Reverse Transcription-polymerase Chain Reaction Assay Research Articles

Impact of relative humidity on SARS-CoV-2 RNA extraction using Nextractor automated extraction system.

This study investigated the influence of relative humidity (RH) on the efficiency of SARS-CoV-2 RNA extraction using the Nextractor automated system. Experiments employing clinical samples demonstrated satisfactory sensitivity and reproducibility for RNA extraction at low humidity (below 50% RH). Conversely, extractions at high humidity (above 70% RH) resulted in complete failure of reverse transcription-polymerase chain reaction assays, with neither SARS-CoV-2 RNA nor the human RNase P gene (internal control) detected. Analysis suggested that residual ethanol, incompletely evaporating due to high humidity, acted as a potent polymerase chain reaction inhibitor in these samples. These findings highlighted the importance of maintaining optimal laboratory humidity (<50% RH) for reliable SARS-CoV-2 RNA extraction using the Nextractor system. Furthermore, laboratories should implement strategies such as regular humidity monitoring, staff training on humidity's impact, and system validation under specific humidity conditions to ensure accurate molecular diagnostic workflows for COVID-19 testing.

Novel 1,3,4-oxadiazole derivatives of naproxen targeting EGFR: Synthesis, molecular docking studies, and cytotoxic evaluation.

The close association between inflammation and cancer inspired the synthesis of a series of 1,3,4-oxadiazole derivatives (compounds H4-A-F) of 6-methoxynaphtalene. The chemical structures of the new compounds were validated utilizing Fourier-transform infrared, proton nuclear magnetic resonance, and carbon-13 nuclear magnetic resonance spectroscopic techniques and CHN analysis. Computer-aided drug design methods were used to predict the compounds biological target, ADMET properties, toxicity, and to evaluate the molecular similarities between the design compounds and erlotinib, a standard epidermal growth factor receptor (EGFR) inhibitor. The antiproliferative effects of the new compounds were evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, cell cycle analysis, apoptosis detection by microscopy, quantitative reverse transcription-polymerase chain reaction, and immunoblotting, and EGFR enzyme inhibition assay. In silico analysis of the new oxadiazole derivatives indicated that these compounds target EGFR, and that compounds H4-A, H4-B, H4-C, and H4-E show similar molecular properties to erlotinib. Additionally, the results indicated that none of the synthesized compounds are carcinogenic, and that compounds H4-A, H4-C, and H4-F are nontoxic. Compound H4-A showed the best-fit score against EGFR pharmacophore model, however, the in vitro studies indicated that compound H4-C was the most cytotoxic. Compound H4-C caused cytotoxicity in HCT-116 colorectal cancer cells by inducing both apoptosis and necrosis. Furthermore, compounds H4-D, H4-C, and H4-B had potent inhibitory effect on EGFR tyrosine kinase that was comparable to erlotinib. The findings of this inquiry offer a basis for further investigation into the differences between the synthesized compounds and erlotinib. However, additional testing will be needed to assess all of these differences and to identify the most promising compound for further research.

First Report of Watermelon Crinkle Leaf-Associated Virus-2 (WCLaV-2) in Watermelon (Citrullus lanatus) in Georgia, USA.

Watermelon (Citrullus lanatus) is a key horticultural crop in Georgia with farmgate value of 142 million USD (2022 Farm Gate Value Report), yet faces challenges from whitefly-transmitted viruses, especially during periods of elevated whitefly populations in the fall. Foliar symptoms on watermelon plants including yellow mottling and chlorosis, wrinkling, bunching, and upward curling, were observed in experimental fields at UGA Tifton and commercial fields in Colquitt County in the fall of 2023. These were similar to those described for watermelon crinkle leaf-associated viruses (WCLaV-1 and WCLaV-2) from Florida (Hendrick et al, 2021) and Texas (Hernandez et al., 2021). The disease incidence reached 100% in both locations. WCLaV-1 was previously identified in Georgia (Adeleke et al., 2022a); however, WCLaV-2 remained undetected in further surveys (Adeleke et al., 2022b). Total nucleic acid was extracted from symptomatic leaf tissues with the MagMAXTM 96 Viral RNA isolation kit (ThermoFisher Scientific, USA), following the manufacturer's guidelines, with the omission of DNAse treatment. The presence of WCLaV-1 was identified through reverse transcription-polymerase chain reaction (RT-PCR) (Hernandez et al., 2021) in 17 out of 24 samples in Tift County, and 12 out of 15 samples from Colquitt County. Within the same set of samples, WCLaV-2 was identified in three samples from Tift County and four samples from Colquitt County by RT-PCR directed at the RdRp gene (Hernandez et al., 2021). WCLaV-1 was not detected in the three samples from Tift County that had WCLaV-2, while mixed infections of WCLaV-2 and WCLaV-1 were observed in the samples form Colquitt County. Two whitefly transmitted viruses, previously reported in Georgia were also identified as mixed infection in these samples (Table S1). The presence of WCLaV-2 was further confirmed by amplifying the movement protein (MP) gene of WCLaV-2 by RT-PCR assays (Hernandez et al., 2021). The amplicons, with expected sizes of 968bp for the RdRp gene and 562bp for the MP gene of WCLaV-2, located on RNA 1 and RNA 2 segments respectively, were directly sequenced from both directions (Genewiz, USA) from a sample collected in Tift County. The resulting data were analyzed via BLASTn search. The MP gene fragment (PP178543) shared 100% identity with isolates from Brazil (LC636074.1), Texas (MW559086.1), and Florida (MZ325858.1). RdRp gene (PP178542) shared >99.7% identity with isolates from Brazil (LC636073.1), Texas (MW559083.1) and Florida (MZ325855.1). WCLaV-1 and WCLaV-2, initially discovered in Asia (Xin et al., 2017), have been assigned to the genus Coguvirus, in the family Phenuiviridae (Walker et al., 2022). Subsequent reports from the USA (Hendrick et al., 2021; Hernandez et al., 2021), Australia (Mulholland et al., 2023), and Brazil (Maeda et al., 2022) indicate the global spread of these viruses. Watermelon is the primary host of WCLaV-2. Despite these findings, biological information, including vector relations, for both viruses and other members of the genus Coguvirus remains elusive. The impact of these viruses on watermelon production and yield in the identified regions remains largely unknown, underscoring the need for further investigations.

Genetic Characterization of Palyam Serogroup Viruses Isolated in Japan from 1984 to 2018 and Development of a Real-Time RT-PCR Assay for Broad Detection of Palyam Serogroup Viruses and Specific Detection of Chuzan (Kasba) and D'Aguilar Viruses.

We performed whole genome sequencing (WGS) of 15 Palyam serogroup virus (PALV) strains isolated from cattle or Culicoides biting midges in Japan from 1984 to 2018. We found that the PALV strains consisted of Chuzan (Kasba) virus (CHUV), D'Aguilar virus (DAGV), Bunyip Creek virus, and another PALV, Marrakai virus (MARV). The Japanese MARV strains isolated in 1997 were closely related to Australian PALV strains isolated in 1968-1976 in genome segments 2 and 10, but they were most closely related to other Japanese PALV strains in the other genome segments. Our data suggest that the Japanese MARV strains were reassortant viruses between Asian and Australian PALVs. In addition to the WGS, we developed a real-time reverse-transcription polymerase chain reaction assay that can broadly detect PALV and specifically detect CHUV and DAGV, utilizing the data obtained by the WGS in this study. We detected the DAGV gene in bovine stillborn fetuses and congenitally abnormal calves in 2019 using the newly developed assay. To our knowledge, this is the first report of isolation of MARV outside of Australia and the first report of detection of PALV in bovine fetuses or calves with congenital abnormality outside of Africa.

COVID-19 Pandemic: Challenges, Controversies and What we have Learned

Introduction: The COVID-19 pandemic put us in a very difficult situation and created a lot of challenges for both diagnostics and follow-up of patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We also had difficult problems understanding the virus-host interactions and the progression of the immune response. Objectives: We aimed to point out our experience in the diagnostics of SARS-CoV-2 in patients admitted to Fundeni Clinical Institute. We have also investigated the healthcare personnel in order to have some epidemiologic data about the transmission of the new coronavirus in our institute. Methods: Three different real-time reverse transcription-polymerase chain reaction (RT-PCR) assays were used to screen for the new coronavirus infection. The immunization rate against SARS-CoV-2 was detected by assessing the IgG antibodies in both patients and clinical staff. We have used the chemiluminescence method to assess the anti-SARS-CoV-2 IgG antibodies. Results: Most of the diagnosed patients with SARS-CoV-2 infections were admitted to the surgery wards for hematology and gastroenterology. Our data showed that all the diagnosed patients developed IgG antibodies against SARS-CoV-2, but we have noticed that the immunization against SARS-CoV-2 did not last. Conclusions: Our experience with the SARS-CoV-2 pandemic emphasized that molecular diagnostics by RT-PCR was essential, together with the study of IgG antibodies against SARS-CoV-2, thus enabling us to better interpret PCR test results.

Effect of Milk Protein-Polyphenol Conjugate on the Regulation of GLP-1 Hormone.

Modern functional foods are designed to provide health benefits beyond basic nutrition. They are enriched with bioactive ingredients like probiotics, vitamins, minerals, and antioxidants. These foods support overall health, enhance immune function, and help prevent chronic diseases. Milk proteins and tea are known to influence satiety and regulate body weight. Studies have shown that green tea polyphenols, namely, (-)-epigallocatechin gallate (EGCG), and whey proteins, predominantly lactoferrin (LF) from milk, play a role in regulating satiety. This study aims to investigate the effect of conjugating EGCG with apo-lactoferrin (Apo-LF) and assessing these effects on satiety through monitoring glucagon-like peptide-1 (GLP-1) regulation in a human colon (NCI-H716) cell line. Apo-LF-EGCG conjugates were synthesized and characterized in terms of structural and functional properties. The effect on GLP-1 regulation was assessed by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) to monitor gene and protein expressions, respectively. The results revealed that the protein-polyphenol interaction occurs through the complex formation of hydrogen bonds at the O-H and carbonyl groups of EGCG. The conjugates also showed a significant up-regulation of gene and protein expression levels of GLP-1 while also preventing EGCG from degradation, thereby preserving its antioxidant properties. The Apo-LF-EGCG conjugates increase satiety via increasing GLP-1 secretion in human colon cells while simultaneously retaining the antioxidant properties of EGCG. Therefore, these conjugates show potential for use as dietary supplements to enhance satiety.

Limited potexvirus diversity in eastern Gulf of Mexico seagrass meadows.

Turtlegrass virus X, which infects the seagrass Thalassia testudinum, is the only potexvirus known to infect marine flowering plants. We investigated potexvirus distribution in seagrasses using a degenerate reverse transcription polymerase chain reaction (RT-PCR) assay originally designed to capture potexvirus diversity in terrestrial plants. The assay, which implements Potex-5 and Potex-2RC primers, successfully amplified a 584 nt RNA-dependent RNA polymerase (RdRp) fragment from TVX-infected seagrasses. Following validation, we screened 74 opportunistically collected, apparently healthy seagrass samples for potexviruses using this RT-PCR assay. The survey examined the host species T. testudinum, Halodule wrightii, Halophila stipulacea, Syringodium filiforme, Ruppia maritima, and Zostera marina. Potexvirus PCR products were successfully generated only from T. testudinum samples and phylogenetic analysis of sequenced PCR products revealed five distinct TVX sequence variants. Although the RT-PCR assay revealed limited potexvirus diversity in seagrasses, the expanded geographic distribution of TVX shown here emphasizes the importance of future studies to investigate T. testudinum populations across its native range and understand how the observed fine-scale genetic diversity affects host-virus interactions.

Abnormal function of EPHA2/p.R957P mutant in congenital cataract.

To identify genetic defects in a Chinese family with congenital posterior polar cataracts and assess the pathogenicity. A four-generation Chinese family affected with autosomal dominant congenital cataract was recruited. Nineteen individuals took part in this study including 5 affected and 14 unaffected individuals. Sanger sequencing targeted hot-spot regions of 27 congenital cataract-causing genes for variant discovery. The pathogenicity of the variant was evaluated by the guidelines of American College of Medical Genetics and InterVar software. Confocal microscopy was applied to detect the subcellular localization of fluorescence-labeled ephrin type-A receptor 2 (EPHA2). Co-immunoprecipitation assay was implemented to estimate the interaction between EphA2 and other lens membrane proteins. The mRNA and protein expression were analyzed by reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assay, respectively. The cell migration was analyzed by wound healing assay. Zebrafish model was generated by ectopic expression of human EPHA2/p.R957P mutant to demonstrate whether the mutant could cause lens opacity in vivo. A novel missense and pathogenic variant c.2870G>C was identified in the sterile alpha motif (SAM) domain of EPHA2. Functional studies demonstrated the variant's impact: reduced EPHA2 protein expression, altered subcellular localization, and disrupted interactions with other lens membrane proteins. This mutant notably enhanced human lens epithelial cell migration, and induced a central cloudy region and roughness in zebrafish lenses with ectopic expression of human EPHA2/p.R957P mutant under differential interference contrast (DIC) optics. Novel pathogenic c.2870G>C variant of EPHA2 in a Chinese congenital cataract family contributes to disease pathogenesis.

Evaluation of self-collected nasal, urine, and saliva samples for molecular detection of SARS-CoV-2 using an EUA approved RT-PCR assay and a laboratory developed LAMP SARS-CoV-2 test.

As the SARS-CoV-2 virus spread throughout the world, millions of positive cases of COVID-19 were registered and, even though there are millions of people already vaccinated against SARS-CoV-2, a large part of the global population remains vulnerable to contracting the virus. Massive nasopharyngeal sample collection in Puerto Rico at the beginning of the pandemic was limited by the scarcity of trained personnel and testing sites. To increase SARS-CoV-2 molecular testing availability, we evaluated the diagnostic accuracy of self-collected nasal, saliva, and urine samples using the TaqPath reverse transcription polymerase chain reaction (RT-PCR) COVID-19 kit to detect SARS-CoV-2. We also created a colorimetric loop-mediated isothermal amplification (LAMP) laboratory developed test (LDT) to detect SARS-CoV-2, as another strategy to increase the availability of molecular testing in community-based laboratories. Automated RNA extraction was performed in the KingFisher Flex instrument, followed by PCR quantification of SARS-CoV-2 on the 7500 Fast Dx RT-PCR using the TaqPath RT-PCR COVID-19 molecular test. Data was interpreted by the COVID-19 Interpretive Software from Applied Biosystems and statistically analyzed with Cohen's kappa coefficient (k). Cohen's kappa coefficient (k) for paired nasal and saliva samples showed moderate agreement (0.52). Saliva samples exhibited a higher viral load. We also observed 90% concordance between LifeGene-Biomarks' SARS-CoV-2 Rapid Colorimetric LAMP LDT and the TaqPath RT-PCR COVID-19 test. Our results suggest that self-collected saliva is superior to nasal and urine samples for COVID-19 testing. The results also suggest that the colorimetric LAMP LDT is a rapid alternative to RT-PCR tests for the detection of SARS-CoV-2. This test can be easily implemented in clinics, hospitals, the workplace, and at home; optimizing the surveillance and collection process, which helps mitigate global public health and socioeconomic upheaval caused by airborne pandemics.

Associations of cold-inducible RNA-binding protein with bacterial load, proinflammatory cytokines and mortality from pneumonia.

Cold-inducible RNA-binding protein (CIRP) is a damage-associated molecular pattern that plays a critical role in triggering inflammatory responses. It remains unknown whether CIRP is strongly associated with bacterial load, inflammatory response, and mortality in sepsis model. Pneumonia was induced in specific pathogen-free 8-9-week old male rats by injecting bacteria via puncture of the tracheal cartilage. The expressions of CIRP and proinflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β] in lung tissues, alveolar macrophages (AMs), plasma, and bronchoalveolar lavage fluid (BALF) were determined by reverse transcription-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. The numbers of bacteria recovered from the lungs were correlated with the bacterial loads injected and mortality. The expressions of CIRP increased sharply as the bacterial loads increased in the lung tissues and AMs. The amounts of TNF-α, IL-6 and IL-1β proteins synthesized were dependent on the bacterial load in the lung tissues. Releases of CIRP, TNF-α, IL-6, and IL-1β increased with the bacterial load in the blood plasma. The proteins confirmed similar patterns in the BALF. CIRP was strongly associated with the releases of TNF-α, IL-6, and IL-1β in the lung tissues, blood plasma, and BALF, and showed a close correlation with mortality. CIRP demonstrated a strong association with bacterial load, which is new evidence, and close correlations with proinflammatory cytokines and mortality of pneumonia in rats, suggesting that it might be an interesting pneumonic biomarker for monitoring host response and predicting mortality, and a promising target for immunotherapy.

Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses

Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

Predicting oxygen needs in COVID-19 patients using chest radiography multi-region radiomics.

The objective is to evaluate the performance of blood test results, radiomics, and a combination of the two data types on the prediction of the 24-h oxygenation support need for the Coronavirus disease 2019 (COVID-19) patients. In this retrospective cohort study, COVID-19 patients with confirmed real-time reverse transcription-polymerase chain reaction assay (RT-PCR) test results between February 2020 and August 2021 were investigated. Initial blood cell counts, chest radiograph, and the status of oxygenation support used within 24h were collected (n = 290; mean age, 45 ± 19years; 125 men). Radiomics features from six lung zones were extracted. Logistic regression and random forest models were developed using the clinical-only, radiomics-only, and combined data. Ten repeats of fivefold cross-validation with bootstrapping were used to identify the input features and models with the highest area under the receiver operating characteristic curve (AUC). Higher AUCs were achieved when using only radiomics features compared to using only clinical features (0.94 ± 0.03 vs. 0.88 ± 0.04). The best combined model using both radiomics and clinical features achieved highest in the cross-validation (0.95 ± 0.02) and test sets (0.96 ± 0.02). In comparison, the best clinical-only model yielded AUCs of 0.88 ± 0.04 in cross-validation and 0.89 ± 0.03 in test set. Both radiomics and clinical data can be used to predict 24-h oxygenation support need for COVID-19 patients with AUC > 0.88. Moreover, the combination of both data types further improved the performance.

Cost-effective in-house COVID-19 reverse transcription-polymerase chain reaction testing with yeast-derived Taq polymerase.

Despite the decline of the COVID-19 pandemic, there continues to be a persistent requirement for reliable testing methods that can be adapted to future outbreaks and areas with limited resources. While the standard approach of using reverse transcription-polymerase chain reaction (RT-PCR) with Taq polymerase is effective, it faces challenges such as limited access to high-quality enzymes and the presence of bacterial DNA contamination in commercial kits, which can impact the accuracy of test results. This study investigates the production of recombinant Taq polymerase in yeast cells and assesses its crude lysate in a multiplex RT-PCR assay for detecting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRP) and N genes, with human Ribonuclease P serving as an internal control. The unpurified yeast Taq polymerase demonstrates sensitivity comparable to commercially purified bacterial Taq polymerase and unpurified bacterial counterparts in detecting the RdRP and N genes. It exhibits the highest specificity, with 100% accuracy, for the N gene. The specificity for the RdRP gene closely aligns with that of commercially purified bacterial Taq polymerase and unpurified bacterial Taq polymerase. The use of unpurified recombinant yeast Taq polymerase shows promise as a cost-effective approach for conducting in-house COVID-19 RT-PCR testing. By eliminating the need for chromatography purification steps, the production of RT-PCR kits can be streamlined, potentially improving accessibility and scalability, especially in resource-limited settings and future pandemics.

TMEM2 suppresses TLR3-mediated IFN-β/ISG56/CXCL10 expression in BEAS-2B bronchial epithelial cells.

Bronchial epithelial cells are at the front line of viral infections. Toll-like receptor 3 (TLR3) cascade causes the expression of interferon (IFN)-β and IFN-stimulated genes (ISGs), which in turn induce an antiviral response. Members of the transmembrane protein (TMEM) family are expressed in various cell types. Although the prognostic value of TMEM2 in various cancers has been reported, its association with infectious diseases remains unknown. In this study, we investigated the effects of TMEM2 on antiviral immunity in BEAS-2B bronchial epithelial cells. TMEM2 protein was found in the cytoplasm of normal human bronchial epithelial cells and differed between organs using immunohistochemistry. Cultured BEAS-2B cells were transfected with TMEM2 siRNA, followed by administration of TLR3 ligand polyinosinic-polycytidylic acid (poly IC) or recombinant human (r(h)) IFN-β. The expression of TMEM2, IFN-β, ISG56, C-X-C motif chemokine ligand 10 (CXCL10) and hyaluronan were evaluated appropriately by western blotting, quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. TMEM2 expression was not altered by poly IC stimulation. Knockdown of TMEM2 increased poly IC-induced expression of IFN-β, CXCL10, and ISG56, while IFN-β-induced expression of ISG56 and CXCL10 were not changed by TMEM2 knockdown. The hyaluronan concentration in the medium was decreased by either TMEM2 knockdown or poly IC, but additive or synergistic effects were not observed. TMEM2 knockdown enhanced TLR3-mediated IFN-β, CXCL10, and ISG56 expression in BEAS-2B cells. This implies that TMEM2 suppresses antiviral immune responses and prevents tissue injury in bronchial epithelial cells.

Research and experimental verification on the mechanisms of cellular senescence in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with high heterogeneity, poor prognosis, and a low 10-year survival rate of less than 50%. Although cellular senescence displays extensive effects on cancer, the comprehensions of cellular senescence-related characteristics in TNBC patients remains obscure. Single-cell RNA sequencing (scRNA-seq) data were analyzed by Seurat package. Scores for cellular senescence-related pathways were computed by single-sample gene set enrichment analysis (ssGSEA). Subsequently, unsupervised consensus clustering was performed for molecular cluster identification. Immune scores of patients in The Cancer Genome Atlas (TCGA) dataset and associated immune cell scores were calculated using Estimation of STromal and Immune cells in MAlignantTumours using Expression data (ESTIMATE) and Microenvironment Cell Populations-counter (MCP-counter), Tumor Immune Estimation Resource (TIMER) and Estimating the Proportion of Immune and Cancer cells (EPIC) methods, respectively. Immunotherapy scores were assessed using TIDE. Furthermore, feature genes were identified by univariate Cox and Least Absolute Shrinkage and Selection Operator (LASSO) regression analyses; these were used to construct a risk model. Additionally, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and transwell assay were conducted for in vitro validation of hub genes. TNBC was classified into three subtypes based on cellular senescence-related pathways as clusters 1, 2, and 3. Specifically, cluster 1 showed the best prognosis, followed by cluster 2 and cluster 3. The levels of gene expression in cluster 2 were the lowest, whereas these were the highest in cluster 3. Moreover, clusters 1 and 3 showed a high degree of immune infiltration. TIDE scores were higher for cluster 3, suggesting that immune escape was more likely in patients with the cluster 3 subtype who were less likely to benefit from immunotherapy. Next, the TNBC risk model was constructed and validated. RT-qPCR revealed that prognostic risk genes (MMP28, ACP5 and KRT6A) were up-regulated while protective genes (CT83) were down-regulated in TNBC cell lines, validating the results of the bioinformatics analysis. Meanwhile, cellular experiments revealed that ACP5 could promote the migration and invasion abilities in two TNBC cell lines. Finally, we evaluated the validity of prognostic models for assessing TME characteristics and TNBC chemotherapy response. In conclusion, these findings help to assess the efficacy of targeted therapies in patients with different molecular subtypes, have practical applications for subtype-specific treatment of TNBC patients, and provide information on prognostic factors, as well as guidance for the revelation of the molecular mechanisms by which senescence-associated genes influence TNBC progression.

TEAD1, MYO7A and NDUFC2 are novel functional genes associated with glucose metabolism in BXD recombinant inbred population.

The liver is an important metabolic organ that governs glucolipid metabolism, and its dysfunction may cause non-alcoholic fatty liver disease, type 2 diabetes mellitus, dyslipidaemia, etc. We aimed to systematic investigate the key factors related to hepatic glucose metabolism, which may be beneficial for understanding the underlying pathogenic mechanisms for obesity and diabetes mellitus. Oral glucose tolerance test (OGTT) phenotypes and liver transcriptomes of BXD mice under chow and high-fat diet conditions were collected from GeneNetwork. QTL mapping was conducted to pinpoint genomic regions associated with glucose homeostasis. Candidate genes were further nominated using a multi-criteria approach and validated to confirm their functional relevance in vitro. Our results demonstrated that plasma glucose levels in OGTT were significantly affected by both diet and genetic background, with six genetic regulating loci were mapped on chromosomes 1, 4, and 7. Moreover, TEAD1, MYO7A and NDUFC2 were identified as the candidate genes. Functionally, siRNA-mediated TEAD1, MYO7A and NDUFC2 knockdown significantly decreased the glucose uptake and inhibited the transcription of genes related to insulin and glucose metabolism pathways. Our study contributes novel insights to the understanding of hepatic glucose metabolism, demonstrating the impact of TEAD1, MYO7A and NDUFC2 on mitochondrial function in the liver and their regulatory role in maintaining in glucose homeostasis.

Differentiating patients admitted primarily due to coronavirus disease 2019 (COVID-19) from those admitted with incidentally detected severe acute respiratory syndrome corona-virus type 2 (SARS-CoV-2) at hospital admission: A cohort analysis of German hospital records.

The number of hospitalized patients with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) does not differentiate between patients admitted due to coronavirus disease 2019 (COVID-19) (ie, primary cases) and incidental SARS-CoV-2 infection (ie, incidental cases). We developed an adaptable method to distinguish primary cases from incidental cases upon hospital admission. Retrospective cohort study. Data were obtained from 3 German tertiary-care hospitals. The study included patients of all ages who tested positive for SARS-CoV-2 by a standard quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay upon admission between January and June 2022. We present 2 distinct models: (1) a point-of-care model that can be used shortly after admission based on a limited range of parameters and (2) a more extended point-of-care model based on parameters that are available within the first 24-48 hours after admission. We used regression and tree-based classification models with internal and external validation. In total, 1,150 patients were included (mean age, 49.5±28.5 years; 46% female; 40% primary cases). Both point-of-care models showed good discrimination with area under the curve (AUC) values of 0.80 and 0.87, respectively. As main predictors, we used admission diagnosis codes (ICD-10-GM), ward of admission, and for the extended model, we included viral load, need for oxygen, leucocyte count, and C-reactive protein. We propose 2 predictive algorithms based on routine clinical data that differentiate primary COVID-19 from incidental SARS-CoV-2 infection. These algorithms can provide a precise surveillance tool that can contribute to pandemic preparedness. They can easily be modified to be used in future pandemic, epidemic, and endemic situations all over the world.

Structural characterization and calcium absorption-promoting effect of sucrose-calcium chelate in Caco-2 monolayer cells and mice.

Sucrose emerges as a chelating agent to form a stable sucrose-metal-ion chelate that can potentially improve metal-ion absorption. This study aimed to analyze the structure of sucrose-calcium chelate and its potential to promote calcium absorption in both Caco-2 monolayer cells and mice. The characterization results showed that calcium ions mainly chelated with hydroxyl groups in sucrose to produce sucrose-calcium chelate, altering the crystal structure of sucrose (forming polymer particles) and improving its thermal stability. Sucrose-calcium chelate dose dependently increased the amount of calcium uptake, retention, and transport in the Caco-2 monolayer cell model. Compared to CaCl2 , there was a significant improvement in the proportion of absorbed calcium utilized for transport but not retention (93.13±1.75% vs. 67.67±7.55%). Further treatment of calcium channel inhibitors demonstrated the active transport of sucrose-calcium chelate through Cav1.3. Cellular thermal shift assay and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays indicated that the ability of sucrose-calcium chelate to promote calcium transport was attributed to its superior ability to bind with PMCA1b, a calcium transporter located on the basement membrane, and stimulate its gene expression compared to CaCl2 . Pharmacokinetic analysis of mice confirmed the calcium absorption-promoting effect of sucrose-calcium chelate, as evident by the higher serum calcium level (44.12±1.90mg/L vs. 37.42±1.88mmol/L) and intestinal PMCA1b gene expression than CaCl2 . These findings offer a new understanding of how sucrose-calcium chelate enhances intestinal calcium absorption and could be used as an ingredient in functional foods to treat calcium deficiency. PRACTICAL APPLICATION: The development of high-quality calcium supplements is crucial for addressing the various adverse symptoms associated with calcium deficiency. This study aimed to prepare a sucrose-calcium chelate and analyze its structure, as well as its potential to enhance calcium absorption in Caco-2 monolayer cells and mice. The results demonstrated that the sucrose-calcium chelate effectively promoted calcium absorption. Notably, its ability to enhance calcium transport was linked to its strong binding with PMCA1b, a calcium transporter located on the basement membrane, and its capacity to stimulate PMCA1b gene expression. These findings contribute to a deeper understanding of how the sucrose-calcium chelate enhances intestinal calcium absorption and suggest its potential use as an ingredient in functional foods for treating calcium deficiency.

Assessment of the yellow fever outbreak in Angola from December 2015 through December 2016: A retrospective study.

The acute tropical infectious disease known as yellow fever (YF) is caused by an arbovirus and is characterized by fever, jaundice, hemorrhage, headache, muscle pain, nausea, vomiting, and fatigue. Angola experienced a yellow fever virus (YFV) outbreak that was documented in December 2015. However, little is known about the outcome of this outbreak. We aimed to demonstrate epidemic features and lessons learned during the YF epidemic in Angola. A total of 4618 blood samples from suspected YF cases were sent to the Instituto Nacional de Investigação em Saúde (INIS), a national referral and public health laboratory, between December 5, 2015, and December 23, 2016. Sample analyses were conducted using enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) assays. Blood samples were sent from 16 out of the 18 provinces of Angola. We detected 884 (19.1%) cases that were positive for ELISA, which were confirmed by RT-PCR assay. Considering the positive cases, the incidence among male patients was around three times higher (n = 223; 10.9%) than in female patients (n = 59; 2.6%) in the 20-29 age group, followed by the age group 10-19 with n = 211 (6.8%) in males versus n = 108 (3.3%) in females; and the age group 30-39 had n = 68 (4.8%) in males versus n = 28 (1.8%) in females. The other groups had an incidence below 3.0%. The case fatality ratio for YF was in young adults in the age group 20-29 with n = 39 cases, followed by the age group 10-19 with n = 16 cases, and finally the age group 0-9 with n = 13 cases. The other age groups had several deaths by YF below 10 cases. This study demonstrates features of the YF epidemic that occurred in Angola. Also, it demonstrates that YF causes deaths in young people but is preventable by high vaccine coverage. Thus, public health laboratory surveillance must be strengthened to reduce the possibility of emerging and re-emerging human infections.

Sensitive and Visual Detection of Brassica Yellows Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification-Coupled CRISPR-Cas12 Assay.

Brassica yellows virus (BrYV) is an economically important virus on cruciferous species. In this study, a one-pot reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was developed for the detection of BrYV. The limit of detection of this method reached 32.8 copies of the BrYV ORF5, which is 100-fold more sensitive than the RT-LAMP method. Moreover, there was no cross-reactivity with other rapeseed-infecting RNA viruses or poleroviruses. We dried the CRISPR/Cas12a reagent in a trehalose and pullulan mixture to retain its efficacy at the RT-LAMP temperature of 63°C in order to allow portable BrYV detection in a water bath. The entire process can be performed in about 1 h, and a positive result can be rapidly and conveniently detected using a handheld UV lamp. In the field, the RT-LAMP-CRISPR/Cas12a assay was accurate and had higher sensitivity than RT-LAMP and reverse transcription-polymerase chain reaction assays. The novel RT-LAMP-CRISPR/Cas12a assay allows convenient, portable, rapid, low-cost, highly sensitive, and specific detection of BrYV and has great potential for on-site monitoring of BrYV.