Specific Quantitative Polymerase Chain Reaction Research Articles

How to distinguish mpox from its mimickers: An observational retrospective cohort study.

During the current global outbreak of mpox (formerly monkeypox), atypical features were frequently described outside endemic areas, raising concerns around differential diagnosis. In this study, we included 372 adult patients who had clinical signs consistent with mpox and who were screened using non-variola orthopoxvirus specific quantitative polymerase chain reaction (PCR) between 15 May and 15 November 2022 at the University Hospital Institute Méditerranée Infection, Marseille, France. At least one clinical sample was positive for 143 (38.4%) of these patients and 229 (61.6%) were negative. Clinically, patients who had mpox presented more frequently with systemic signs (69.9% vs. 31.0%, p < 10-6 ) including fever (51.0% vs. 30.1%, p < 10-3 ), myalgia (33.5% vs. 17.9%, p = 0.002), and lymphadenopathy (38.5% vs. 13.1%, p < 10-6 ). Among the patients who were negative for the non-variola orthopoxvirus, an alternative diagnosis was identified in 58 of them (25.3%), including chickenpox (n = 30, 13.1%), syphilis (n = 9, 4%), bacterial skin infection (n = 8, 3.5%), gonococcus (n = 5, 2.2%), HSV infection (n = 5, 2.2%), and histoplasmosis (n = 1, 0.4%). Overall, in the current outbreak, we show that mpox has a poorly specific clinical presentation. This reinforces the importance of microbiological confirmation. In symptomatic patients who are negative for the monkeypox virus by PCR, a broad differential diagnosis should be maintained.

Borrelia persica infection in wild carnivores in Israel: molecular characterization and new potential reservoirs

BackgroundBorrelia persica causes tick-borne relapsing fever in Israel, the eastern Mediterranean basin, and Asia. Relapsing fever is associated with severe illness and potentially death in humans and animals. Since B. persica infection has rarely been described in wild animals, the aim of this study was to evaluate the prevalence of infection with B. persica in wild carnivores in Israel.MethodsSpleen and blood clot samples from wild carnivores, which underwent necropsy, were tested for the presence of Borrelia DNA by real-time polymerase chain reaction (PCR). PCR products were sequenced, and the spirochete loads were quantified using a specific quantitative PCR (qPCR).ResultsA total of 140 samples from 74 wild carnivores were analyzed for the presence of Borrelia DNA. Six out of the 74 (8.1%) animals were found positive for B. persica by PCR and sequencing of the flagellin B gene, of which 4/74 (5.4%) were also positive by PCR for the glycerophosphodiester phosphodiesterase (glpQ) gene. Positive samples were obtained from three European badgers, and one striped hyena, golden jackal, and red fox each. All B. persica-positive animals were young males (P < 0.0001). Quantifiable results were obtained from 3/5 spleen and 4/5 blood samples. The spirochete loads in the blood were significantly higher than those found in the spleen (P = 0.034).ConclusionsThe prevalence of B. persica infection found in wild carnivores brought for necropsy was unexpectedly high, suggesting that this infection is widespread in some wild animal species in Israel. This is the first report of B. persica infection in the European badger and striped hyena. These carnivores have a wide geographical range of activity, and the results of this survey raise the possibility that they may serve as reservoir hosts for B. persica.Graphical

Effects of company and season on blood fluke (Cardicola spp.) infection in ranched Southern Bluefin Tuna: preliminary evidence infection has a negative effect on fish growth.

Aporocotylid blood flukes Cardicola forsteri and C. orientalis are an ongoing health concern for Southern Bluefin Tuna (SBT), Thunnus maccoyii, ranched in Australia. Therapeutic application of praziquantel (PZQ) has reduced SBT mortalities, however PZQ is not a residual treatment therefore reinfection can occur after the single treatment application. This study documents the epidemiology of Cardicola spp. infection in ranched SBT post treatment over three ranching seasons (2018, 2019 and 2021). Infection prevalence (percentage of SBT affected) and intensity (parasite load) was determined by adult fluke counts from heart, egg counts from gill filaments and the use of specific quantitative polymerase chain reaction (qPCR) for detection of C. forsteri and C. orientalis ITS-2 DNA in SBT hearts and gills. SBT Condition Index decreased as intensity of Cardicola spp. DNA in SBT gills increased, suggesting blood fluke infection had a negative effect on SBT growth (Spearman's r = -0.2426, d.f. = 138, p = 0.0041). Prevalence and intensity of infection indicated PZQ remained highly effective at controlling Cardicola spp. infection in ranched SBT, 10 years after PZQ administration began in this industry. Company A had the highest prevalence and intensity of Cardicola spp. infection in 2018, and Company G had the highest in 2019. No consistent pattern was seen in 2021. Overall, intensity of infection did not increase as ranching duration increased post treatment. Results from this study improve our knowledge of the biology of blood flukes and helps the SBT industry to modify or design new blood fluke management strategies to reduce health risks and improve performance of SBT.

Insights into Overlappings of Fibrosis and Cancer: Exploring the Tumor-related Cardinal Genes in Idiopathic Pulmonary Fibrosis.

The pathogenesis of idiopathic pulmonary fibrosis (IPF) is quite similar to that of cancer pathogenesis, and several pathways appear to be involved in both disorders. The mammalian target of the rapamycin (mTOR) pathway harbors several established oncogenes and tumor suppressors. The same signaling molecules and growth factors, such as vascular endothelial growth factor (VEGF), contributing to cancer development and progression play a part in fibroblast proliferation, myofibroblast differentiation, and the production of extracellular matrix in IPF development as well. The expression of candidate genes acting upstream and downstream of mTORC1, as well as Vegf and low-density lipoprotein receptor related protein 1(Lrp1), was assessed using specific primers and quantitative polymerase chain reaction (qPCR) within the lung tissues of bleomycin (BLM)-induced IPF mouse models. Lung fibrosis was evaluated by histological examinations and hydroxyproline colorimetric assay. BLM-exposed mice developed lung injuries characterized by inflammatory manifestations and fibrotic features, along with higher levels of collagen and hydroxyproline. Gene expression analyses indicated a significant elevation of regulatory associated protein of mTOR (Raptor), Ras homolog enriched in brain (Rheb), S6 kinase 1, and Eukaryotic translation initiation factor 4E-binding protein 1 (4Ebp1), as well as a significant reduction of Vegfa, Tuberous sclerosis complex (Tsc2), and Lrp1; no changes were observed in the Tsc1 mRNA level. Our findings support the elevation of S6K1 and 4EBP1 in response to the TSC/RHEB/mTORC1 axis, which profoundly encourages the development and establishment of IPF and cancer. In addition, this study suggests a possible preventive role for VEGF-A and LRP1 in the development of IPF.

Expression profile of miRNAs computationally predicted to target PDL-1 in cervical tissues of different histology groups.

Background: Human papilloma virus (HPV) is considered a successful pathogen as it has the ability to evade host immune responses and establish long-term persistent infection. It has been reported that programmed death ligand 1 (PDL-1) expression is correlated with HPV-positivity and is increased with lesion progression or tumor metastasis in cervical cancer. The expression of microRNAs (miRNAs) is often deregulated in cancer, and their potential targets are affected. Methods: RNA was extracted from formalin-fixed paraffin-embedded (FFPE) cervical samples of different histological types, previously typed for the presence of HPV. A specific quantitative polymerase chain reaction (qPCR) protocol with SYBR Green was used to check for the expression of four miRNAs that were computationally predicted to target PDL-1. Results and conclusion: hsa-miR-20a-5p and hsa-miR-106b-5p showed an expression increase with the severity of the lesions, while hsa-miR-125b-5p depicted a significant decrease in its expression in cancerous samples when compared to normal samples.

Oral Abundance of Actinomyces spp. in Breast Cancer Patients

Objectives: Pathophysiology of medication-related osteonecrosis of the jaw (MRONJ) is still unclear, and disease development is associated with adverse reaction of bisphosphonates and denosumab, and Actinomyces spp. as well. In this study, we evaluated the abundance of Actinomyces spp. in breast cancer patients undergoing chemotherapy compared to healthy controls. Methods: Oropharyngeal samples were collected from treatment-naive early-stage breast cancer patients, who were scheduled for standard of care therapy (eight samples throughout chemotherapy, one prior to radiotherapy and one after a year of start), as well as from healthy controls at matched timepoints. We quantified Actinomyces spp. in the samples with a highly sensitive and specific quantitative polymerase chain reaction. Results: Twenty-one patients and 16 healthy subjects were enrolled. Forty-eight percent of patients suffered from estrogen receptor-positive/progesterone receptor-positive or -negative/human epidermal growth factor receptor 2 (HER2)-negative disease, 38% were HER2-positive, and 14% were triple-negative. Comparison of Actinomyces spp. loads in cancer patients and healthy controls did not reveal significant difference. Fluctuations on bacterial quantity were observed in both groups over time. Tumor receptor status or different chemotherapy schemes of patients were not correlated with a particular pattern on abundance of Actinomyces spp. Conclusions: We suggest that Actinomyces spp. are not the initiative factors in MRONJ development. These bacteria are not altered in abundance during chemotherapy, but they behave opportunistic when there is a bone disruption in the oropharynx in the first place caused by antiresorptive drugs or dental trauma and proliferate in their new niche. Thus, Actinomyces spp. plays a latter role in MRONJ development, rather than a primary causative one.

Rapid Diagnosis of Central Nervous System Scedosporiosis by Specific Quantitative Polymerase Chain Reaction Applied to Formalin-Fixed, Paraffin-Embedded Tissue.

Scedosporium (S.) apiospermum is a typical mold causing cerebral abscesses, often after near-drowning. Infections are associated with high morbidity and mortality due to diagnostic challenges including the need for prolonged incubation of cultures. In addition, histopathological differentiation from other filamentous fungi, including Aspergillus fumigatus, may not be possible, excluding early specific diagnosis and targeted therapy. Polymerase chain reaction (PCR) on tissue samples can rapidly identify fungi, leading to an earlier adequate treatment. Due to an extensive spectrum of causative fungi, broad-range PCRs with amplicon sequencing have been endorsed as the best DNA amplification strategy. We herein describe a case with brain abscesses due to S. apiospermum in a 66-year-old immunocompromised female patient. While broad-range PCR failed to identify a fungal pathogen from a cerebral biopsy demonstrating hyaline mold hyphae, specific quantitative PCR (qPCR) identified Scedosporium and ruled out Aspergillus, the most prevalent agent of central nervous system mold infection. A panel of specific qPCR assays, guided by the morphology of fungal elements in tissue or as a multiplex assay, may be a successful molecular approach to identify fungal agents of brain abscesses. This also applies in the presence of negative broad-range fungal PCR, therefore providing diagnostic and therapeutic potential for early specific management and improvement of patient clinical outcome.

Occurrence, Characteristics, and PCR-Based Detection of Pectobacterium polaris Causing Soft Rot of Chinese Cabbage in China.

Bacterial soft rot is an important disease of Chinese cabbage (Brassica rapa L. ssp. pekinensis) in China and many other countries. Four pectinolytic bacterial strains (WBC1, WBC6, WBC9, and WBC11) were isolated from soft-rotted Chinese cabbage in Beijing, China. Based on 16S rDNA and pmrA gene sequence analyses, multilocus sequence analysis (MLSA), and genomic average nucleotide identity (ANI) analysis, these four strains were identified as Pectobacterium polaris. This species, previously reported from potato in countries not including China, is a new soft rot pathogen of Chinese cabbage in China. Biochemical characteristics of these P. polaris strains tested by Biolog were mostly consistent with those of P. polaris NIBIO1006T. Their pathogenicity on Chinese cabbage is temperature dependent, with all four strains as well as the type strain exhibiting high pathogenicity at 23°C and 28°C. These four strains infected Lactuca sativa, Daucus carota, Solanum tuberosum, and Capsicum annuum by artificial inoculation. Specific polymerase chain reaction (PCR) and quantitative PCR (qPCR) primers for P. polaris were developed on the basis of its specific gene sequences (determined by genome comparison methods). Both PCR and qPCR detected not only genomic DNA of P. polaris but also the pathogen from diseased plant tissues even before external symptoms appeared. Their detection sensitivities were as low as 1 pg and 100 pg genomic DNA of P. polaris, respectively. To our knowledge, this study is the first to both report the emergence of P. polaris on Chinese cabbage in China and provide rapid and accurate PCR/qPCR-based detection systems specific for P. polaris.

A Multiplex TaqMan qPCR Assay for Detection and Quantification of Clade 1 and Clade 2 Isolates of Pseudoperonospora cubensis and Pseudoperonospora humuli

The ability to detect and quantify aerially dispersed plant pathogens is essential for developing effective disease control measures and epidemiological models that optimize the timing for control. There is an acute need for managing the downy mildew pathogens infecting cucurbits and hop incited by members of the genus Pseudoperonospora (Pseudoperonospora cubensis clade 1 and 2 isolates and Pseudoperonospora humuli, respectively). A highly specific multiplex TaqMan quantitative polymerase chain reaction (PCR) assay targeting unique sequences in the pathogens' mitochondrial genomes was developed that enables detection of all three taxa in a single multiplexed amplification. An internal control included in the reaction evaluated whether results were influenced by PCR inhibitors that can make it through the DNA extraction process. Reliable quantification of inoculum as low as three sporangia in a sample was observed. The multiplexed assay was tested with DNA extracted from purified sporangia, infected plant tissue, and environmental samples collected on impaction spore traps samplers. The ability to accurately detect and simultaneously quantify all three pathogens in a single multiplexed amplification should improve management options for controlling the diseases they cause.

Developing species-specific quantitative real-time polymerase chain reaction primers for detecting Lautropia mirabilis

This study aimed to develop Lautropia mirabilis -specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis . The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/ RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.

Prevalence and Intensity of Cardicola spp. Infection in Ranched Southern Bluefin Tuna and a Comparison of Diagnostic Methods.

The parasitic blood flukes Cardicola forsteri and C. orientalis are an ongoing health concern for Southern Bluefin Tuna Thunnus maccoyii (SBT) ranched in Australia. In this study we compared the effect of treatment, company, and ranching year on blood fluke infections in ranched SBT. SBT were sampled during the 2018 and 2019 ranching seasons from praziquantel (PZQ) treated pontoons and untreated pontoons managed by two companies. Severity of infection was diagnosed by several criteria including adult fluke counts from hearts, egg counts from gill filaments and the use of specific quantitative polymerase chain reaction (qPCR) for detection of C. forsteri and C. orientalis ITS-2 DNA in SBT hearts and gills. PZQ treatment remains highly effective against C. forsteri infection. Prevalence and intensity of Cardicola spp. infection was lower in 2019 than 2018 for Company A in treated pontoons at week 12 and week 17 of ranching, and lower for Company A than Company B in untreated pontoons at month 5 of ranching. Results indicate re-infection may be less likely in the environment near Company A pontoons, and consistent years of treatment may have lowered the parasite load in the environment. qPCR demonstrated higher sensitivity when comparing diagnostic methods for C. forsteri in heart, and higher specificity when comparing diagnostic methods for Cardicola spp. in gills. Continuing to monitor blood fluke infections in ranched SBT can help to detect changes in drug efficacy over time and help industry to develop a best practice for treatment.

Chloramine Concentrations within Distribution Systems and Their Effect on Heterotrophic Bacteria, Mycobacterial Species, and Disinfection Byproducts

Chloramine is a secondary disinfectant used to maintain microbial control throughout public water distribution systems. This study investigated the relationship between chloramine concentration, heterotrophic bacteria, and specific Mycobacterium species. Sixty-four water samples were collected at four locations within the utility's distribution network on four occasions. Water samples were analyzed for total chlorine and monochloramine. Traditional culture methods were applied for heterotrophic bacteria and nontuberculous mycobacteria (NTM), and specific quantitative polymerase chain reaction (qPCR) assays were used to detect and quantify Mycobacterium avium, M. intracellulare, and M. abscessus. Total chlorine and monochloramine concentrations decreased between the distribution entry point (4.7 mg/L and 3.4 mg/L as Cl2, respectively) to the maximum residence time location (1.7 mg/L and 1.1 mg/L as Cl2, respectively). Results showed that heterotrophic bacteria and NTM counts increased by two logs as the water reached the average residence time (ART) location. Microbiological detection frequencies among all samples were: 86% NTMs, 66% heterotrophic bacteria, 64% M. abscessus, 48% M. intracellulare, and 2% M. avium. This study shows that heterotrophic bacteria and NTM are weakly correlated with disinfectant residual concentration, R2=0.18 and R2=0.04, respectively. Considering that specific NTMs have significant human health effects, these data fill a critical knowledge gap regarding chloramine's impact on heterotrophic bacteria and Mycobacterial species survival within public drinking water distribution systems.

PCSK9 genetic (rs11591147) and epigenetic (DNA methylation) modifications associated with PCSK9 expression and serum proteins in CAD patients.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) genetic polymorphisms play a significant role in cholesterol homeostasis. Therefore, we aimed to investigate the association of PCSK9 genetic variations NM_174936.3:c.137G>T (R46L, rs11591147) and NM_174936.3:c.1120G>T (D374Y, rs137852912), as well as promoter DNA methylation status, with mRNA expression and circulating serum protein levels in coronary artery disease (CAD) patients. The present study includes 300 CAD cases and 300 controls from South India. Biochemical assays were performed using commercially available kits. PCSK9 rs11591147 and rs137852912 polymorphisms were analyzed by the polymerase chain reaction (PCR)-restriction fragment length polymorphism method, whereas promoter DNA methylation status and gene expression were determined using methylation specific PCR and quantitative PCR respectively. The genotypic distribution of PCSK9 rs11591147 revealed that individuals with the TT-genotype and T-allele have a reduced risk for CAD. Furthermore, patients with the PCSK9 rs11591147 TT genotype have a significantly lower total cholesterol and low-density lipoprotein-cholesterol levels and also higher high-density lipoprotein-cholesterol levels than individuals with the GG genotype. Logistic regression analysis has shown that the GG and GT (p =1.51 × 10-8 , p =1.47 × 10-9 ) genotypes predicted the risk for CAD with an odds ratio of 5.8 and 7.3 respectively. In addition, individuals with the TT genotype were hypermethylated at promoter DNA of PCSK9, resulting in lower mRNA expression and circulating serum proteins than in individuals with the GG genotype. In silico analyses revealed that rs11591147 T-allele has protein destabilizing capacity. In conclusion, the present study indicates that the PCSK9 gene expression and circulating serum protein levels are not only associated with rs11591147 genotype, but also with promoter DNA methylation. Furthermore, the findings with respect to both single nucleotide polymorphism and promoter DNA methylation may open avenues for novel treatment possibilities targeting PCSK9 for CAD management.

Epidemiological evaluation of sewage surveillance as a tool to detect the presence of COVID-19 cases in a low case load setting

In low prevalence settings the development of sensitive and specific quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) tests to detect SARS-CoV-2 (the virus causing COVID-19) in sewage presents the possibility of using sewage sampling as a diagnostic test for the presence of infected people in the catchment of the sampled sewer. However, the usefulness of such surveillance has not been quantified. In this study in the Australian state of Victoria between August and October 2020 the location of each known SARS-CoV-2-infected person was determined on each day from two days before onset to 55 days after, in 46 metropolitan and rural sewer catchments sampled weekly – a total of 71 positive and 275 negative samples, and 354,155 person-days of location data. These were categorised by time since onset and distance from the sampling site. The odds of detection in sewage were between 5 and 20 times higher where known cases were present, with less effect of distance than time since onset. Using positive qRT-PCR in a sewage sample as a diagnostic test not just for viral RNA in the sample, but for the presence of known infected people in the catchment on the same day, the sensitivity was moderate (31% to 76%) and the specificity high (87% to 94%). The odds of detection were increased with increased numbers of known infected people but decreased with increased distance and time since onset. The probability of detection of the viral subgenome in sewage samples was about 10% when one known infected person was present, and this increased with higher numbers of known infected people and greater proximity to the sampling site. Sewage surveillance can be used to detect people infected with SARS-CoV-2 in the catchment, directing a search for infectious clinical cases and other public health actions. However, detection at least eight weeks after onset may be due to existing cases rather than new ones, and, although not zero, the probability of detecting a single case is low.

Methylome and transcriptome profiling revealed epigenetic silencing of LPCAT1 and PCYT1A associated with lipidome alterations in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine diseases of fertile women and a major cause of infertility. The regulatory effects of DNA methylation on gene transcription and downstream lipid metabolism have not been explored in PCOS. In this study, MBD-seq and RNA-seq were performed on ovarian granulosa cells of PCOS patients and controls, and methylation specific PCR and quantitative polymerase chain reaction were used to validate the results. Then lipidomic profiling was conducted on serum of PCOS patients and controls using UPLC-MS. We identified 73 genes with differently methylated promoters and 830 differently expressed genes. The promoter regions of LPCAT1 and PCYT1A were hypermethylated, accompanied by downregulation of their messenger RNA expression, which may be involved in the regulation of PCOS through downstream glycerophospholipid metabolism and phosphatidylcholine synthesis. The lipid profiling results showed significant changes in 21 lipids, which demonstrated the disturbance in glycerophospholipid metabolism and glycerolipid metabolism pathways. Furthermore, the metabolites-genes interaction network was constructed to illustrate the association of aberrant methylome and transcriptome with lipidome alterations in glycerolipid and glycerophospholipid metabolism pathways. Our study suggested that the methylation silencing of LPCAT1 and PCYT1A may promote glycerophospholipids metabolism dysregulation, which provided a novel genetic and lipometabolic basis for the pathogenesis of PCOS.

Correlation between goose circovirus and goose parvovirus with gosling feather loss disease and goose broke feather disease in southern Taiwan.

BackgroundGoslings in several Taiwanese farms experienced gosling feather loss disease (GFL) at 21–35 days and goose broke feather disease (GBF) at 42–60 days. The prevalence ranges from a few birds to 500 cases per field. It is estimated that about 12,000 geese have been infected, the morbidity is 70–80% and the mortality is 20–30%.ObjectivesThis study aims to investigate the pathogens that cause GFL and GBF. Focus on the study of the correlation between goose circovirus (GoCV) and goose parvovirus (GPV) with the goose feather loss in southern Taiwan. Furthermore, a phylogenetic tree was established to align the differences between southern and northern Taiwan and compare with virus strains from China and Europe.MethodsSamples were collected from animal hospitals. Molecular and microscopy diagnostics were used to examine 92 geese. Specific quantitative polymerase chain reaction (Q-PCR) assays are performed to evaluate GPV and GoCV viral loads and simultaneously evaluated the feather loss conditions in geese with the scoring method.ResultsHigh prevalence of GoCV and GPV infection in geese showing signs of GFL and GBF. Inclusion body was detected in the feather follicles and Lieberkühn crypt epithelial cells. The Q-PCR showed the high correlation between feather loss and viruses during 3rd–5th week. However, the infection was not detected using the same test in 60 healthy geese.ConclusionsThus, GFL and GBF appear to be significantly closely related to GoCV and GPV. The geese feathers showed increasing recovery after being quarantined and disinfected.

In Vivo Validation of Alternative FDXR Transcripts in Human Blood in Response to Ionizing Radiation.

Following cell stress such as ionising radiation (IR) exposure, multiple cellular pathways are activated. We recently demonstrated that ferredoxin reductase (FDXR) has a remarkable IR-induced transcriptional responsiveness in blood. Here, we provided a first comprehensive FDXR variant profile following DNA damage. First, specific quantitative real-time polymerase chain reaction (qPCR) primers were designed to establish dose-responses for eight curated FDXR variants, all up-regulated after IR in a dose-dependent manner. The potential role of gender on the expression of these variants was tested, and neither the variants response to IR nor the background level of expression was profoundly affected; moreover, in vitro induction of inflammation temporarily counteracted IR response early after exposure. Importantly, transcriptional up-regulation of these variants was further confirmed in vivo in blood of radiotherapy patients. Full-length nanopore sequencing was performed to identify other FDXR variants and revealed the high responsiveness of FDXR-201 and FDXR-208. Moreover, FDXR-218 and FDXR-219 showed no detectable endogenous expression, but a clear detection after IR. Overall, we characterised 14 FDXR transcript variants and identified for the first time their response to DNA damage in vivo. Future studies are required to unravel the function of these splicing variants, but they already represent a new class of radiation exposure biomarkers.

Extraction of norovirus based on nanomagnetic beads and establishment of a fluorescence quantitative detection method

Norovirus infection is the main cause of epidemic acute gastroenteritis outbreaks. As a result, norovirus merits careful attention. Acute gastroenteritis caused by norovirus is very harmful, necessitating effective prevention and monitoring. This study extracted norovirus using the Fe3O4 nanomagnetic bead method while establishing a rapid and sensitive quantitative norovirus detection method for emergency use. Norovirus RNA was extracted from norovirus-positive stool samples, norovirus-specific primers were designed, and SYBR Green I fluorescent dye was used to construct specific fluorescence quantitative reverse-transcription polymerase chain reaction (RT-PCR) method. Norovirus in the sample was successfully detected with the quantitative RT-PCR method established in this study, with a measured cycle threshold value of 28.73; the melting temperature value of the PCR product was approximately 83.5 °C. Additionally, by analyzing melting curve with quantitative PCR and evaluating the repeatability of the detection method, the performance of the reaction system in terms of specificity and repeatability was determined to be good. The fluorescence quantitative detection method established here can rapidly and effectively detect norovirus, and the method has good specificity and high repeatability. Currently, there is a trend of norovirus infection becoming increasingly severe. Thus, this rapid and sensitive fluorescence quantitative detection method will play a significant role in the monitoring and detection of norovirus and is thus worthy of promotion and application.

Association of Interleukin 7 Immunotherapy With Lymphocyte Counts Among Patients With Severe Coronavirus Disease 2019 (COVID-19)

Cytokine storm–mediated organ injury continues to dominate current thinking as the primary mechanism for coronavirus disease 2019 (COVID-19). Although there is an initial hyper-inflammatory phase, mounting evidence suggests that virus-induced defective host immunity may be the real cause of death in many patients.1,2 COVID-19 has been called a serial lymphocyte killer because profound and protracted lymphopenia is a near uniform finding among patients with severe COVID-19 and correlates with morbidity and mortality.1,3 Autopsies demonstrate a devastating depletion of lymphocytes in the spleen and other organs.2 CD4, CD8, and natural killer cells, which play important antiviral roles, are depleted and have reduced function, leading to immune collapse.1 Clinical and pathological findings in patients with COVID-19 indicate that immunosuppression is a critical determinant of outcomes. Secondary hospital-acquired infections occur in 50% of patients who die.1 Cell inclusion bodies, consistent with viral persistence, have been found in lungs, kidneys, and other organs.1,2 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific quantitative polymerase chain reaction has revealed viral load in the lungs of most patients at autopsy, consistent with an inability to eliminate the pathogen.1,2 Collectively, these studies indicate that impaired immune competence is an important pathogenic mechanism in COVID-19. Interleukin 7 (IL-7) is a pleiotropic cytokine essential for lymphocyte survival and expansion.4,5 Administration of IL-7 invariably increases circulating and tissue lymphocytes and has been administered to more than 450 patients with an excellent safety profile.4,5 IL-7 is currently in multiple randomized clinical trials for oncologic and infectious disorders, and a trial in the United Kingdom is evaluating its use among patients with severe COVID-19. Importantly, IL-7 has documented efficacy as an antiviral agent.4,5 IL-7 therapy has been shown to restore lymphocyte counts and functional activity, leading to decreased viral load and clinical improvement in several life-threating viral infections.4,5 It has been shown to increase CD4 and CD8 T-cells 3-fold, to improve T-cell activation, to not increase proinflammatory cytokines, and to be well tolerated in patients with bacterial sepsis.6 Thus, a compelling scientific rationale exists for examining whether IL-7 is associated with restored host protective immunity in patients with COVID-19 and immunosuppression and improve outcomes.1

ATF4, DLX3, FRA1, MSX2, C/EBP-ζ, and C/EBP-α Shape the Molecular Basis of Therapeutic Effects of Zoledronic Acid in Bone Disorders.

Zoledronic Acid (ZA) is one of the common treatment choices used in various boneassociated conditions. Also, many studies have investigated the effect of ZA on Osteoblastic-Differentiation (OSD) of Mesenchymal Stem Cells (MSCs), but its clear molecular mechanism(s) has remained to be understood. It seems that the methylation of the promoter region of key genes might be an important factor involved in the regulation of genes responsible for OSD. The present study aimed to evaluate the changes in the mRNA expression and promoter methylation of central Transcription Factors (TFs) during OSD of MSCs under treatment with ZA. MSCs were induced to be differentiated into the osteoblastic cell lineage using routine protocols. MSCs received ZA during OSD and then the methylation and mRNA expression levels of target genes were measured by Methylation Specific-quantitative Polymerase Chain Reaction (MS-qPCR) and real-time PCR, respectively. The osteoblastic differentiation was confirmed by Alizarin Red Staining and the related markers to this stage. Gene expression and promoter methylation level for DLX3, FRA1, ATF4, MSX2, C/EBPζ, and C/EBPa were up or down-regulated in both ZA-treated and untreated cells during the osteodifferentiation process on days 0 to 21. ATF4, DLX3, and FRA1 genes were significantly up-regulated during the OSD processes, while the result for MSX2, C/EBPζ, and C/EBPa was reverse. On the other hand, ATF4 and DLX3 methylation levels gradually reduced in both ZA-treated and untreated cells during the osteodifferentiation process on days 0 to 21, while the pattern was increasing for MSX2 and C/EBPa. The methylation pattern of C/EBPζ was upward in untreated groups while it had a downward pattern in ZA-treated groups at the same scheduled time. The result for FRA1 was not significant in both groups at the same scheduled time (days 0-21). The results indicated that promoter-hypomethylation of ATF4, DLX3, and FRA1 genes might be one of the mechanism(s) controlling their gene expression. Moreover, we found that promoter-hypermethylation led to the down-regulation of MSX2, C/EBP-ζ and C/EBP-α. The results implicate that ATF4, DLX3 and FRA1 may act as inducers of OSD while MSX2, C/EBP-ζ and C/EBP-α could act as the inhibitor ones. We also determined that promoter-methylation is an important process in the regulation of OSD. However, yet there was no significant difference in the promoter-methylation level of selected TFs in ZA-treated and control cells, a methylation- independent pathway might be involved in the regulation of target genes during OSD of MSCs.