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Wastewater-based epidemiology (WBE) has become a valuable tool for monitoring the prevalence of SARS-CoV-2 on university campuses. However, concerns about effectiveness of raw sewage as a COVID-19 early warning system still exist, and its not clear how useful normalization by simultaneous comparison of Pepper Mild Mottle Virus (PMMoV) is in addressing variations resulting from fecal discharge dilution. This study aims to contribute insights into these aspects by conducting an academic-year field trial at the student residences on the University of Tennessee, Knoxville campus, raw sewage. This was done to investigate the correlations between SARS-CoV-2 RNA load, both with and without PMMoV normalization, and various parameters, including active COVID-19 cases, self-isolations, and their combination among all student residents. Significant positive correlations between SARS-CoV-2 RNA load a week prior, during the monitoring week, and the subsequent week with active cases. Despite these correlations, normalization by PMMoV does not enhance these associations. These findings suggest the potential utility of SARS-CoV-2 RNA load as an early warning indicator and provide valuable insights into the application and limitations of WBE for COVID-19 surveillance specifically within the context of raw sewage on university campuses.

The 4T1 model is extensively employed in murine studies to elucidate the mechanisms underlying the carcinogenesis of triple-negative breast cancer. Molecular biology serves as a cornerstone in these investigations. However, accurate gene expression analyses necessitate data normalization employing housekeeping genes (HKGs) to avert spurious results. Here, we initially delve into the characteristics of the tumor evolution induced by 4T1 in mice, underscoring the imperative for additional tools for tumor monitoring and assessment methods for tracking the animals, thereby facilitating prospective studies employing this methodology. Subsequently, leveraging various software platforms, we assessed ten distinct HKGs (GAPDH, 18 S, ACTB, HPRT1, B2M, GUSB, PGK1, CCSER2, SYMPK, ANKRD17) not hitherto evaluated in the 4T1 breast cancer model, across tumors and diverse tissues afflicted by metastasis. Our principal findings underscore GAPDH as the optimal HKG for gene expression analyses in tumors, while HPRT1 emerged as the most stable in the liver and CCSER2 in the lung. These genes demonstrated consistent expression and minimal variation among experimental groups. Furthermore, employing these HKGs for normalization, we assessed TNF-α and VEGF expression in tissues and discerned significant disparities among groups. We posit that this constitutes the inaugural delineation of an ideal HKG for experiments utilizing the 4T1 model, particularly in vivo settings. [ABSTRACT FROM AUTHOR]

Background: MADS-box transcription factors have been shown to be involved in multiple developmental processes, including the regulation of floral organ formation and pollen maturation. However, the role of the MADS-box gene family in floral development of the alpine plant species Coptis teeta Wall, which is widely used in Traditional Chinese Medicine (TCM), is unknown. Results: Sixty-six MADS-box genes were identified in the C. teeta genome. These genes were shown to be unevenly distributed throughout the genome of C. teeta. The majority of which (49) were classified as type I MADS-box genes and were further subdivided into four groups (Mα, Mβ, Mγ and Mδ). The remainder were identified as belonging to the type II MADS-box gene category. It was observed that four pairs of segmental and tandem duplication had occurred in the C. teeta MADS-box gene family, and that the ratios of Ka/Ks were less than 1, suggesting that these genes may have experienced purifying selection during evolution. Gene expression profiling analysis revealed that 38 MADS-box genes displayed differential expression patterns between the M and F floral phenotypes. Sixteen of these MADS-box genes were further verified by RT-qPCR. The 3D structure of each subfamily gene was predicted, further indicating that MADS-box genes of the same type possess structural similarities to the known template. Conclusions: These data provide new insights into the molecular mechanism of dichogamy and herkogamy formation in C. teeta and establish a solid foundation for future studies of the MADS-box genes family in this medicinal plant species. [ABSTRACT FROM AUTHOR]

Background: The COVID-19 pandemic has been the most significant health challenge of the last century. Multiple and successive waves of COVID-19 cases, driven particularly by the emergence of new SARS-CoV-2 variants, have kept the world in a constant state of alert. Methods: We present an observational, descriptive, cross-sectional study aimed at identifying SARS-CoV-2 variants circulating during two local waves of COVID-19 cases in southern Bahia, Brazil (late 2021 and late 2022), and analyzing the association between the detected variants and the epidemiological and clinical characteristics of the disease. For this purpose, data and nasopharyngeal samples from individuals in southern Bahia, Brazil, with suspected COVID-19 were included. Viral detection was performed by RT-qPCR, and SARS-CoV-2 variants were identified by next-generation viral sequencing. Results: A total of 368 nasopharyngeal samples were tested. Approximately 23% of the samples from late 2021 tested positive for SARS-CoV-2, while in 2022, the positivity rate was about 56%. All sequenced samples from 2021 were identified as the Delta variant, while in 2022, all samples were classified as the Omicron variant. Overall, individuals who tested positive for SARS-CoV-2 in 2022 were younger than those who tested positive in 2021. Moreover, we observed significant differences in the clinical spectrum of SARS-CoV-2 infection when comparing the two periods. Individuals who presented with anosmia/ageusia were more likely to test positive for SARS-CoV-2 infection in 2021 but not in 2022. Additionally, fever, dry cough, pharyngalgia, headache, and rhinorrhea were more frequent among individuals infected with the Omicron variant than among those infected with the Delta variant. Conclusions: The profile of COVID-19 in southern Bahia differed when analyzing two distinct waves of the pandemic in the region. These differences are likely related to the variants, which may differ in transmissibility and virulence, thereby altering the dynamics of the pandemic. This underscores the importance of genomic surveillance in better understanding the behavior of viral infections. [ABSTRACT FROM AUTHOR]

Introduction: Severe acute respiratory infection (SARI) is mainly caused by viral pathogens, with a high prevalence in high-risk populations such as infants and older adults. Coinfections by different viruses are frequent and, in some cases, associated with severe disease outcomes. Purpose: The main purpose of this study was to identify respiratory viruses circulating in Barranquilla during the peaks of the COVID-19 pandemic and estimate the prevalence of viral coinfections in samples from individuals with different degrees of respiratory infection. Methods: We received 5083 samples between epidemiological weeks 33–42 of 2021 submitted by the District Health Laboratory of Barranquilla and four local healthcare institutions during COVID-19 surveillance. Among them, we analyzed 101 samples from individuals presenting with influenza-like illness (ILI). Eighteen respiratory viruses, including SARS-CoV-2, were evaluated via qRT-PCR using nasal swabs or nasopharyngeal aspirate samples. Results: Of the 101 study individuals, 56 were male and 45 were female (55.5% and 44.5%, respectively); 25.7% of individuals were infected with at least one of the evaluated viruses. Respiratory syncytial virus (RSV) and human rhinovirus (HRV) were the two most frequently detected viruses (30.7% and 15.4% of total positives, respectively). Coinfections with two or more respiratory viruses accounted for 42% of the total positive cases. Discussion: Our findings indicate the presence of different respiratory viruses in swab or nasopharyngeal aspirate samples from individuals with ILI, including coinfections. These results reveal the circulation of several respiratory viruses in the city of Barranquilla, confirming their importance as potential causes of SARI in Colombia and the need for their active surveillance. [ABSTRACT FROM AUTHOR]

The study of the pathogenesis of febrile seizures and their consequences frequently necessitates gene expression analysis. The primary methodology employed for such analysis is reverse transcription with quantitative polymerase chain reaction (RT-qPCR). To ensure the accuracy of data obtained by RT-qPCR, it is crucial to utilize stably expressed reference genes. The objective of this study was to identify the most suitable reference genes for use in the analysis of mRNA production in various brain regions of rats following prolonged neonatal febrile seizures. The expression stability of eight housekeeping genes was evaluated using the online tool RefFinder in the dorsal and ventral hippocampal regions and in the temporal and medial prefrontal cortex of the brain. The Ppia gene exhibited the greatest stability of expression. Conversely, the genes with the least stable expression levels were Actb and Ywhaz; thus, it is not recommended to use them for normalization in a febrile seizure model. Additionally, the majority of housekeeping genes demonstrate age-related, region-specific fluctuations. Therefore, it is crucial to employ the appropriate housekeeping genes for each brain structure under investigation when examining the expression dynamics of genes of interest in a febrile seizure model. [ABSTRACT FROM AUTHOR]

Enterococci are ubiquitous microorganisms in almost all environments, from the soil we step on to the food we eat. They are frequently found in naturally fermented foods, contributing to ripening through protein, lipid, and sugar metabolism. On the other hand, these organisms are also leading the current antibiotic resistance crisis. In this study, we performed whole-genome sequencing and comparative genomics of an Enterococcus faecium strain isolated from an artisanal Mexican Cotija cheese, namely QD-2. We found clear genomic differences between commensal and pathogenic strains, particularly in their carbohydrate metabolic pathways, resistance to vancomycin and other antibiotics, bacteriocin production, and bacteriophage and CRISPR content. Furthermore, a bacteriocin transcription analysis performed by RT-qPCR revealed that, at the end of the log phase, besides enterocins A and X, two putative bacteriocins not reported previously are also transcribed as a bicistronic operon in E. faecium QD-2, and are expressed 1.5 times higher than enterocin A when cultured in MRS broth. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a ubiquitous and psychrotrophic foodborne pathogen commonly found in raw materials, ready-to-eat products, and food environments. We previously demonstrated that L. monocytogenes can grow faster at low temperature when unsaturated fatty acids (UFA) are present in its environment. This could question the maintenance of food safety for refrigerated foods, especially those reformulated with a higher ratio of UFA versus saturated fatty acids (SFA) to fit with nutritional recommendations. In this study, we used transcriptomics to understand the impact of UFA on the behavior of L. monocytogenes at low temperature. We first demonstrated that fabK, a key gene in SFA synthesis, is up-regulated in the presence of UFA but not SFA at low temperature. L. monocytogenes can thus regulate the synthesis of SFA in its membrane according to the type of FA available in its environment. Interestingly, we also observed up-regulation of genes involved in chemotaxis and flagellar assembly (especially cheY and flaA) in the presence of UFA but not SFA at low temperature. TEM observations confirmed that L. monocytogenes acquired a remarkable phenotype with numerous and long-looped flagella only in the presence of UFA at 5°C but not at 37°C. As flagella are well known to be involved in biofilm formation, this new finding raises questions about the structure and persistence of biofilms settled in refrigerated environments using unsaturated lipid-rich products. [ABSTRACT FROM AUTHOR]

AbstractCaralluma europaea Guss. (C. europaea) is a medicinal plant used for cancer treatment. However, these treatments may be associated with complications that need to be investigated. This work aims to evaluate not only the chemical composition but also the hepatoprotective and anticancer properties of C. europaea extracts. The chemical constitution of the hydroethanolic extract was explored using gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). The hydroethanolic extract, flavonoids, and polyphenols-rich extract at 100, 15, and 50 mg/kg, respectively, were administered to acetaminophen-treated rats for seven days. We used Western blotting and Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) to determine the protein and the mRNA levels of cancer stemness markers in pancreatic cancer cell lines MIA PaCa-2 and BxPC-3 exposed to increasing doses of C. europaea extracts. In silico analysis was used to evaluate the effects of phenolic compounds revealed in C. europaea on caspase-3 and HSP90, and on liver damage on CYP2E1. The primary phenolics detected by GC-MS and HPLC were ferulic acid and benzofurazan. The positive control group showed an increase in AST, ALT, ALP, triglycerides, and VLDL levels. C. europaea extracts demonstrated hepatoprotective effects by ameliorating acetaminophen-induced alterations of biochemical and hispathological parameters. Immunoblotting and RT-qPCR profiling of cancer stemness markers indicated a reduction in the expression levels of Oct-4 and Nanog proteins, as well as a reduction in the mRNA levels of CD133 by 50–60% and Sox2 by 80–90% in pancreatic cancer cells. Molecular docking showed that naringenin presented the highest docking Gscore on CYP2E1 (−8.199) and HSP90 (−7.742). In conclusion, C. europaea extracts could be considered as a safe and promising therapeutic strategy to sensitize pancreatic cancer cells to chemotherapy. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has underscored the significance of monitoring genetic variations in pathogenic viruses to manage its transmission and identify emerging variants. Nonetheless, sequencing every positive patient case is impractical for large-scale testing of the population. Here, we introduce a cost-effective tool for identifying SARS-CoV-2 mutations associated with variants in wastewater. This approach relies on a wastewater surveillance system and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), utilizing mutation-specific probes. Wastewater samples were analyzed for SARS-CoV-2 mutations corresponding to variants under monitoring (VUM) (P.2, alpha, beta, gamma, delta, and omicron) as part of a wastewater-based SARS-CoV-2 monitoring program. Our findings validate the efficacy of this method in detecting SARS-CoV-2 mutations linked to variants in wastewater, facilitating early detection of emerging variants and informing decision-making on control measures. [ABSTRACT FROM AUTHOR]

Yellow Sigatoka, caused by the fungus Pseudocercospora musae, represents one of the most challenging diseases in bananas, which is aggravated due to the genetic variability of this pathogen. The main objective of this study was to characterize the infection process of P. musae in two banana cultivars with different levels of resistance and to quantify the expression of resistance genes. Inoculated and non-inoculated leaf samples of the two cultivars, Yangambi km 5—resistant and Grande Naine—susceptible, were investigated by light microscopy, histochemistry, scanning electron microscopy, and RT-qPCR. The cultivar Grande Naine showed early signs of P. musae infection, including the production of lipophilic substances and phenolic compounds, while the cultivar Yangambi km 5 was not affected. In the Grande Naine cultivar, the protein kinase gene was upregulated, while in the Yangambi km 5, it was repressed. The WAK2 gene was initially upregulated in both cultivars; however, in Grande Naine, it was subsequently repressed, while in Yangambi km 5, it remained downregulated. These findings have the potential to contribute to more effective management strategies in the control of yellow Sigatoka and the development of banana varieties resistant to yellow Sigatoka. [ABSTRACT FROM AUTHOR]

Objectives: Acute temperature stress was explored in Hippocampus abdominalis through a comprehensive RNA-seq analysis. Methods: RNA-seq was conducted on 20-day-old H. abdominalis after 24 h of temperature stress. Four experimental conditions were established: a control group (18 °C) and three temperature treatment groups (21, 24, and 27 °C). Results: Seahorse larvae were found to be unaffected by 21 °C and 24 °C and were able to survive for short periods of time during 24 h of incubation, whereas mortality approached 50% at 27 °C. The sequencing process produced 75.63 Gb of high-quality clean data, with Q20 and Q30 base percentages surpassing 98% and 96%, respectively. A total of 141, 333, and 1598 differentially expressed genes were identified in the 21, 24, and 27 °C groups vs. a control comparison group, respectively. Notably, the number of up-regulated genes was consistently higher than that of down-regulated genes across all comparisons. Gene Ontology functional annotation revealed that differentially expressed genes were predominantly associated with metabolic processes, redox reactions, and biosynthetic functions. In-depth KEGG pathway enrichment analysis demonstrated that down-regulated genes were significantly enriched in pathways related to steroid biosynthesis, terpenoid backbone biosynthesis, spliceosome function, and DNA replication. Up-regulated genes were enriched in pathways associated with the FoxO signaling pathway and mitophagy (animal). The results indicated that temperature stress induced extensive changes in gene expression in H. abdominalis, involving crucial biological processes such as growth, biosynthesis, and energy metabolism. Conclusions: This study provided key molecular mechanisms in the response of H. abdominalis to temperature stress, offering a strong basis for future research aimed at understanding and mitigating the effects of environmental stressors on marine species. [ABSTRACT FROM AUTHOR]

Studies have demonstrated the susceptibility of companion animals to natural infection with SARS-CoV-2. Using quantitative reverse transcription polymerase chain reaction and sequencing analyses, this study investigated SARS-CoV-2 RNA excretion in pets in households with infected owners. Oropharyngeal and rectal swabs were collected from dogs and cats in Parana, Southern Brazil, between October 2020 and April 2021. Viral RNA was detected in 25% of cats and 0.98% of dog oropharyngeal swabs; however, systemic, respiratory, and gastrointestinal signs were absent. Complete viral genomes belonged to the Gamma lineage. Phylogenetic analyses indicated that pet samples were probably derived from human-positive cases in Parana. Viral excretion in the oropharynx was more frequent in cats than in dogs. Mutations in the S protein characteristic of Gamma strains were present in all sequenced SARS-CoV-2 strains. The receptor-binding domain of these Brazilian strains did not show any additional mutations not reported in the Gamma strains. Mutations in NSP6, NSP12, and N proteins previously mapped to strains that infect deer or minks were detected. This study highlights the importance of actively monitoring the SARS-CoV-2 strains that infect pets with continued viral exposure. Monitoring genetic changes is crucial because new variants adapted to animals may pose human health risks. [ABSTRACT FROM AUTHOR]

Most eukaryotic genes generate multiple messenger RNA (mRNA) transcript variants by alternative splicing. The incomplete annotation of gene transcripts in genomic databases can result in improper primer design, adversely affecting the reliability of gene expression measurements by reverse transcriptase quantitative polymerase chain reaction (RT‐qPCR). Hence, we present a workflow combining bioinformatics analyses, to select two to three evolutionarily conserved constitutive exons in rats, mice and humans as target sequences for PCR primer design, with experimental RT‐PCR amplification and amplicon sequencing to confirm the expression and identity of gene transcript targets. The application of this workflow to the characterization of neurodevelopmental biomarker genes identified an unannotated exon in the rat Map2 gene, illustrating the importance of target sequence validation for the development of translational mRNA biomarkers for toxicological and biomedical studies. [ABSTRACT FROM AUTHOR]

Small non-coding RNAs (sRNAs, also called sncRNAs) known as gene expression regulatory factors are capable of modulating mRNA functions through complementary base pairing. A number of studies has shown that when exposed to radiation, the expression of drug resistance genes increases in some cells. Here, in Escherichia coli subjected to 12C6+ heavy ion beams or X-ray exposure, five sRNAs (sRNA120, sRNA127, sRNA109, sRNA72, sRNA53) with elevated expression were identified by deep sequencing and sRNAscanner prediction. To investigate whether they have a potential role in drug resistance, we engineered strains overexpressing these sRNAs, and assessed their survival rate under sub-lethal antibiotic concentrations. It was noteworthy that under Gentamicin, Norfloxacin, Chloramphenicol and Cfotaxime, the survival rate of ::sRNA53 strain was 1.0667, 1.0251, 1.3797 and 3.9327 times higher, respectively, than for the control and strains overexpressing other sncRNAs. TargetRNA2 software identified lsrA as a likely target gene regulated by sRNA53 based on binding free energy calculations. We analyzed the interaction sites between sRNA53 and lsrA and measured the expression of these molecules in various mutants using RT-qPCR. We also investigated the regulation of Luxs/AI-2 system by the lsr operon and the biofilm formation of mutants. In the ::sRNA53 strain, the log2FC for sRNA53 and lsrA concurrently escalated by 1.8533 and 1.7367-fold. Additionally, the biofilm formation ability of ::sRNA53, ::lsrA, and ::sRNA53::lsrA (co-expression) strains was increased 5.4542, 3.946, and 7.1758-fold, respectively, compared to wild-type MG1655. Based on these data, we can conclude that sRNA53 plays a critical role in the development of antibiotic resistance in E. coli. Apparently, the action of sRNA53 targets the lsrA gene, which, by modulating the Luxs/AI-2 system, affects the ability to biofilm formation and drug resistance of the bacterium. The study shows that a new sRNA, named sRNA53, is involved in the formation of tolerance to sub-lethal doses of various antibiotics. [ABSTRACT FROM AUTHOR]

Breeding abiotic stress-tolerant varieties of Rosa chinensis is a paramount goal in horticulture. WRKY transcription factors, pivotal in plant responses to diverse stressors, offer potential targets for enhancing stress resilience in R. chinensis. Using bioinformatics and genomic data, we identified RcWRKY transcription factor genes, characterised their chromosomal distribution, phylogenetic relationships, structural attributes, collinearity, and expression patterns in response to saline stress. Leveraging bidirectional database searches, we pinpointed 66 RcWRKY genes, categorised into three groups. All except RcWRKY60 encoded DNA Binding Domain and Zinc Finger Motif regions of the WRKY domain. Expansion of the RcWRKY gene family was propelled by 19 segmental, and 2 tandem, duplications. We unveiled 41 and 15 RcWRKY genes corresponding to 50 AtWRKY and 17 OsWRKY orthologs respectively, indicating postdivergence expansion. Expression analyses under alkaline stress pinpointed significant alterations in 54 RcWRKY genes. Integration of functional roles from their Arabidopsis orthologs and cis -acting elements within their promoters, along with quantitative reverse transcription PCR validation, underscored the importance of RcWRKY27 and 29 in R. chinensis ' alkaline stress response. These findings offer insights into the biological roles of RcWRKY transcription factors, as well as the regulatory dynamics governing R. chinensis ' growth, development, and stress resilience. The China rose (Rosa chinensis) is known as the 'queen of flowers', and is commercially cultivated globally. To enable breeders to develop varieties tolerant to alkaline soils, we investigated the genes involved in their stress response. This research will also contribute to the broader understanding of plant stress responses and offer potential avenues for enhancing stress tolerance in crop species. [ABSTRACT FROM AUTHOR]

Neuroinflammation is critical to the pathogenesis and progression of epileptic seizures, and the inflammatory pathway is controlled by an interplay of highly regulated mediators such as cytokines. The combination of treadmill exercise program and curcumin administration has been shown to offer neuroprotection against epileptic seizure but without detailed mechanism of action being elucidated. This study therefore evaluated the responses of pro-inflammatory (tumor necrosis factor alpha [TNFα] and interleukin 6 [IL-6]) and anti-inflammatory (interleukin 10 [IL-10]) cytokines, as well as the transcription factor regulator (nuclear factor kappa B [NFκB]) gene expressions to combinatory therapy of curcumin and treadmill exercise in cortex and hippocampus of pilocarpine-induced epileptic seizure in rats. The rats were grouped into control, pilocarpine-induced, pilocarpine + curcumin, pilocarpine + curcumin + exercise for 14 days thereafter; total ribonucleic acid (RNA) was extracted from the cortex and hippocampus and used for reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay for TNFα, IL-6, IL-10, and NFκB. The results revealed that in pilocarpine-induced rats' cortex and hippocampus, the expressions of TNFα, IL-6, and NFκB were significantly upregulated while that of IL-10 was significantly downregulated. However, treatment with either curcumin or curcumin plus exercise significantly ameliorated these impairments in the inflammatory markers 'expression levels. Nevertheless, the combinatory therapy of curcumin and exercise showed significantly higher ameliorative effects in the cortical and hippocampal TNFα and IL-10, as well as cortical NFκB and IL-6. These superior therapeutic properties of the combination of curcumin and treadmill exercise further support the therapeutic potentials of this combinatory regimen in the management of epileptic seizures. Nonetheless, further studies including clinical observations are highly recommended. [ABSTRACT FROM AUTHOR]

Urothelial carcinoma (UC) is the most common urogenital cancer in dogs. With early diagnosis, the disease can be controlled, to reduce progression of disease, in most dogs with a good quality of life. MicroRNAs (miRNAs) have been identified as a potential diagnostic and prognostic tool due to their stability and presence in both bodily fluids and tissues. MiRNAs have been frequently researched in human medicine and human UC; however, few manuscripts regarding miRNAs in canine UC are available. A search was performed on both PubMed and Google Scholar evaluating original research manuscripts with experimentally validated results for the terms "canine" or "dog"; "urothelial carcinoma," "bladder cancer," "transitional cell carcinoma," "TCC," "MIBC," "IMBUC," or "BLCA"; and "miRNA" or "microRNA." We identified 3 peer-reviewed manuscripts evaluating miRNA expression in canine UC and compared the reported miRNA expression studies to human UC to identify experimentally validated targets of the dysregulated miRNA. In this review, we highlight the similarities and differences between what is reported in canine UC and human UC and discuss the literature gaps that call for further evaluation. [ABSTRACT FROM AUTHOR]

Probiotics, which are prevalent in camel milk (CM) and naturally fermented camel milk (FCM), can regulate the intestinal ecological structure to alleviate alcoholic liver disease (ALD) through the "gut–liver" axis. The protective effects and mechanisms of CM and FCM interventions on alcohol-induced acute liver injury were investigated by combining the behavior observed in rats following alcohol exposure. The results revealed that CM and FCM effectively controlled the increased levels of alcohol-induced ALT, AST, TG, MDA, and proinflammatory cytokines. Alcohol-induced oxidative depletion of hepatic CAT, GPX, GSH, and ALDH was reversed, diminishing lipid accumulation, ameliorating severe pathological damage, increasing antioxidant capabilities, and postponing oxidative stress. Additionally, the abundance of the phylum Bacteroidota (which reduces the F/B ratio); the family Prevotellaceae; the genera Clostridia_vadinBB60_group, parabacteroides, Alloprevotella, and Prevotellaceae_UC_G001; the gastrointestinal barrier; and the microbiological environment was increased. The steroid hormone biosynthesis pathway was altered to reduce alcohol-induced predominant steroid metabolites such as 17-hydroxyprogesterone, cortisol, and dehydroepiandrosterone, preventing alcoholic liver impairment. Taken together, CM could be a therapeutic dietary supplement for preventing alcoholic liver injury by ameliorating the intestinal ecology and hepatic metabolism. [ABSTRACT FROM AUTHOR]

Oral cancer holds the sixth position in malignancies worldwide; 90% correspond to oral squamous cell carcinoma (OSCC). Diverse reports suggest that NTRK genes and their receptors are key oncogenesis regulators to tumor progression in human cancers. Objective: To analyze the NTRK and Trk expression and their association with clinicopathological features of OSCC in Mexican patients' samples. Material and Methods: We analyzed 95 OSCC cases of pan-trk immunoexpression through a software-assisted method. Gene expression was analyzed by RT-qPCR employing the ΔΔCT method. Kruskal–Wallis and Spearman's correlation tests were performed. Results: Our mean age was 62.4 (±16.9) years. A total of 37 cases were tumors in the lateral border of the tongue. Age was significantly associated with the anatomical site. 42% (40 of 95) cases were pan-trk positive. A total of 21 cases showed intense immunoexpression predominantly in poorly differentiated OSCC, with a significant correlation between immunoexpression and age and gender. Gene expression showed that poorly differentiated cases exhibited higher NTRK2 expression, while well-differentiated cases demonstrated NTRK3 significantly higher expression. Conclusions: Our results suggest that NTRK family expression is present in OSCC, with differential expression related to differentiation degree. Additional information about their activation or mutational status could reinforce their potential as a possible primary or adjuvant treatment target. [ABSTRACT FROM AUTHOR]

Wastewater surveillance for SARS-CoV-2 provides a broad assessment of community health since wastewater represents all community members, regardless of health care access and status of health (i.e., symptomatic and asymptomatic). Wastewater surveillance also provides early detection of disease transmission since the virus can be present in human waste before the presentation of clinical symptoms. We analyzed wastewater from Lehigh University (Bethlehem, PA) as well as the greater Bethlehem community for SARS-CoV-2 (N1, N2, and E genes) from August 2020 through May 2021. Total RNA was extracted and analyzed for SARS-CoV-2 and pepper mild mottle virus (PMMoV) by RT-qPCR. Of 73 Bethlehem wastewater samples, the number of positive samples depended on which SARS-CoV-2 gene was screened: 62 (84.9%) for N1, 52 (71.2%) for E, and 28 (38.4%) for N2. Of 67 university wastewater samples, the numbers of positive samples were 59 (88.1%) for N1, 51 (76.1%) for E, and 31 (46.3%) for N2. Temporal trends of SARS-CoV-2 in wastewater mirrored trends in COVID-19 positive cases in the Bethlehem community. Normalizing SARS-CoV-2 RNA concentrations to PMMoV (a human fecal indicator) increased the correlation between both N1 (ρ increased from 0.37 to 0.72) and E (ρ increased from 0.32 to 0.61) concentrations in wastewater with COVID-19 cases in the community. [ABSTRACT FROM AUTHOR]

During the COVID-19 pandemic, wastewater surveillance was used to monitor community transmission of SARS-CoV-2. As new genetic variants emerged, the need for timely identification of these variants in wastewater became an important focus. In response to increased reports of Omicron transmission across the United States, the Oklahoma Wastewater Surveillance team utilized allele-specific RT-qPCR assays to detect and differentiate variants, such as Omicron, from other variants found in wastewater in Oklahoma. The PCR assays showed presence of the Omicron variant in Oklahoma on average two weeks before official reports, which was confirmed through genomic sequencing of selected wastewater samples. Through continued surveillance from November 2021 to January 2022, we also demonstrated the transition from prevalence of the Delta variant to prevalence of the Omicron variant in local communities. We further assessed how this transition correlated with certain demographic factors characterizing each community. Our results highlight RT-qPCR assays as a rapid, simple, and cost-effective method for monitoring the community spread of SARS-CoV-2 genetic variants in wastewater. Additionally, they demonstrate that specific demographic factors such as ethnic composition and household income can correlate with the timing of SARS-CoV-2 variant introduction and spread. [ABSTRACT FROM AUTHOR]

Background: SET domain-containing histone lysine methyltransferases (HKMTs) and JmjC domain-containing histone demethylases (JHDMs) are essential for maintaining dynamic changes in histone methylation across parasite development and infection. However, information on the HKMTs and JHDMs in human pathogenic piroplasms, such as Babesia duncani and Babesia microti, and in veterinary important pathogens, including Babesia bigemina, Babesia bovis, Theileria annulata and Theileria parva, is limited. Results: A total of 38 putative KMTs and eight JHDMs were identified using a comparative genomics approach. Phylogenetic analysis revealed that the putative KMTs can be divided into eight subgroups, while the JHDMs belong to the JARID subfamily, except for BdJmjC1 (BdWA1_000016) and TpJmjC1 (Tp Muguga_02g00471) which cluster with JmjC domain only subfamily members. The motifs of SET and JmjC domains are highly conserved among piroplasm species. Interspecies collinearity analysis provided insight into the evolutionary duplication events of some SET domain and JmjC domain gene families. Moreover, relative gene expression analysis by RT‒qPCR demonstrated that the putative KMT and JHDM gene families were differentially expressed in different intraerythrocytic developmental stages of B. duncani, suggesting their role in Apicomplexa parasite development. Conclusions: Our study provides a theoretical foundation and guidance for understanding the basic characteristics of several important piroplasm KMT and JHDM families and their biological roles in parasite differentiation. [ABSTRACT FROM AUTHOR]

Rodents are commonly used as animal models in studies investigating various experimental conditions, often requiring gene expression analysis. Quantitative real-time reverse transcription PCR (RT-qPCR) is the most widely used tool to quantify target gene expression levels under different experimental conditions in various biological samples. Relative normalization with reference genes is a crucial step in RT-qPCR to obtain reliable quantification results. In this work, the main reference genes used in gene expression studies among the three rodents commonly employed in scientific research—hamster, rat, and mouse—are analyzed and described. An individual literature search for each rodent was conducted using specific search terms in three databases: PubMed, Scopus, and Web of Science. A total of 157 articles were selected (rats = 73, mice = 79, and hamsters = 5), identifying various reference genes. The most commonly used reference genes were analyzed according to each rodent, sample type, and experimental condition evaluated, revealing a great variability in the stability of each gene across different samples and conditions. Classic genes, which are expected to be stably expressed in both samples and conditions analyzed, demonstrated greater variability, corroborating existing concerns about the use of these genes. Therefore, this review provides important insights for researchers seeking to identify suitable reference genes for their validation studies in rodents. [ABSTRACT FROM AUTHOR]

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Plant defence mechanisms, including physical barriers like toughened bark and chemical defences like allelochemicals, are essential for protecting them against pests. Trees allocate non-structural carbohydrates (NSCs) to produce secondary metabolites like monoterpenes, which increase during biotic stress to fend off pests like the Eurasian spruce bark beetle, ESBB (Ips typographus). Despite these defences, the ESBB infests Norway spruce, causing significant ecological damage by exploiting weakened trees and using pheromones for aggregation. However, the mechanism of sensing and resistance towards host allelochemicals in ESBB is poorly understood. We hypothesised that the exposure of ESBB to spruce allelochemicals, especially monoterpenes, leads to an upsurge in the important detoxification genes like P450s, GSTs, UGTs, and transporters, and at the same time, genes responsible for development must be compromised. The current study demonstrates that exposure to monoterpenes like R-limonene and sabiene effectively elevated detoxification enzyme activities. The differential gene expression (DGE) analysis revealed 294 differentially expressed (DE) detoxification genes in response to R-limonene and 426 DE detoxification genes in response to sabiene treatments, with 209 common genes between the treatments. Amongst these, genes from the cytochrome P450 family 4 and 6 genes (CP4 and CP6), esterases, glutathione S-transferases family 1 (GSTT1), UDP-glucuronosyltransferase 2B genes (UDB), and glucose synthesis-related dehydrogenases were highly upregulated. We further validated 19 genes using RT-qPCR. Additionally, we observed similar high expression levels of detoxification genes across different monoterpene treatments, including myrcene and α-pinene, suggesting a conserved detoxification mechanism in ESBB, which demands further investigation. These findings highlight the potential for molecular target-based beetle management strategies targeting these key detoxification genes. [ABSTRACT FROM AUTHOR]

Background: Plant biostimulants constitute a promising environmentally friendly alternative for increasing crop yield and tolerance to unfavorable conditions. Among various types of such formulations, botanical extracts are gaining more recognition as products supporting plant performance. Moreover, novel tools such as cold-plasma or low-pressure microwave plasma discharge are being proposed as techniques that might improve their efficacy. Elucidation of the biostimulant's mode of action requires complex research at a molecular level. Transcriptional changes occurring after biostimulant spraying might be investigated using RT-qPCR. However, this technique requires data normalization against stable endogenous controls. Results: Here, we tested the expression stability of ten candidate genes in soybean plants exposed to various biostimulants treatment. Selection of the best-performing reference genes was conducted using four algorithms (geNorm, NormFinder, BestKeeper, and ΔCt method). According to the obtained results, Bic-C2 (RNA-binding protein Bicaudal-C) and CYP (cyclophilin type peptidyl-prolyl cis–trans isomerase) showed highest expression stability, while expression of EF1B (elongation factor 1-beta) fluctuated the most among a tested set of candidate genes. Conclusions: Overall, we recommend using Bic-C2 together with CYP for the RT-qPCR data normalization in soybean biostimulation experiments. To our best knowledge, this is the first comprehensive study of reference genes stability in plants subjected to biostimulant treatment. The results of this study will aid in further biostimulant research in crop plants, facilitating analyses performed on the transcriptional level. [ABSTRACT FROM AUTHOR]

Amaranthus dubius, an African leafy vegetable (ALV), is an easy‐to‐grow, annual shrub and a highly nutritious food source, containing elevated levels of essential nutrients in the leaves. Many ALVs, including A. dubius, can tolerate salinity stress, enabling their cultivation on marginal land. However, the widespread propagation of A. dubius as a stable food source has thus far not been realised due partially to the high frequency at which hybridisation occurs, resulting in high genotypic and phenotypic variability. Therefore, to increase the agricultural output capacity of this species on salt‐affected marginal lands, it is important to screen, select and then clonally propagate the identified salinity‐tolerant genotypes to ensure true‐to‐type fidelity in the regenerated population. It is also important, thereafter, to elucidate their underlying gene expression of the stress response. The present study exposed 4‐week‐old A. dubius seedlings to 100, 200 and 400 mM NaCl to determine their degree of salt tolerance. Genotypes were then screened, selected and clonally propagated through cuttings, based on high growth rates and biomass, and salt tolerance. Generally, growth and physiological parameters decreased as substrate salinity increased. However, individual salt‐stressed genotypes demonstrated similar vigour to nonstressed plants and were able to maintain total protein and chlorophyll concentrations despite increasing salinity. The relative expression of an NHX1‐like transcript was quantified in 15 genotypes using degenerately primed real‐time qPCR. The relative expression of the putative NHX1 gene was 6.7 times greater in root tissues of seedlings treated with 400 mM NaCl (10.7 ± 1.8) compared to the roots of untreated seedlings (1.6 ± 1.3), and 2.8‐fold more than leaf tissues harvested from seedlings treated with 400 mM NaCl. Furthermore, the relative electrical conductivity (EC) of root tissues was 10 times greater than the EC of leaf tissues from the same 400 mM NaCl treatment. Numerous genotypes yielded similar chlorophyll content between 200 and 400 mM NaCl treatments, with genotypes salinity‐1 (S1) (3.5 ± 0.2 μg/cm2) and S34 (4.0 ± 0.4 μg/cm2) having the highest concentrations of chlorophyll in the 400 mM group, which was positively correlated with total protein content. Following micropropagation through direct organogenesis, selected clones maintained true‐to‐type traits such as similar chlorophyll, protein and NHX1‐like expression as their parent plants when exposed to 400 mM NaCl. This study revealed that some genotypes demonstrated salt stress tolerance capabilities rivalling established halophytes by regulating the constitutive or inducible expression of an NHX1‐like protein in roots and leaves. The correlation between protein content and NHX1‐like expression was nonlinear and nonproportional, demonstrating the complexity of this response and necessitating further exploration of specific protein families or functional groups conferring salinity tolerance in this species. [ABSTRACT FROM AUTHOR]

To explore the molecular mechanisms of the Litopenaeus vannamei response to infection by Photobacterium damselae, reveal its immune response and energetic metabolic effect, and provide a valuable genetic data source for the scientific prevention and control of Vibrio infection, transcriptomic analysis, RT-qPCR, and physiological and biochemical tests were conducted. The results showed that the expression of key genes involved in lipid and carbohydrate transport, such as apolipoprotein and TPS, was upregulated after pathogenic infection, which brought the accumulation of triacylglycerol and trehalose into the hemolymph. Additionally, the pathogenic infection selectively triggered an immune response in infected L. vannamei, activating certain immune pathways, such as the serpins and MAPK pathways. The pathogenic infection suppressed the activity of phenoloxidase (PO), and the prophenoloxidase (PPO) cascade responses were suppressed by the invasive bacteria. This paper will help us understand the energetic metabolism, immune response, and activation of the immune recognition response after pathogenic infection by P. damselae, and it lays a theoretical foundation for the biological prevention and control of P. damselae infection. [ABSTRACT FROM AUTHOR]

Background: RT-qPCR is a powerful strategy for recognizing the most appropriate reference genes, which can successfully minimize experimental mistakes through accurate normalization. Ludisia discolor, recognized for its ornamental value, features little, distinctive blossoms with twisted lips and gynostemium showing chiral asymmetry, together with striking blood-red fallen leaves periodically marked with golden blood vessels. Methods and Results: To ensure the accuracy of qRT-PCR, selecting appropriate reference genes for quantifying target gene expression levels is essential. This study aims to identify stable reference genes during the development of L. discolor. In this study, the entire floral buds, including the lips and gynostemium from different development stages, were taken as materials. Based upon the transcriptome information of L. discolor, nine housekeeping genes, ACT, HIS, EF1-α1, EF1-α2, PP2A, UBQ1, UBQ2, UBQ3, and TUB, were selected in this research study as prospect interior referral genes. The expression of these nine genes were found by RT-qPCR and afterwards comprehensively examined by four software options: geNorm, NormFinder, BestKeeper, and ΔCt. The outcomes of the analysis showed that ACT was the most steady gene, which could be the most effective inner referral gene for the expression evaluation of flower advancement in L. discolor. Conclusions: The results of this study will contribute to the molecular biology research of flower development in L. discolor and closely related species. [ABSTRACT FROM AUTHOR]

Beneficial fungi of the genus Trichoderma are among the most widespread biocontrol agents that induce a plant's defense response against pathogens. Fusarium solani is one of the main pathogens that can negatively affect Astragalus mongholicus production and quality. To investigate the impact of Trichoderma harzianum on Astragalus mongholicus defense responses to Fusarium solani, A. mongholicus roots under T. harzianum + F. solani (T + F) treatment and F. solani (F) treatment were sampled and subjected to transcriptomic analysis. A differential expression analysis revealed that 6361 differentially expressed genes (DEGs) responded to T. harzianum induction. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the 6361 DEGs revealed that the genes significantly clustered into resistance-related pathways, such as the plant–pathogen interaction pathway, phenylpropanoid biosynthesis pathway, flavonoid biosynthesis pathway, isoflavonoid biosynthesis pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and plant hormone signal transduction pathway. Pathway analysis revealed that the PR1, formononetin biosynthesis, biochanin A biosynthesis, and CHIB, ROS production, and HSP90 may be upregulated by T. harzianum and play important roles in disease resistance. Our study further revealed that the H2O2 content was significantly increased by T. harzianum induction. Formononetin and biochanin A had the potential to suppress F. solani. Weighted gene coexpression network analysis (WGCNA) revealed one module, including 58 DEGs associated with T. harzianum induction. One core hub gene, RPS25, was found to be upregulated by T. harzianum, SA (salicylic acid) and ETH (ethephon). Overall, our data indicate that T. harzianum can induce induced systemic resistance (ISR) and systemic acquired resistance (SAR) in A. mongholicus. The results of this study lay a foundation for a further understanding of the molecular mechanism by which T. harzianum induces resistance in A. mongholicus. [ABSTRACT FROM AUTHOR]

Context: Heat shock proteins play a vital role in cellular homeostasis by protecting proteins against various environmental stresses, which facilitates the survival of plants under unfavourable conditions. Aims: We aimed to provide the first comprehensive genomic and expression analysis of the HSP70 gene family in betel palm (Areca catechu) to elucidate its role in heat stress response. Methods: Genomic analysis revealed 34 putative HSP70 genes distributed across 13 chromosomes. These were renamed AcatHSP70 and classified into five subfamilies (A–E) based on phylogenetic analysis. These genes are mostly localised in the chloroplast, cytoplasm, and nucleus. Gene ontology revealed that these genes are mostly involved in heat stress. The gene duplication events of HSP70 genes involved only segmental duplications. We subjected betel palm seedlings (2 years old) to heat stress under controlled conditions for 30 days at high, low, and room temperatures for expression analyses of HSP70 genes. Key results: Expression analysis revealed eight putative candidate genes (AcatHSP70-3 , AcatHSP70-13 , AcatHSP70-22 , AcatHSP70-19 , AcatHSP70-21 , AcatHSP70-24 , AcatHSP70-25 , and AcatHSP70-26) that showed significantly higher expression under high-temperature stress. AcatHSP70-5 showed higher expression under low-temperature treatment, and AcatHSP70-16 was responsive at room temperature treatment. Conclusion: We conclude that the majority of AcatHSP70 genes play a crucial role under thermal stress conditions, and respond to high-temperature stress as shown by the quantitative reverse transcription polymerase chain reaction analysis. Implications: This comprehensive characterisation of the HSP70 gene family provides novel insights into the thermal protection mechanisms of betel palms in changing climates. We investigate the role of some genes in betel palm under thermal stress, an increasing problem for the plant's survival as climate change progresses. This gene family plays a fundamental role in tolerating heat stress. We identified 34 genes, of which eight exhibited enhanced expression under heat stress. The role of this gene family in betel palm's heat tolerance mechanisms is revealed, providing valuable insights into the plant's ability to withstand heat stress in a changing climate. This article belongs to the Collection Abiotic Stress Tolerance vs Climate Change. [ABSTRACT FROM AUTHOR]

Doxorubicin (DOX), a commonly used anticancer agent, causes cardiotoxicity that begins with the first dose and may progress to heart failure years after treatment. An inflammatory response associated with neutrophil recruitment has been recognized as a mechanism of DOX-induced cardiotoxicity. This study aimed to validate mRNA expression of the previously identified biomarkers of DOX-induced cardiotoxicity, PGLYRP1, CAMP, MMP9, and CEACAM8, and to assay their protein expression in the peripheral blood of breast cancer patients. Blood samples from 40 breast cancer patients treated with DOX-based chemotherapy were collected before and after the first chemotherapy cycle and > 2 years after treatment. The protein and gene expression of PGLYRP1/Tag7, CAMP/LL37, MMP9/gelatinase B, and CEACAM8/CD66b were determined using ELISA and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of each candidate biomarker. Patients with cardiotoxicity (n = 20) had significantly elevated levels of PGLYRP1, CAMP, MMP9, and CEACAM8 at baseline, after the first dose of DOX-based chemotherapy, and at > 2 years after treatment relative to patients without cardiotoxicity (n = 20). The first dose of DOX induced significantly higher levels of all examined biomarkers in both groups of patients. At > 2 years post treatment, the levels of all but MMP9 dropped below the baseline. There was a good correlation between the expression of mRNA and the target proteins. We demonstrate that circulating levels of PGLYRP1, CAMP, MMP9, and CEACAM8 can predict the cardiotoxicity of DOX. This novel finding may be of value in the early identification of patients at risk for cardiotoxicity. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic highlighted the crucial role of diagnostic testing in managing infectious diseases, particularly through the use of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) tests. RT-qPCR has been pivotal in detecting and quantifying viral RNA, enabling the identification and management of SARS-CoV-2 infections. However, despite its widespread use, there remains a notable gap in understanding fundamental diagnostic metrics such as sensitivity and specificity among many scientists and healthcare practitioners. This gap is not merely academic; it has profound implications for interpreting test results, making public health decisions, and affecting patient outcomes. This review aims to clarify the distinctions between laboratory- and field-based metrics in the context of RT-qPCR testing for SARS-CoV-2 and summarise the global efforts that led to the development and optimisation of these tests during the pandemic. It is intended to enhance the understanding of these fundamental concepts among scientists and healthcare professionals who may not be familiar with the nuances of diagnostic test evaluation. Such knowledge is crucial for accurately interpreting test results, making informed public health decisions, and ultimately managing infectious disease outbreaks more effectively. [ABSTRACT FROM AUTHOR]

Simple Summary: Investigating the proliferation and differentiation of skeletal muscle satellite cells (MuSCs) in vitro is crucial for elucidating the underlying mechanisms of skeletal muscle development in goats. Real-time quantitative PCR (RT-qPCR) is a widely utilized technique for quantifying the expression levels of target genes. A significant challenge associated with this method is the identification of optimal reference genes for accurate normalization. In this study, we evaluated ten candidate reference genes for the standardization of gene expression. Our results indicated that RPL14 and RPS15A constituted the most stable reference gene combination during the proliferation and differentiation of goat MuSCs. The process of skeletal muscle development is intricate and involves the regulation of a diverse array of genes. Accurate gene expression profiles are crucial for studying muscle development, making it essential to choose the right reference genes for real-time quantitative PCR (RT-qPCR). In the present study, eight candidate reference genes were identified from our previous transcriptome sequencing analysis of caprine skeletal muscle satellite cells (MuSCs), and two traditional reference genes (ACTB and GAPDH) were assessed. The quantitative levels of the candidate reference genes were determined through the RT-qPCR technique, while the stability of their expression was evaluated utilizing the GeNorm, NormFinder, BestKeeper, and RefFinder programs. Furthermore, the chosen reference genes were utilized for the normalization of the gene expression levels of PCNA and Myf5. It was determined that conventional reference genes, including ACTB and GAPDH, were not appropriate for normalizing target gene expression. Conversely, RPL14 and RPS15A, identified through RNA sequencing analysis, exhibited minimal variability and were identified as the optimal reference genes for normalizing gene expression during the proliferation and differentiation of goat MuSCs. Our research offers a validated panel of optimal reference genes for the detection of differentially expressed genes in goat muscle satellite cells using RT-qPCR. [ABSTRACT FROM AUTHOR]

Globally, there is a move towards using local, native species for ecotoxicological risk assessments. Anthropogenic stressors from urban, agricultural, and industrial activities can impact the health of receiving ecosystems. Biomarkers can provide valuable insights as early warning signals of the potential environmental impacts of stressors. The aim of this study was to develop biomarkers in the green-lipped mussel (Perna canaliculus), a potential bioindicator of environmental health for coastal marine ecosystems in New Zealand. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays targeting the expression of genes involved in oxidative stress, xenobiotic transfer, membrane transportation, cellular and DNA response/repair, and endocrine disruption were developed and validated for P. canaliculus. We found significant modulation of genes associated with oxidative stress, xenobiotic transfer, membrane transport, endocrine disruption, and genotoxicity in P. canaliculus following 48-hour exposures to copper and benzo[α]pyrene. These results demonstrate the potential of P. canaliculus as a bioindicator species for environmental risk assessment. The gene expression assays showed potential as early indicators of exposure to the chemicals tested but require additional validation to assess their ability to predict effects at higher levels of biological organisation. [ABSTRACT FROM AUTHOR]

Honey bees are vital pollinators in agriculture and important model insects. To understand the genetic and molecular aspects in their development, a reverse transcription quantitative polymerase chain (RT-qPCR) is used to investigate the target genes. However, it is essential to use the appropriate reference genes as endogenous controls for accurate normalization of target genes. To identify stable reference genes in two honey bee species, [Apis mellifera (Am) and Apis cerana (Ac)], we evaluated eight candidate reference genes including, actin, atub, ef1α, gapdh, rpl13a, rpl32, rps18 and tif. Worker bees belonging to the two species were collected at each developmental day during the embryonic and postembryonic developmental stages. The tyrosine hydroxylase (th) gene was used as the target gene to validate the selected reference genes. Our results revealed that rpl13a was the most stable reference gene at all developmental stages of Am and Ac. In addition, gene combinations, including Amrpl13a & Amrps18 & Amactin, Amrpl13a & Amrpl32, Acrpl13a & Acrpl32, Acrpl13a & Acrpl32 & Acef1α followed by other combinations effectively normalized the expression of the target genes during the embryonic and postembryonic developmental stages of Am and Ac, respectively. Our findings provide a foundation for standardized RT-qPCR analysis to improve the accuracy of genes normalization during the different developmental stages of honey bees. [ABSTRACT FROM AUTHOR]

Siraitia siamensis is a traditional Chinese medicinal herb. In this study, using S. siamensis cultivated in vitro, twelve candidate reference genes under various treatments were analyzed for their expression stability by using algorithms such as GeNorm, NormFinder, BestKeeper, Delta CT, and RefFinder. The selected reference genes were then used to characterize the gene expression of cucurbitadienol synthase, which is a rate-limiting enzyme for mogroside biosynthesis. The results showed that CDC6 and NCBP2 expression was the most stable across all treatments and are the best reference genes under the tested conditions. Utilizing the validated reference genes, we analyzed the expression profiles of genes related to the synthesis pathway of mogroside in S. siamensis in response to a range of abiotic stresses. The findings of this study provide clear standards for gene expression normalization in Siraitia plants and exploring the rationale behind differential gene expression related to mogroside synthesis pathways. [ABSTRACT FROM AUTHOR]

Main conclusion: Transcriptome analysis in potato varieties revealed genes associated with tuber yield-related traits and developed gene expression markers. This study aimed to identify genes involved in high tuber yield and its component traits in test potato varieties (Kufri Frysona, Kufri Khyati, and Kufri Mohan) compared to control (Kufri Sutlej). The aeroponic evaluation showed significant differences in yield-related traits in the varieties. Total RNA sequencing was performed using tuber and leaf tissues on the Illumina platform. The high-quality reads (QV > 25) mapping with the reference potato genomes revealed statistically significant (P < 0.05) differentially expressed genes (DEGs) into two categories: up-regulated (> 2 Log2 fold change) and down-regulated (< -2 Log2 fold change). DEGs were characterized by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Collectively, we identified genes participating in sugar metabolism, stress response, transcription factors, phytohormones, kinase proteins, and other genes greatly affecting tuber yield and its related traits. A few selected genes were UDP-glucose glucosyltransferase, glutathion S-transferase, GDSL esterase/lipase, transcription factors (MYB, WRKY, bHLH63, and BURP), phytohormones (auxin-induced protein X10A, and GA20 oxidase), kinase proteins (Kunitz-type tuber invertase inhibitor, BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1) and laccase. Based on the selected 17 peptide sequences representing 13 genes, a phylogeny tree and motifs were analyzed. Real time–quantitative polymerase chain reaction (RT-qPCR) analysis was used to validate the RNA-seq results. RT-qPCR based gene expression markers were developed for the genes such as 101 kDa heat shock protein, catechol oxidase B chloroplastic, cysteine protease inhibitor 1, Kunitz-type tuber invertase inhibitor, and laccase to identify high yielding potato genotypes. Thus, our study paved the path for potential genes associated with tuber yield traits in potato under aeroponics. [ABSTRACT FROM AUTHOR]

The COVID-19 epidemic had a profound impact on global health and the economy and Ghana was no exception to its far-reaching consequences. Regarding detection of the causative agent—the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), reverse-transcription–qPCR (RT–qPCR) is widely recognized as a very sensitive and reliable diagnostic technique used globally. There are, however, high operational costs in acquiring test kits, equipment, and accessories for RT–qPCR testing, which pose significant challenges in resource-limited settings. Hence, this proof-of-concept study set out to develop a more affordable COVID-19 protocol for use in low or lower-middle-income settings, such as Ghana, that would bypass the traditional extraction process using inexpensive reagents and evaluate the possibility of processing samples collected using wooden shaft swabs. Several less expensive media were used for the extraction-free process. Results demonstrated that direct RT–qPCR assay after 5 min heat inactivation of virus at 95 °C in 0.1× PBS or molecular grade water resulted in viral detection with quantification cycle (Cq) values that are comparable to results obtained following the extraction process. Also, wooden shaft swabs could be used for sampling if incubation times are kept to less than 6 h. The study demonstrates that extraction-free protocols are one way to minimize the cost of COVID-19 testing by RT–qPCR. [ABSTRACT FROM AUTHOR]

Crassostrea hongkongensis (C. hongkongensis) is one of the three most commonly cultivated oyster species in China. Seasonal hypoxia is one of the most serious threats to its metabolism, reproductive behavior, and survival. To investigate the effects of hypoxia stress on the antioxidant capacity and energy metabolism of C. hongkongensis, the total antioxidant capacity (T-AOC), glycogen content, and enzyme activities (phosphofructokinase, PFK; pyruvate kinase, PK; phosphoenolpyruvate carboxykinase, PEPCK) of oysters were determined under normoxic (DO 6 ± 0.2 mg/L) and hypoxic (DO 1.5 mg/L) conditions at 0 h, 6 h, 48 h, and 72 h. We also determined the T-AOC, glycogen content, and enzyme activities of oysters under reoxygenation (recovered to normoxia for 24 h). To further examine the potential molecular regulatory mechanism of hypoxic adaptation, a transcriptome analysis was conducted on the gill of C. hongkongensis under normoxia (N, 72 h), hypoxia (H, 72 h), and reoxygenation (R). After being exposed to hypoxia for 6 h, the T-AOC, glycogen content, and enzyme activities of PK, PFK, and PEPCK in C. hongkongensis were significantly decreased. However, after prolonging the duration of hypoxia exposure for 72 h, the T-AOC, glycogen content, and enzyme activities increased compared to that of 48 h. After 24 h reoxygenation, the T-AOC, glycogen content, and enzyme activity of PK and PFK returned to close to initial levels. In addition, a transcriptome analysis discovered 6097 novel genes by mapping the C. hongkongensis genome with the clean reads. In total, 352 differentially expressed genes (DEGs) were identified in the H vs. N comparison group (235 upregulated and 117 downregulated genes). After recovery to normoxia, 292 DEGs (134 upregulated and 158 downregulated genes) were identified in the R vs. N comparison group, and 632 DEGs were identified (253 upregulated and 379 downregulated genes) in the R vs. H comparison group. The DEGs included some hypoxia-tolerant genes, such as phosphoenolpyruvate carboxykinase (PEPCK), mitochondrial (AOX), tyramine beta-hydroxylase (TBH), superoxide dismutase (SOD), glutathione S-transferase (GST), and egl nine homolog 1 isoform X2 (EGLN1). Additionally, DEGs were significantly enriched in the KEGG pathways that are involved in hypoxia tolerance, including the metabolism of xenobiotics by cytochrome P450 pathways and the HIF-1 signaling pathway. Then, we selected the five hypoxic-tolerant candidate DEGs for real-time quantitative polymerase chain reaction (RT-qPCR) validation, and the results were consistent with the transcriptome sequencing data. These discoveries have increased our understanding of hypoxia tolerance, recovery ability after reoxygenation, and molecular mechanisms governing the responses to hypoxia in C. hongkongensis. [ABSTRACT FROM AUTHOR]

Abstract The 4T1 model is extensively employed in murine studies to elucidate the mechanisms underlying the carcinogenesis of triple-negative breast cancer. Molecular biology serves as a cornerstone in these investigations. However, accurate gene expression analyses necessitate data normalization employing housekeeping genes (HKGs) to avert spurious results. Here, we initially delve into the characteristics of the tumor evolution induced by 4T1 in mice, underscoring the imperative for additional tools for tumor monitoring and assessment methods for tracking the animals, thereby facilitating prospective studies employing this methodology. Subsequently, leveraging various software platforms, we assessed ten distinct HKGs (GAPDH, 18 S, ACTB, HPRT1, B2M, GUSB, PGK1, CCSER2, SYMPK, ANKRD17) not hitherto evaluated in the 4T1 breast cancer model, across tumors and diverse tissues afflicted by metastasis. Our principal findings underscore GAPDH as the optimal HKG for gene expression analyses in tumors, while HPRT1 emerged as the most stable in the liver and CCSER2 in the lung. These genes demonstrated consistent expression and minimal variation among experimental groups. Furthermore, employing these HKGs for normalization, we assessed TNF-α and VEGF expression in tissues and discerned significant disparities among groups. We posit that this constitutes the inaugural delineation of an ideal HKG for experiments utilizing the 4T1 model, particularly in vivo settings.

Abstract Background MADS-box transcription factors have been shown to be involved in multiple developmental processes, including the regulation of floral organ formation and pollen maturation. However, the role of the MADS-box gene family in floral development of the alpine plant species Coptis teeta Wall, which is widely used in Traditional Chinese Medicine (TCM), is unknown. Results Sixty-six MADS-box genes were identified in the C. teeta genome. These genes were shown to be unevenly distributed throughout the genome of C. teeta. The majority of which (49) were classified as type I MADS-box genes and were further subdivided into four groups (Mα, Mβ, Mγ and Mδ). The remainder were identified as belonging to the type II MADS-box gene category. It was observed that four pairs of segmental and tandem duplication had occurred in the C. teeta MADS-box gene family, and that the ratios of Ka/Ks were less than 1, suggesting that these genes may have experienced purifying selection during evolution. Gene expression profiling analysis revealed that 38 MADS-box genes displayed differential expression patterns between the M and F floral phenotypes. Sixteen of these MADS-box genes were further verified by RT-qPCR. The 3D structure of each subfamily gene was predicted, further indicating that MADS-box genes of the same type possess structural similarities to the known template. Conclusions These data provide new insights into the molecular mechanism of dichogamy and herkogamy formation in C. teeta and establish a solid foundation for future studies of the MADS-box genes family in this medicinal plant species.

This study was conducted to investigate the effects and mechanisms of glutamine (Gln) on the repair of acid-induced injury in dairy cow ruminal epithelial cells (RECs) in vitro. Dairy cow RECs were cultured in a medium with pH of 5.5 for 3 h and subsequently treated with various concentrations of Gln (4, 8, 12, 32 mmol/l) for 12 h. The messenger ribonucleic acid (mRNA) expression levels of occludin (OCLN), claudin 1 (CLDN1), toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4) and genes for inflammatory factors were measured using reverse transcription-quantitative real-time PCR (RT-qPCR). The results showed that cellular activity and OCLN expression were significantly highest at 8 mmol/l Gln (P < 0.05). CLDN1 expression was significantly higher at 4 mmol/l Gln compared to the other groups (P < 0.05). The relative expression levels of tumour necrosis factor (TNF), interleukin 1B (IL1B), C-X-C motif chemokine ligand 8 (CXCL8), TLR2 and TLR4 in the acid treatment group were significantly higher than those in the control group (P < 0.05), but they were lower in the Gln-treated groups than in the acid treatment group (P < 0.05). These findings demonstrate that Gln promotes the proliferation of RECs, enhances the expression of epithelial cell junction proteins, and inhibits the expression of inflammatory factors and surface receptors. In conclusion, Gln shows a potential for repairing acid-induced injury in RECs.

Abstract Background The COVID-19 pandemic has been the most significant health challenge of the last century. Multiple and successive waves of COVID-19 cases, driven particularly by the emergence of new SARS-CoV-2 variants, have kept the world in a constant state of alert. Methods We present an observational, descriptive, cross-sectional study aimed at identifying SARS-CoV-2 variants circulating during two local waves of COVID-19 cases in southern Bahia, Brazil (late 2021 and late 2022), and analyzing the association between the detected variants and the epidemiological and clinical characteristics of the disease. For this purpose, data and nasopharyngeal samples from individuals in southern Bahia, Brazil, with suspected COVID-19 were included. Viral detection was performed by RT-qPCR, and SARS-CoV-2 variants were identified by next-generation viral sequencing. Results A total of 368 nasopharyngeal samples were tested. Approximately 23% of the samples from late 2021 tested positive for SARS-CoV-2, while in 2022, the positivity rate was about 56%. All sequenced samples from 2021 were identified as the Delta variant, while in 2022, all samples were classified as the Omicron variant. Overall, individuals who tested positive for SARS-CoV-2 in 2022 were younger than those who tested positive in 2021. Moreover, we observed significant differences in the clinical spectrum of SARS-CoV-2 infection when comparing the two periods. Individuals who presented with anosmia/ageusia were more likely to test positive for SARS-CoV-2 infection in 2021 but not in 2022. Additionally, fever, dry cough, pharyngalgia, headache, and rhinorrhea were more frequent among individuals infected with the Omicron variant than among those infected with the Delta variant. Conclusions The profile of COVID-19 in southern Bahia differed when analyzing two distinct waves of the pandemic in the region. These differences are likely related to the variants, which may differ in transmissibility and virulence, thereby altering the dynamics of the pandemic. This underscores the importance of genomic surveillance in better understanding the behavior of viral infections.

Sugar beet hybrids are essential in modern agriculture due to their superior yields, disease resistance, and adaptability. This study investigates the role of the Rz2 gene in conferring resistance to beet necrotic yellow vein virus (BNYVV) in 14 sugar beet hybrids cultivated in Kazakhstan, including local and European varieties. The Rz2 gene, encoding a CC-NB-LRR protein, is a known resistance factor against BNYVV. Using RT-qPCR, we assessed Rz2 expression and detected BNYVV in bait plants inoculated with virus-infested soil. Our findings identified two highly resistant varieties: the Kazakh cultivar ‘Abulhair’ and the French line 22b5006. Additionally, the Kazakh cultivar ‘Pamyati Abugalieva’ and the French hybrid ‘Bunker’ exhibited increased resistance, suggesting involvement of other resistance loci. Notably, the Danish hybrid ‘Alando’, despite resistance to rhizomania, did not effectively resist BNYVV, highlighting possible evasion of its genetic factors by local virus strains. Our results emphasize the importance of Rz2 in resistance breeding programs and advocate for further research on additional resistance genes and the genetic variability of BNYVV in Kazakhstan. This work pioneers the molecular evaluation of BNYVV resistance in sugar beet in Kazakhstan, contributing to sustainable disease management and improved sugar beet production.

The determination of transcript accumulation values significantly affects gene expression in oil palm. Various genes are involved in pathogen infection, including probable 2‐oxoglutarate‐dependent dioxygenase At5g05600 (EgUnk3), zinc finger protein 2‐like (EgZFP2), and inositol polyphosphate multikinase beta‐like (EgIPK2b). Gene expression is typically measured using relative quantitative methods to calculate differences in quantitative values in the expression levels of targeted genes compared to a reference gene. However, the effectiveness of these methods in assessing the expression of EgUnk3, EgZFP2, and EgIPK2b, which are involved in Ganoderma boninense infection in oil palm seedlings, requires evaluation. This study aimed to establish an effective and straightforward method for analyzing the expression of EgUnk1, EgZFP2, and EgIPK2b genes in oil palm seedlings infected with G. boninense, utilizing Ct value correction through regression coefficients on the 2‐ΔΔCt and E‐ΔΔCt approaches. A correlation regression revealed values of 0.28, ‐0.32, and 0.29 for delta Ct of EgUnk1, EgZFP2, and EgIPK2b, respectively. However, a negative correlation in the Ct mean was corrected by linear regression for the targeted genes: ‐0.55, ‐0.81, and ‐0.29 for EgUnk1, EgZFP2, and EgIPK2b, respectively. The amplification factor (E) and efficiency value (R) using the EgActin gene were 1.95 and 94.92%, respectively. Normalization of log10 on the fold change value 2‐ΔΔCt and 1.95‐ΔΔCt approaches using the regression coefficient yielded consistent results for the EgUnk1, EgZFP2, and EgIPK2b genes. Overall, EgUnk3 and EgIPK2b genes exhibited downregulated expression in susceptible oil palm seedlings (‐0.60 for 2(‐ΔΔCt) and ‐0.58 for 1.95(‐ΔΔCt)), whereas EgIPK2b gene showed up‐regulated and the highest value in inoculated resistant seedlings (1.39 for 2(‐ΔΔCt) and 1.34 for 1.95(‐ΔΔCt)). Basal stem rot disease (BSR) in oil palm decreased EgUnk1 and EgIPK2b expression in susceptible seedlings but increased EgZFP2 gene expression in resistant ones. The results of this research provide valuable corrections to Ct values obtained directly from RT‐qPCR machines using simple linear regression. Consequently, the Ct values of target genes and reference genes exhibit smaller bias values, rendering gene expression levels more reliable.

Reliable gene expression analysis in bone remodeling studies requires an appropriate selection of internal controls, i.e. stable reference genes for the normalization of quantitative real-time PCR (RT-qPCR), the most common method used for quantifying gene expression measurements. Even the most widely used reference genes can have variable expression under different experimental conditions, or in different tissue types or treatment regimes, so selecting appropriate controls is a key step in ensuring reliable results. The aim of this research was to identify the most stable reference gene(s) for the study of olanzapine modulated bone remodeling in rats. RNA was isolated from the maxillary alveolar and femoral bones of olanzapine or placebo-treated Wistar rats and transcribed to cDNA. The expression of 12 candidate reference genes was assessed by RT-qPCR. Their expressions were analysed using GeNorm, NormFinder, BestKeeper and delta Ct algorithms, and by the comprehensive ranking method. PPIA, HRPT1 and PGK1 were the most stably expres sed reference genes and the combination of the three genes was optimal for normalization. This study is the first to identify the optimal reference genes for research in olanzapine-exposed rats, which serve as a pivotal benchmark for enhancing the accuracy and reliability of future RT-qPCR expression in bone studies.

Abstract Background Plant biostimulants constitute a promising environmentally friendly alternative for increasing crop yield and tolerance to unfavorable conditions. Among various types of such formulations, botanical extracts are gaining more recognition as products supporting plant performance. Moreover, novel tools such as cold-plasma or low-pressure microwave plasma discharge are being proposed as techniques that might improve their efficacy. Elucidation of the biostimulant’s mode of action requires complex research at a molecular level. Transcriptional changes occurring after biostimulant spraying might be investigated using RT-qPCR. However, this technique requires data normalization against stable endogenous controls. Results Here, we tested the expression stability of ten candidate genes in soybean plants exposed to various biostimulants treatment. Selection of the best-performing reference genes was conducted using four algorithms (geNorm, NormFinder, BestKeeper, and ΔCt method). According to the obtained results, Bic-C2 (RNA-binding protein Bicaudal-C) and CYP (cyclophilin type peptidyl-prolyl cis–trans isomerase) showed highest expression stability, while expression of EF1B (elongation factor 1-beta) fluctuated the most among a tested set of candidate genes. Conclusions Overall, we recommend using Bic-C2 together with CYP for the RT-qPCR data normalization in soybean biostimulation experiments. To our best knowledge, this is the first comprehensive study of reference genes stability in plants subjected to biostimulant treatment. The results of this study will aid in further biostimulant research in crop plants, facilitating analyses performed on the transcriptional level. Graphical Abstract

Circulating plasma miRNAs have emerged as potential early predictors of glucometabolic disorders. However, their biomarker potential remains unvalidated in populations with diverse genetic backgrounds, races, and ethnicities. This study aims to validate the biomarker potential of plasma miR-9, miR-29a, miR-192, and miR-375 for early detection of prediabetes and type 2 diabetes mellitus (T2DM) in Nepali populations that represent distinct genetic backgrounds, races, and ethnicities. A total of 46 adults, categorized into healthy controls (n = 25), prediabetes (n = 9), and T2DM (n = 12) groups, were enrolled. Baseline sociodemographic, anthropometric, and clinical characteristics were collected. Fold change in plasma expression of all four miRNAs was quantified using RT-qPCR against the RNU6B reference gene. Their biomarker potential was determined by receiver operating characteristic (ROC) curve analysis. Multivariate discriminant function and hierarchical cluster analyses were used to evaluate the effectiveness of the miRNA panel in reclassifying study participants who were initially categorized according to their glucose tolerance status. Plasma expression of all four miRNAs was significantly upregulated in T2DM patients compared to normoglycemic controls. Furthermore, the expression of only miR-29a and miR-375 was upregulated in T2DM patients than in prediabetic individuals. Notably, only miR-192 expression was significantly upregulated in prediabetic individuals than in the normoglycemic controls. The miRNA expression profiles had the potential of reclassifying the participants into three original groups with an accuracy of 69.6 %. ROC curve analysis identified miR-192 as the predictor for both prediabetes and T2DM, while miR-9, miR-29a, miR-192, and miR-375 were predictive only for T2DM. The specific set of miRNA combinations significantly improved their predictive accuracy. This study validates the early predictive biomarker potential of plasma miR-9, miR-29a, miR-192, and miR-375 also in the Nepali population and paves the way for future translational studies to validate their utility in clinical laboratories.