (Ubc) and proposed a set of reliable reference genes for future studies of gene expression profiles in MP treated mouse models.

FN1 Promotes Thyroid Carcinoma Cell Proliferation and Metastasis by Activating the NF-Κb Pathway.

Thyroid cancer (THCA) is a common endocrine tumor. This study aims to identify the THCA-related key gene Fibronectin 1 (FN1) by bioinformatics methods and explore its function and regulatory mechanism. Gene Expression Omnibus database (GSE3678, GSE33630, and GSE53157 datasets) was searched for the analysis of differentially expressed genes (DEGs) in THCA tissues v.s. (normal tissues). The enrichment of DEGs was investigated by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways using the DAVID database. Screening the hub gene was performed with the STRING database and Cytoscape software. The expression and survival analyses of these hub genes in THCA were studied with the Gene Expression Profiling Interactive Analysis database. LinkedOmics database was searched for the related signaling pathways regulated by FN1 in THCA. Real-time quantitative reverse transcriptase polymerase chain reaction was adopted to detect the mRNA expression of Fibromodulin, microfibril-associated protein 4, Osteoglycin, and FN1. The cell viability, growth, migration and aggressiveness were examined by Cell counting kit-8, 5-Ethynyl-2 '- deoxyuridine assay, scratch assay, and Transwell assay. The expression levels of NF-κB signaling pathway-related proteins (p-IκB-α, p-IKK-β, NF-κB p65) were detected by Western blot. FN1 mRNA was up-regulated in THCA tissues and cell lines (MDA-T85 and MDA-T41). The high expression of FN1 is relevant to larger tumor diameters and lymph node metastasis in sufferers with THCA. Functional experiments showed that overexpression of FN1 in the MDA-T85 cell line promoted growth, migration and aggressiveness; knockdown of FN1 in MDA-T41 cells inhibited these malignant behaviors. In mechanism, FN1 promoted the expression levels of proteins related with NF-κB signaling pathway and activated NF-κB signaling pathway. FN1 is up-regulated in THCA and facilitates cell growth, migration and invasion by activating the NF-κB signaling pathway. FN1 will be a promising biomarker of THCA and may become a molecular target for THCA treatment.

An Estimate of Time-Dependent Transmission of COVID-19 Pandemic in Katsina State from June to November 2021

This paper investigates the spread of COVID-19 pandemic in Katsina state from 23rd June to 27th November, 2021, using graphical tools to visually analyse infection over time and time-dependent reproduction number to quantify the transmissibility of the disease. Study data consist of diagnostic results of reverse transcriptase real-time quantitative polymerase chain reaction assays (RT-PCR) on nine hundred and eighty-nine nasal swab samples from suspected COVID-19 cases, collected from public and private health facilities across Local government areas of the State and analysed by the Molecular Laboratory of the Federal Medical Centre Katsina, recording a total of 137 positive cases. Our investigation revealed that, over the study period, COVID-19 transmission reaches its peak on 17th October 2021 (Epidemic Week 41), with a mean Reproduction number (Rt) of 3.22 and a standard deviation of 0.5976 (95% CI: 2.6193 - 3.8145), culminating in 19 new infections on Wednesday the 27th (Epidemic Week 43). The combined age groups 21-30 and 31-40 constituted the most affected cases with 29 (21.17%) and 19 (13.87%) positive cases, respectively. This was closely followed by age group 51-60 with 19 (13.87%) positive cases. On gender basis, 70 (51%) females tested positive compared to 67 (49%) males. We therefore conclude that for the third wave of COVID-19 pandemic in Katsina State, disease transmission reaches its peak in Epidemic Week 41 in October, then rapidly diminishes to Rt values of less than one, indicating that no new infections were expected by the end of November, with female gender and age groups in the range 21 to 40 years being most infected. We recommend that mitigation strategies that take into consideration features inherent in female gender and more pronounced in young (21 to 40 years) and middle-aged (51-60 years) adults should be adopted in case of future waves.

Clinical and virological characteristics of viral shedding in children with norovirus gastroenteritis.

The correlation between the clinical manifestations and fecal viral load of norovirus (NoV) infection remains unknown. We established a SYBR® Green-based real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) method to quantify NoV and then sequenced its genomes from the feces of patients admitted at the Chang Gung Memorial Hospital from 2017 to 2018. NoV GII.4 Sydney (n=21, 36.2%) and GII.P16-GII.2 (n=19, 32.8%), the two predominant genotypes found among 58 isolates, were closely related to the Taiwan variant 2012a cluster in the VP1 region and genotypes of China strain. An increase in viral load could be observed on Day 3 following the onset of NoV infection. The viral load then declined rapidly from days 10-15 but remained high for >1 month in a severe combined immunodeficiency patient. Significantly longer shedding was found in patients with fever (p=0.03) or infected by the GII.4 Sydney strain (p<0.01). The qRT-PCR-mediated method proposed in this work could quantify the viral load in patients with NoV infection. Significant viral shedding over a period of 2 weeks in children with acute gastroenteritis and >1 month in an immunodeficient patient was observed. Significantly longer shedding could be correlated with infection by the GII.4 Sydney strain and febrile patients.

PD36 Outcomes Of Centenaries Hospitalized Due To COVID-19 In A Private Healthcare System

IntroductionLife expectancy is increasing worldwide. However, during the COVID-19 pandemic, people 100 years or more (centenaries) were challenged by a potentially fatal disease. We evaluated the outcome of centenaries hospitalized due to COVID-19 in a private healthcare system of Belo Horizonte/Brazil (Unimed-BH).MethodsAdministrative data were collected from the hospital database. Patients were included if they had a severe adult respiratory syndrome due to coronavirus type 2 (SARS-CoV-2) ribonucleic acid identified by quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) or by the International Code of Disease-10th review (ICD-10) hospitalization codes U07.1, B34.2, or B97.2.ResultsFrom March 1 2020 to October 31 2021, 316.4 ± 12.9 centenaries/month were registered. Eighteen hospitalizations due to COVID-19 were identified. Median age was 101.8 years (inter-quartile range [IQR]:100.7,103.0). Most patients were female (83%). There was a median of 6.0 morbidities per patient (IQR:5.3,7.8), range 2-12 morbidities, among 71 possible morbidities. The most described morbidities were systemic arterial hypertension (94%), dementia (61%), and congestive heart failure (61%). Median length of hospitalization was 6.5 days (IQR:3.3,8.0). No patient was dialyzed. Seven (39%) patients died during hospitalization, of whom 3 (17%) were admitted to the Intensive Care Unit and 2 (11%) were oxygenated by invasive mechanical ventilation. No other patients were admitted to the Intensive Care Unit or invasively mechanically ventilated.ConclusionsAlthough the hospitalization rate was low, the mortality rate during hospitalization was high among centenaries. Further research is required to evaluate the actual risks of centenaries to be infected by SARS-CoV-2 and the subsequent outcomes.