Dopamine alters phage morphology to exert an anti-infection effect.

Antiviral drug development is important for human health, and the emergence of novel COVID-19 variants has seriously affected human lives and safety. A bacteriophage-a bacterial virus with a small and simple structure-is an ideal experimental candidate for studying the interactions between viruses and their hosts. In this study, the effects and mechanisms of catecholamines on phages were explored, and dopamine (DA) was found to have general and efficient anti-infection effects. A clear dose-dependent effect was observed when different phages were treated with DA, with higher DA concentrations exhibiting stronger anti-phage activity. The half maximal inhibitory concentration values of DA for vB-EcoS-IME167, T4 Phage, and VMY22 were determined as 0.26, 0.12, and 0.73 mg mL-1, respectively. The anti-phage effect of DA increased with treatment duration. In addition, the anti-infection activities of DA against vB-EcoS-IME167, T4 Phage, and VMY22 were increased by 105, 104, and 104 folds compared to that of the control. This ability of DA was observed only in phages and not in the host bacteria. Morphological changes of phages were observed under transmission electron microscopy following their treatment with DA, and considerable changes in adsorption were confirmed via quantitative reverse transcription polymerase chain reaction. These results suggest that the anti-phage effect of DA is primarily due to the destruction of the external structure of the phage. This study, to the best of our knowledge, is the first to report the universal anti-phage infection effect of dopamine, which provides novel information regarding DA and forms a basis for further research and development of antiviral drugs. Moreover, it provides a new perspective for the research about the defense and counter-defense of bacteria and bacteriophages.

Development of a Quadruplex RT-qPCR for the Detection of Porcine Rotaviruses and the Phylogenetic Analysis of Porcine RVH in China.

Rotavirus A species (RVA), RVB, RVC, and RVH are four species of rotaviruses (RVs) that are prevalent in pig herds, and co-infections occur frequently. In this study, a quadruplex real-time quantitative RT-PCR (RT-qPCR) for the simultaneous detection of four porcine RVs was developed by designing specific primers and probes based on the VP6 gene of RVA, RVB, RVC, and RVH, respectively. The method showed high specificity and could only detect RVA, RVB, RVC, and RVH, without cross-reaction with other porcine viruses; showed excellent sensitivity, with a limit of detection (LOD) of 1.5 copies/µL for each virus; showed good repeatability, with intra-assay coefficients of variation (CVs) of 0.15-1.14% and inter-assay CVs of 0.07-0.96%. A total of 1447 clinical fecal samples from Guangxi province in China were tested using the developed quadruplex RT-qPCR. The results showed that RVA (42.71%, 618/1447), RVB (26.95%, 390/1447), RVC (42.92%, 621/1447), and RVH (13.68%, 198/1447) were simultaneously circulating in the pig herds, and the co-infection rate of different species of rotaviruses was found to be up to 44.01% (579/1447). The clinical samples were also detected using one previously reported method, and the coincidence rate of the detection results using two methods was more than 99.65%. The phylogenetic tree based on the VP6 gene sequences of RVH revealed that the porcine RVH strains from Guangxi province belonged to the genotype I5, which was closely related to Japanese and Vietnamese strains. In summary, an efficient, sensitive, and accurate method for the detection and differentiation of RVA, RVB, RVC, and RVH was developed and applied to investigate the prevalence of porcine RVs in Guangxi province, China. This study is the first to report the prevalence of porcine RVH in China.

A Revised Diagnostic Quantitative RT-PCR for the Detection of Nipah Virus Infection.

From its discovery in Malaysia in the late 1990s, the spillover of the Nipah virus from its pteropid reservoir into the human population has resulted in sporadic outbreaks of fatal encephalitis and respiratory disease. In this chapter, we revise a previously described quantitative reverse transcription polymerase chain reaction method, which now utilizes degenerate nucleotides at certain positions in the probe and the reverse primer to accommodate the sequence heterogeneity observed within the Nipah henipavirus species.

Blood Plasma Small Non-Coding RNAs as Diagnostic Molecules for the Progesterone-Receptor-Negative Phenotype of Serous Ovarian Tumors.

The expression level of the progesterone receptor (PGR) plays a crucial role in determining the biological characteristics of serous ovarian carcinoma. Low PGR expression is associated with chemoresistance and a poorer outcome. In this study, our objective was to explore the relationship between tumor progesterone receptor levels and RNA profiles (miRNAs, piwiRNAs, and mRNAs) to understand their biological characteristics and behavior. To achieve this, we employed next-generation sequencing of small non-coding RNAs, quantitative RT-PCR, and immunohistochemistry to analyze both FFPE and frozen tumor samples, as well as blood plasma from patients with benign cystadenoma (BSC), serous borderline tumor (SBT), low-grade serous ovarian carcinoma (LGSOC), and high-grade serous ovarian carcinoma (HGSOC). Our findings revealed significant upregulation of MMP7 and MUC16, along with downregulation of PGR, in LGSOC and HGSOC compared to BSC. We observed significant correlations of PGR expression levels in tumor tissue with the contents of miR-199a-5p, miR-214-3p, miR-424-3p, miR-424-5p, and miR-125b-5p, which potentially target MUC16, MMP7, and MMP9, as well as with the tissue content of miR-16-5p, miR-17-5p, miR-20a-5p, and miR-93-5p, which are associated with the epithelial-mesenchymal transition (EMT) of cells. The levels of EMT-associated miRNAs were significantly correlated with the content of hsa_piR_022437, hsa_piR_009295, hsa_piR_020813, hsa_piR_004307, and hsa_piR_019914 in tumor tissues. We developed two optimal logistic regression models using the quantitation of hsa_piR_020813, miR-16-5p, and hsa_piR_022437 or hsa_piR_004307, hsa_piR_019914, and miR-93-5p in the tumor tissue, which exhibited a significant ability to diagnose the PGR-negative tumor phenotype with 93% sensitivity. Of particular interest, the blood plasma levels of miR-16-5p and hsa_piR_022437 could be used to diagnose the PGR-negative tumor phenotype with 86% sensitivity even before surgery and chemotherapy. This knowledge can help in choosing the most effective treatment strategy for this aggressive type of ovarian cancer, such as neoadjuvant chemotherapy followed by cytoreduction in combination with hyperthermic intraperitoneal chemotherapy and targeted therapy, thus enhancing the treatment's effectiveness and the patient's longevity.

Flow Cytometry Identification of Cell Compartments in the Murine Brain.

Glioma can be modelled in the murine brain through the induction of genetically engineered mouse models or intracranial transplantation. Gliomas (oligodendroglioma and astrocytoma) are thought to arise from neuronal and glial progenitor populations in the brain and are poorly infiltrated by immune cells. An improved understanding of oligodendrocytes, astrocytes, and the immune environment throughout tumor development will enhance the analysis and development of brain cancer models. Here, we describe the isolation and analysis of murine brain cell types using a combination of flow cytometry and quantitative RT-PCR strategies to analyze these individual cell populations in vivo.

Development of a Crystal Digital RT-PCR for the Detection of Atypical Porcine Pestivirus.

Atypical porcine pestivirus (APPV), a newly discovered virus, is associated with the type A-II congenital tremor (CT) in neonatal piglets. APPV distributes throughout the world and causes certain economic losses to the swine industry. The specific primers and probe were designed targeting the 5' untranslated region (UTR) of APPV to amplify a 90 bp fragment, and the recombinant standard plasmid was constructed. After optimizing the concentrations of primers and probe, annealing temperature, and reaction cycles, a crystal digital RT-PCR (cdRT-PCR) and real-time quantitative RT-PCR (qRT-PCR) were successfully established. The results showed that the standard curves of the qRT-PCR and the cdRT-PCR had R2 values of 0.999 and 0.9998, respectively. Both methods could specifically detect APPV, and no amplification signal was obtained from other swine viruses. The limit of detection (LOD) of the cdRT-PCR was 0.1 copies/µL, and that of the qRT-PCR was 10 copies/µL. The intra-assay and inter-assay coefficients of variation of repeatability and reproducibility were less than 0.90% for the qRT-PCR and less than 5.27% for the cdRT-PCR. The 60 clinical tissue samples were analyzed using both methods, and the positivity rates of APPV were 23.33% by the qRT-PCR and 25% by the cdRT-PCR, with a coincidence rate of 98.33%. The results indicated that the cdRT-PCR and the qRT-PCR developed here are highly specific, sensitive methods for the rapid and accurate detection of APPV.

Claudin-10 is a new candidate prognostic marker in metastatic high-grade serous carcinoma.

The objective of this study was to analyze the expression and prognostic role of the tight junction protein claudin-10 in high-grade serous carcinoma (HGSC). Claudin-10 protein expression by immunohistochemistry was analyzed in 588 HGSC (414 effusions, 174 surgical specimens). Expression in mesotheliomas (n = 97; 47 effusions, 50 surgical specimens) was studied for comparative purposes. CLDN10 mRNA expression by quantitative RT-PCR (qRT-PCR) was analyzed in 40 HGSC effusions. Claudin-10 protein expression was found in 360/588 (61%) HGSC vs. 19/97 (20%) mesotheliomas (p < 0.001), and was higher in HGSC surgical specimens compared to effusions (p < 0.001). qRT-PCR confirmed the presence of CLDN10 mRNA in HGSC effusions. High (> 25%) claudin-10 expression in HGSC effusions was significantly associated with shorter overall survival (OS; p = 0.036) and progression-free survival (PFS; p = 0.045) in univariate analysis, and was an independent prognosticator of OS in multivariate analysis (p = 0.045). In conclusion, claudin-10 protein expression is higher in HGSC compared to mesothelioma, although the diagnostic power of this marker appear to be lesser than other claudin family members. Claudin-10 expression in HGSC effusions is marker of more aggressive disease.

Quantifying red complex bacteria, oral hygiene condition, and inflammation status in elderly: A pilot study.

Introduction: Periodontitis is an inflammation of the periodontal apparatus leads to destruction of connective tissue attachment and tooth loss. Red complex bacteria may contribute to disease initiation. Bacterial infection in periodontitis leads to a low-grade chronic infection and inflammation in distant organs. Notably, aging can affect the immune response. Objectives: The aim of this study was to analyze the effect of aging on oral hygiene and inflammation condition. Moreover, to evaluate the correlation between the oral hygiene condition and red complex bacterial load in subgingival plaque. Materials and methods: In this cross-sectional study, we examined 20 adult and 20 elderly subjects with periodontitis. Clinical parameters included Oral Hygiene Index Simplified (OHI-S) and Papillary Bleeding Index (PBI) were recorded. Subgingival plaque was collected from the tooth with a probing depth of 5-7 mm and analyzed with a reverse transcription polymerase chain reaction (RT-PCR) for red complex bacteria quantification. Statistical analysis was performed, respectively. Results: Both groups had poor oral hygiene conditions, reflected by high OHI-S and PBI. The quantity of red complex bacteria (P. gingivalis, T. denticola, T. forsythia) in the elderly group was significantly higher in comparison to the adult group. There was significant strong linear relationship between OHI-S and red complex bacteria (r < 1, p < 0.05). Only P. gingivalis bacteria with PBI values had a strong linear relationship and statistically significant. (r < 1, p < 0.05). P. gingivalis load was significantly higher than T. denticola and T. forsythia load, and it correlated with poor oral hygiene in the adult and elderly groups and with PBI in the elderly group. Conclusions: Aging affects to the red complex bacterial load and oral hygiene condition, but not the inflammation. These findings contribute to the development of novel treatment strategies focusing on bacterial aspect for periodontitis in the elderly.

Development of a Multiplex Crystal Digital RT-PCR for Differential Detection of Classical, Highly Pathogenic, and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) type 1 (European genotype) and PRRSV type 2 (North American genotype) are prevalent all over the world. Nowadays, the North American genotype PRRSV (NA-PRRSV) has been widely circulating in China and has caused huge economic losses to the pig industry. In recent years, classical PRRSV (C-PRRSV), highly pathogenic PRRSV (HP-PRRSV), and NADC30-like PRRSV (NL-PRRSV) have been the most common circulating strains in China. In order to accurately differentiate the circulating strains of NA-PRRSV, three pairs of specific primers and corresponding probes were designed for the Nsp2 region of C-PRRSV, HP-PRRSV, and NL-PRRSV. After optimizing the annealing temperature, primer concentration, and probe concentration, a multiplex real-time quantitative RT-PCR (qRT-PCR) and a multiplex Crystal digital RT-PCR (cdRT-PCR) for the differential detection of C-PRRSV, HP-PRRSV, and NL-PRRSV were developed. The results showed that the two assays illustrated high sensitivity, with a limit of detection (LOD) of 3.20 × 100 copies/µL for the multiplex qRT-PCR and 3.20 × 10-1 copies/µL for the multiplex cdRT-PCR. Both assays specifically detected the targeted viruses, without cross-reaction with other swine viruses, and indicated excellent repeatability, with coefficients of variation (CVs) of less than 1.26% for the multiplex qRT-PCR and 2.68% for the multiplex cdRT-PCR. Then, a total of 320 clinical samples were used to evaluate the application of these assays, and the positive rates of C-PRRSV, HP-PRRSV, and NL-PRRSV by the multiplex qRT-PCR were 1.88%, 21.56%, and 9.69%, respectively, while the positive rates by the multiplex cdRT-PCR were 2.19%, 25.31%, and 11.56%, respectively. The high sensitivity, strong specificity, excellent repeatability, and reliability of these assays indicate that they could provide useful tools for the simultaneous and differential detection of the circulating strains of C-PRRSV, HP-PRRSV, and NL-PRRSV in the field.

An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5.

A duplex real-time quantitative reverse transcription-polymerase chain reaction (dqRT-PCR) assay was successfully developed to simultaneously detect canine parainfluenza virus 5 (CPIV5) and a canine endogenous internal positive control (EIPC) in canine clinical samples. Two sets of primers and probes for the CPIV5 L and canine 16S rRNA genes were included in the dqRT-PCR assay to detect CPIV and monitor invalid results throughout the qRT-PCR process. The developed dqRT-PCR assay specifically detected CPIV5 but no other canine pathogens. Furthermore, 16S rRNA was stably amplified by dqRT-PCR assay in all samples containing canine cellular materials. The assay's sensitivity was determined as below ten RNA copies per reaction, with CPIV5 L gene standard RNA and 1 TCID50/mL with the CPIV5 D008 vaccine strain, which was 10-fold higher than that of the previous HN gene-specific qRT-PCR (HN-qRT-PCR) assays and was equivalent to that of the previous N gene-specific qRT-PCR (N-qRT-PCR) assays, respectively. Moreover, the Ct values of the CPIV5-positive samples obtained using the dqRT-PCR assay were lower than those obtained using the previous HN- and N-qRT-PCR assays, indicating that the diagnostic performance of the dqRT-PCR assay was superior to those of previous HN- and N-qRT-PCR assays. The calculated Cohen's kappa coefficient values (95% confidence interval) between dqRT-PCR and the HN- or N-specific qRT-PCR assays were 0.97 (0.90-1.03) or 1.00 (1.00-1.00), respectively. In conclusion, the newly developed dqRT-PCR assay with high sensitivity, specificity, and reliability will be a promising diagnostic tool for the detection of CPIV5 in clinical samples and useful for etiological and epidemiological studies of CPIV5 infection in dogs.

Unhatched bovine blastocysts express all transcripts of the estrogen biosynthetic pathway, but steroid hormone synthesis could not yet be demonstrated.

Early embryos of rodent species and rabbits but also farm animals such as pigs, horses and cattle produce estrogens, which are considered important regulators of the implantation process. In cattle, the exact stage at which embryonic estrogen synthesis commences is yet unknown. However, this information is regarded as important to consider a possible role of embryonic estrogens in preimplantation development. Therefore, in this study, we first used quantitative reverse transcription PCR to examine the mRNA expression of the enzymes required for the conversion of cholesterol into free and sulfonated estrogens (CYP11A1, CYP17A1, HSD3B, CYP19A1, and SULT1E1), the cholesterol carrier protein STAR, and the estrogen receptors ESR1 and ESR2 in in vitro produced morulae and unhatched blastocysts (d 6-9). Only in the blastocysts, were the mRNAs of the entire estrogen biosynthesis chain and of both estrogen receptors clearly present, whereas mRNA specific to ESRs was already detectable in the morulae. We also examined the expression of the corresponding enzymes in blastocysts at the protein level. None of the enzymes were detectable by capillary-based western analysis. Immunofluorescence methods were established for the detection of CYP17A1, CYP19A1, and SULT1E1. CYP17A1 was observed in the inner cell mass and trophectoderm, whereas CYP19A1 and SULT1E1 were present only in trophectoderm. An attempt to detect estrogen sulfotransferase activity was unsuccessful. Despite clear evidence that some elements of the estrogen biosynthetic pathway are also present at the protein level, it remains to be clarified whether the enzyme cascade underlying estrogen production is already functional in unhatched blastocysts.

Analysis of the Function of CCN2 in Tubular Epithelium Cells with a Focus on Renal Fibrogenesis.

Renal interstitial fibrosis is the final common pathway in the process of all kidney diseases, and it results in chronic kidney disease. CCN2 is an important factor in the pathogenesis of renal interstitial fibrosis, and analysis of its function can lead to treatments for chronic kidney disease. Since CCN2 knockout mice are developmentally lethal, generation of conditional knockout mice is essential for in vivo analysis. Since CCN2 is expressed in a variety of cells in the kidney, including podocytes, mesangial cells, pericytes, and tubular epithelial cells, it is necessary to perform cell-specific verification of the cells that play a central role in fibrosis. However, cell-specific validation using the Cre/loxP system in vivo has only been performed in mesangial cells. In our research program, we are focusing on the role of CCN2 in tubular epithelial cells in renal fibrogenesis. In this report, we introduce the creation of a tubular epithelial cell-specific knockout model and method of its analysis.

Establishment of qRT-PCR for absolute quantitative detection of Chikungunya virus / 中国热带医学

@#Abstract: Objective　To develop a real-time fluorescent quantitative RT-PCR (qRT-PCR) method for qualitative and quantitative Chikungunya virus (CHIKV) analysis. Methods Based on the systematic analysis of the genomic sequences of Chikungunya and its related arboviruses, the specific nucleic acid sequences for Chikungunya virus were screened and identified, and then the primers and TaqMan probe were designed. Meanwhile, the human GAPDH gene was used as an internal reference. The reaction system for qRT-PCR was systematically optimized by L9(34) orthogonal design, and a rapid detection method for Chikungunya by qRT-PCR based on TaqMan probe methods was established. The sensitivity, specificity, reproducibility, and coverage of the established method were analyzed in detail. The standard curve was made, and the absolute quantitative method was established using the cloned nucleic acid fragments as positive samples. Results　A real-time fluorescent quantitative RT-PCR assay was developed for the qualitative and quantitative analysis of Chikungunya virus. The reaction system included Chikungunya virus and reference internal gene specific primers and probe, RT/Taq enzyme mixture, reaction buffer, and negative and positive reference. The established method obtained positive results with the ROSS strain of ECSA subtype, LR2006 strain of IOL branch, 181/25 strain of Asian type and Dongguan 2010 epidemic strains of Chikungunya virus, but there was no cross-reaction with other 18 arboviruses belonging to Flaviviruses, Alphaviruses and Bunyavirus. The minimum detection limit of the established method was 5.80 copies/mL, and a linear relationship was observed between the amount of input plasmid DNA and fluorescence signal value over a range of 5.80×102 copies/mL to 5.80×1010 copies/mL, and the correlation coefficient was 0.999 5. The qRT-PCR amplification efficiency was 91%, and the intra-assay variations and inter-assay variations were 0.01-0.07 and 0.03-0.11, respectively. Conclusions　The TaqMan qRT-PCR method developed in this study can qualitatively and quantitatively detect Chikungunya virus rapidly with specificity and sensitivity, providing a technical method for the prevention and control of this viral disease.

COVID-19 surveillance via wastewater:Introduction of wastewater-based epidemiology / 西安交通大学学报(医学版)

Since the outbreak of the COVID-19 global pandemic in 2019, monitoring COVID-19 infection status and trend through wastewater, known as wastewater-based epidemiology (WBE), has been widely used in many countries and regions. WBE consists of five steps:wastewater sample collection, viral concentration, viral nucleic acid extraction, quantification of virus using quantitative RT-PCR, and dissemination of the wastewater surveillance results. This method could be used for early warning of COVID-19 outbreak in a population, monitoring COVID-19 distributions and epidemic trend, prediction of COVID-19 prevalence rate, understanding of temporal trend of SARS-COV-2 variants, and simultaneous surveillance of multiple pathogens. WBE and clinical surveillance can be used concurrently and the former is a good complement to the latter.

Search for New Participants in the Pathogenesis of High-Grade Serous Ovarian Cancer with the Potential to Be Used as Diagnostic Molecules.

Recent studies have attempted to develop molecular signatures of epithelial ovarian cancer (EOC) based on the quantitation of protein-coding and non-coding RNAs to predict disease prognosis. Due to the heterogeneity of EOC, none of the developed prognostic signatures were directly applied in clinical practice. Our work focuses on high-grade serous ovarian carcinoma (HGSOC) due to the highest mortality rate relative to other types of EOC. Using deep sequencing of small non-coding RNAs in combination with quantitative real-time PCR, we confirm the dualistic classification of epithelial ovarian cancers based on the miRNA signature of HGSOC (type 2), which differs from benign cystadenoma and borderline cystadenoma-precursors of low-grade serous ovarian carcinoma (type 1)-and identified two subtypes of HGSOC, which significantly differ in the level of expression of the progesterone receptor in the tumor tissue, the secretion of miR-16-5p, miR-17-5p, miR-93-5p, miR-20a-5p, the level of serum CA125, tumor size, surgical outcome (optimal or suboptimal cytoreduction), and response to chemotherapy. It was found that the combined determination of the level of miR-16-5p, miR-17-5p, miR-20a-5p, and miR-93-5p circulating in blood plasma of patients with primary HGSOC tumors makes it possible to predict optimal cytoreduction with 80.1% sensitivity and 70% specificity (p = 0.022, TPR = 0.8, FPR = 0.3), as well as complete response to adjuvant chemotherapy with 77.8% sensitivity and 90.9% specificity (p = 0.001, TPR = 0.78, FPR = 0.09). After the additional verification of the obtained data in a larger HGSOC patient cohort, the combined quantification of these four miRNAs is proposed to be used as a criterion for selecting patients either for primary cytoreduction or neoadjuvant chemotherapy followed by interval cytoreduction.

Evaluation of Rapid Lateral-Flow Tests Directed against the SARS-CoV-2 Nucleoprotein Using Viral Suspensions Belonging to Different Lineages of SARS-CoV-2.

Within the successive waves that occurred during the SARS-CoV-2 pandemic, recommendations arose to test symptomatic and contact subjects by using rapid antigen devices directed against the viral nucleocapsid protein with the aim to isolate contagious patients without delay. The objective of this study was to evaluate the ability of four rapid lateral-flow tests (RLFT) that were commercially available on the French market in 2022 to recognize various strains of SARS-CoV-2. Series of five-fold dilutions of seven viral suspensions belonging to different lineages of SARS-CoV-2 (19A, 20A, Alpha, Beta, Gamma, Delta and Omicron) were used to evaluate the analytical sensitivity of four commercially available RLFTs (manufacturers: Abbott, AAZ, Becton-Dickinson and Biospeedia). Cell culture and quantitative RT-PCR were used as references. Excellent correlations were observed for each lineage strain between the viral titer obtained via cell culture and the number of RNA copies measured by quantitative RT-PCR. Although the four tests were able to recognize all the tested variants, significant differences in terms of sensitivity were observed between the four RLFTs. Despite the limitation represented by the small number of devices and clinical isolates that were tested, this study contributed by rapidly comparing the sensitivity of SARS-CoV-2 RLFTs in the Omicron era.

Novel RT-PCR Using Sugar Chain-Immobilized Gold-Nanoparticles Correlates Patients' Symptoms: The Follow-Up Study of COVID-19 Hospitalized Patients.

Background: The transmissible capacity and toxicity of SARS-CoV-2 variants are continually changing. We report here the follow-up study of hospitalized COVID-19 patients from 2020 to 2022. It is known that the PCR diagnosis for hospitalized patients sometimes causes confusion because of the incompatibility between their diagnosis and symptoms. We applied our sugar chain-immobilized gold-nanoparticles for the extraction and partial purification of RNA from specimens for quantitative RT-PCR assay and evaluated whether the results correlate with patients' symptoms. Methods and Results: Saliva specimens were taken from hospitalized patients with mild or moderate symptoms every early morning. At the time of RT-PCR diagnosis, two methods for the extraction and partial purification of RNA from the specimen were performed: a commonly used Boom (Qiagen) method and our original sugar chain-immobilized gold nanoparticle (SGNP) method. For symptoms, body temperature and oxygen saturation (SpO2) of patients were monitored every 4 h. Conclusions: It was clear that patients infected with the Delta variant needed more time to recover than those with the Omicron variant, and that the SGNP method showed more realistic correlation with the symptoms of patients compared with the common Qiagen method.

Very low likelihood that cultivated oysters are a vehicle for SARS-CoV-2: 2021-2022 seasonal survey at supermarkets in Kyoto, Japan.

The pandemic caused by novel coronavirus disease of 2019 (COVID-19) is a global threat. Wastewater surveillance in Japan and abroad has led to the detection of SARS-CoV-2, causing concern that SARS-CoV-2 in the feces of infected persons may contaminate the aquatic environment. Bivalves such as oysters cultivated in coastal areas are known to filter and concentrate viruses such as norovirus present in seawater in their bodies; however, whether they do so with SARS-CoV-2 is unknown. Therefore, we examined cultivated oysters sold in Japan for the presence of SARS-CoV-2 between October 2021 and April 2022 to clarify the extent of viral contamination and evaluate the risk of food-borne transmission of SARS-CoV-2. Porcine epidemic diarrhea virus (PEDV), known as pig coronavirus, was used to spike midgut-gland samples as a whole process control. The presence of SARS-CoV-2 and PEDV was investigated using a modified polyethylene glycol precipitation method and RT-qPCR. While all samples spiked with the whole process control were positive, no SARS-CoV-2 was detected in any of the 145 raw oyster samples surveyed, despite a marked increase in infections caused by the Omicron variant from January to April 2022 in Japan. Therefore, our results suggest that with well-developed sewage treatment facilities, consumption of oysters cultivated in coastal areas may not be a risk factor for SARS-CoV-2 outbreaks.

Quantification of changes in human islet G protein-coupled receptor mRNA expression in obesity.

BACKGROUND: G protein-coupled receptors (GPCRs) play crucial roles in regulating islet function, with Gαs- and Gαq-coupled receptors being linked to the stimulation of insulin secretion. We have quantified the mRNA expression of 384 non-olfactory GPCRs in islets isolated from lean and obese organ donors to determine alterations in islet GPCR mRNA expression in obesity. METHODS: RT-qPCR was used to quantify GPCR mRNAs relative to five reference genes (ACTB, GAPDH, PPIA, TBP, and TFRC) in human islets isolated from lean (BMI = 22.6 ± 0.5) and obese (BMI = 32.0 ± 0.8) donors. RESULTS: Overall, 197 and 256 GPCR mRNAs were detected above trace level in islets from lean and obese donors, respectively, with 191 GPCR mRNAs being common to the lean and obese groups. 40.9% (n = 157) and 27.1% (n = 104) of the mRNAs were expressed at trace level whilst 7.8% and 6.3% were absent in islets from lean and obese donors, respectively. Hundred and seventeen GPCR mRNAs were upregulated at least twofold in islets from obese donors, and there was >twofold downregulation of 21 GPCR mRNAs. Of particular interest, several receptors signalling via Gαs or Gαq showed significant mRNA upregulation in islets from obese donors (fold increase: PTH2R: 54.0 ± 14.6; MC2R: 34.3 ± 11.5; RXFP1: 8.5 ± 2.1; HTR2B: 6.0 ± 2.0; GPR110: 3.9 ± 1.2; PROKR2: 3.9 ± 0.7). CONCLUSIONS: Under conditions of obesity, human islets showed significant alterations in mRNAs encoding numerous GPCRs. The increased expression of Gαs- and Gαq-coupled receptors that have not previously been investigated in ß-cells opens up possibilities of novel therapeutic candidates that may lead to the potentiation of insulin secretion and/or ß-cell mass to regulate glucose homeostasis.

Midnight/midday-synchronized expression of cryptochrome genes in the eyes of three teleost species, zebrafish, goldfish, and medaka.

Photoperiodic responses are observed in many organisms living in the temperate zones. The circadian clock is involved in photoperiodic time measurement; however, the underlying molecular mechanism for detection of the day length remains unknown. We previously compared the expression profiles of the Cryptochrome(Cry) genes in the zebrafish eye and reported that Cry1ab has a double peak with variable expression duration depending on the photoperiod. In this study, to understand commonalities and differences in the photoperiodic responses of ocular Cry genes, we identified Cryptochrome genes in two other teleost species, goldfish and medaka, living in temperate zones, and measured ocular Cry mRNA levels in all of the three species, under different photoperiods (long-day [14 h light: 10 h dark] and short-day [10 h light: 14 h dark] and in constant darkness. Cry1ab mRNA levels did not show dual peaks in goldfish or medaka under the examined conditions; however, the mRNA expression profiles of many Crys were altered in all three species, depending on the day length and light condition. Based on their expression profiles, Cry mRNA peaks were classified into three groups that better synchronize to sunrise (light-on), midnight/midday (middle points of the dark/light periods), or sunset (light-off). These results suggest the presence of multiple oscillators that oscillate independently or a complex oscillator in which Cry expression cycles change in a photoperiod-dependent manner in the eye.