Mapping the Periostin splice isoforms in atopic dermatitis and an in vitro asthma model - A multi-platform analysis using mass spectrometry and RT-qPCR.

Periostin is a matricellular protein known to be alternatively spliced to produce ten isoforms with a molecular weight of 78-91 kDa. Within the extracellular matrix, periostin attaches to cell surfaces to induce signaling via integrin-binding and actively participates in fibrillogenesis, orchestrating the arrangement of collagen in the extracellular environment. In atopic diseases such as atopic dermatitis (AD) and asthma, periostin is known to participate in driving the disease-causing type 2 inflammation. The periostin isoforms expressed in these diseases and the implication of the alternative splicing events are unknown. Here, we present two universal assays to map the expression of periostin isoforms at the mRNA (RT-qPCR) and protein (PRM-based mass spectrometry) levels. We use these assays to study the splicing profile of periostin in AD lesions as well as in in vitro models of AD and asthma. In these conditions, periostin displayed overexpression with isoforms 3 and 5 standing out as highly overexpressed. Notably, isoforms 9 and 10 exhibited a divergent pattern relative to the remaining isoforms. Isoforms 9 and 10 are often overlooked in periostin research and this paper presents the first evidence of their expression at the protein level. This underlines the necessity to include isoforms 9 and 10 in future research addressing periostin splice isoforms. The assays presented in this paper hold the potential to improve our insight into the splicing profile of periostin in tissues and diseases of interest. The application of these assays to AD lesions and in vitro models demonstrated their potential for identifying isoforms of particular significance, warranting a further in-depth investigation.

Identification of circulating miRNA biomarkers in Leptospirosis for early detection: A Promising Diagnostic Approach.

Leptospirosis is a zoonotic disease of global significance, contributing to morbidity and mortality worldwide. It is endemic to tropical regions, with outbreaks during monsoons. The disease manifestations are similar to that of other febrile illness such as dengue, malaria hence often misdiagnosed and underreported. The zoonoses if undetected, progresses to cause severe life-threatening complications also known as Weil's disease. Routine diagnostic tests are based on the detection of antibodies in patient serum and are not accurate during the initial phase of the infection. Therefore, it is necessary to detect novel biomarkers that can be used in early detection of leptospirosis. Circulating miRNAs are known to be promising biomarkers for various diseases including cancer, tuberculosis, influenza; hence in this study the potential of miRNAs as biomarkers for leptospirosis was evaluated. A total of 30 leptospirosis cases were screened for the differential expression of 10 miRNA by RT-qPCR assay. The differential expression was calculated by relative quantification using healthy individuals as controls. Among the 10 miRNA,3 miRNA, miR-28-5p, miR-302c-3p and miR-302a-3p were reported to exhibit a significant trend of upregulation. Further their role in immune pathways and biological processes was investigated by KEGG analysis and Gene Ontology. The 3 miRNAs were observed to target various immune response pathways, thus confirming their role in host immune response. Based on the results obtained in this study, miR-28-5p, miR-302c-3p and miR-302a-3p can be considered as potential biomarkers for the detection of leptospirosis.

Prenatal tetrahydrocannabinol and cannabidiol exposure produce sex-specific pathophysiological phenotypes in the adolescent prefrontal cortex and hippocampus.

Clinical and preclinical evidence has demonstrated an increased risk for neuropsychiatric disorders following prenatal cannabinoid exposure. However, given the phytochemical complexity of cannabis, there is a need to understand how specific components of cannabis may contribute to these neurodevelopmental risks later in life. To investigate this, a rat model of prenatal cannabinoid exposure was utilized to examine the impacts of specific cannabis constituents (Δ9-tetrahydrocannabinol [THC]; cannabidiol [CBD]) alone and in combination on future neuropsychiatric liability in male and female offspring. Prenatal THC and CBD exposure were associated with low birth weight. At adolescence, offspring displayed sex-specific behavioural changes in anxiety, temporal order and social cognition, and sensorimotor gating. These phenotypes were associated with sex and treatment-specific neuronal and gene transcriptional alterations in the prefrontal cortex, and ventral hippocampus, regions where the endocannabinoid system is implicated in affective and cognitive development. Electrophysiology and RT-qPCR analysis in these regions implicated dysregulation of the endocannabinoid system and balance of excitatory and inhibitory signalling in the developmental consequences of prenatal cannabinoids. These findings reveal critical insights into how specific cannabinoids can differentially impact the developing fetal brains of males and females to enhance subsequent neuropsychiatric risk.

An improved alphaviruses-specific RT-qPCR facilitates monitoring and prevention of alphaviruses.

Molecular surveillance is vital for monitoring arboviruses, often employing genus-specific quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Despite this, an overlooked chikungunya fever outbreak occurred in Yunnan province, China, in 2019 and false negatives are commonly encountered during alphaviruses screening practice, highlighting the need for improved detection methods. In this study, we developed an improved alphaviruses-specific RT-qPCR capable of detecting chikungunya virus, eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, Sindbis virus, Mayaro virus, and Ross River virus with high sensitivity and specificity. The assay identified three chikungunya virus-positive cases out of 188 sera retrospectively. Later genetic characterization suggested that imported cases from neighboring countries may be responsible for the neglected chikungunya fever outbreak of 2019 in Yunnan. Our findings underscore the value of improved alphaviruses-specific RT-qPCR in bolstering alphaviruses surveillance and informing preventive strategies.

Spatial-temporal comparison of Eph/Ephrin gene expression in ocular lenses from aging and knockout mice.

Cataracts, defined as any opacity in the transparent ocular lens, remain the leading cause of blindness and visual impairment in the world; however, the etiology of this pathology is not fully understood. Studies in mice and humans have found that the EphA2 receptor and the ephrin-A5 ligand play important roles in maintaining lens homeostasis and transparency. However, due to the diversity of the family of Eph receptors and ephrin ligands and their promiscuous binding, identifying functional interacting partners remains a challenge. Previously, 12 of the 14 Ephs and 8 of 8 ephrins in mice were characterized to be expressed in the mouse lens. To further narrow down possible genes of interest in life-long lens homeostasis, we collected and separated the lens epithelium from the fiber cell mass and isolated RNA from each compartment in samples from young adult and middle-aged mice that were either wild-type, EphA2-/- (knockout), or ephrin-A5 -/- . Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was implemented to compare transcript levels of 33 Eph and ephrin gene variants in each tissue compartment. Our results show that, of the Eph and ephrin variants screened, 5 of 33 showed age-related changes, and 2 of 33 showed genotype-related changes in lens epithelium. In the isolated fibers, more dynamic gene expression changes were observed, in which 12 of 33 variants showed age-related changes, and 6 of 33 showed genotype-related changes. These data allow for a more informed decision in determining mechanistic leads in Eph-ephrin-mediated signaling in the lens.

Mild Hyperthermia-Induced Thermogenesis in the Endoplasmic Reticulum Defines Stress Response Mechanisms.

Previous studies reported that a mild, non-protein-denaturing, fever-like temperature increase induced the unfolded protein response (UPR) in mammalian cells. Our dSTORM super-resolution microscopy experiments revealed that the master regulator of the UPR, the IRE1 (inositol-requiring enzyme 1) protein, is clustered as a result of UPR activation in a human osteosarcoma cell line (U2OS) upon mild heat stress. Using ER thermo yellow, a temperature-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we detected significant intracellular thermogenesis in mouse embryonic fibroblast (MEF) cells. Temperatures reached at least 8 °C higher than the external environment (40 °C), resulting in exceptionally high ER temperatures similar to those previously described for mitochondria. Mild heat-induced thermogenesis in the ER of MEF cells was likely due to the uncoupling of the Ca2+/ATPase (SERCA) pump. The high ER temperatures initiated a pronounced cytosolic heat-shock response in MEF cells, which was significantly lower in U2OS cells in which both the ER thermogenesis and SERCA pump uncoupling were absent. Our results suggest that depending on intrinsic cellular properties, mild hyperthermia-induced intracellular thermogenesis defines the cellular response mechanism and determines the outcome of hyperthermic stress.

Identification of HOXC Gene Family as Prognostic and Immune-Related Biomarkers in Breast Cancer Through mRNA Transcriptional Profile and Experimental Validation.

Breast cancer (BC) is the most common malignancy in women worldwide, and more effective biomarkers are urgently needed for the prevention and treatment of BC. Our study aimed to investigate the role of the HOXC gene family (HOXCs) and its relationship with the immune response in BC. The differential expression of HOXCs and its clinical prognostic significance in BC were explored using bioinformatics analysis, and the cBioPortal database was used to evaluate the genetic mutation profile of the HOXCs in BC. The results indicated that the expression levels of HOXC4, 10, 11, 12, and 13 were significantly increased in BC tissues compared with the normal tissues, and expressions of these genes were closely associated with BC stage, among them, high expression levels of HOXC10 and HOXC13 predicted poor outcome in BC patients. In addition, to elucidate the essential role of HOXCs in the tumor microenvironment and immunotherapeutic response of BC, the impact of HOXCs on the regulation of immune infiltration in BC was comprehensively assessed. The result showed that HOXC10 and HOXC13 expressions were significantly positively linked with the infiltration levels of CD8+T cell and M1 macrophage, while they were negatively related to Mast and Natural killer cells, suggesting the important influence of HOXCs on regulating tumor immunity in BC patients. Lastly, the RT-qPCR assay was employed to validate HOXCs expression in samples of BC patients. In conclusion, HOXCs may be a promising prognostic indicator and could regulate the immune infiltration in BC patients, thus being a promising targeted immunotherapy for BC.

Evaluation and comparison of one-step real-time PCR and one-step RT-LAMP methods for detection of SARS-CoV-2.

BACKGROUND: There is an increasing disease trend for SARS-COV-2, so need a quick and affordable diagnostic method. It should be highly accurate and save costs compared to other methods. The purpose of this research is to achieve these goals. METHODS: This study analyzed 342 samples using TaqMan One-Step RT-qPCR and fast One-Step RT-LAMP (Reverse Transcriptase Loop-Mediated Isothermal Amplification). The One-Step LAMP assay was conducted to assess the sensitivity and specificity. RESULTS: The research reported positive samples using two different methods. In the RT-LAMP method, saliva had 92 positive samples (26.9%) and 250 negative samples (73.09%) and nasopharynx had 94 positive samples (27.4%) and 248 negative samples (72.51%). In the RT-qPCR method, saliva had 86 positive samples (25.1%) and 256 negative samples (74.8%) and nasopharynx had 93 positive samples (27.1%) and 249 negative samples (72.8%). The agreement between the two tests in saliva and nasopharynx samples was 93% and 94% respectively, based on Cohen's kappa coefficient (κ) (P < 0.001). The rate of sensitivity in this technique was reported at a dilution of 1 × 101 and 100% specificity. CONCLUSIONS: Based on the results of the study the One-Step LAMP assay has multiple advantages. These include simplicity, cost-effectiveness, high sensitivity, and specificity. The One-Step LAMP assay shows promise as a diagnostic tool. It can help manage disease outbreaks, ensure prompt treatment, and safeguard public health by providing rapid, easy-to-use testing.

Epidemiological, virological and clinical characterization of a Dengue/Zika outbreak in the Caribbean region of Costa Rica 2017-2018.

The increase in incidence and geographical expansion of viruses transmitted by the Aedes mosquitoes, such as dengue (DENV) and zika (ZIKV) in the Americas, represents a burden for healthcare systems in tropical and subtropical regions. These and other under-detected arboviruses co-circulate in Costa Rica, adding additional complexity to their management due to their shared epidemiological behavior and similarity of symptoms in early stages. Since diagnostics of febrile illness is mostly based on clinical symptoms alone, we gathered acute-phase serum and urine from 399 samples of acute dengue-like cases from two healthcare facilities of Costa Rica, during an outbreak of arboviruses from July 2017 to May 2018, and tested them using molecular and serological methods. The analyses showed that of the clinically presumptive arbovirus cases that were reported, only 39.4% (n=153) of the samples were confirmed positive by RT-PCR to be DENV (DENV (10.3%), CHIKV (0.2%), ZIKV (27.3%), or mixed infections (1.5%). RT-PCR for other alphaviruses and flaviviruses, and PCR for Leptospira sp were negative. Furthermore, to assess flavivirus positivity in post-acute patients, the negative sera were tested against Dengue-IgM. 20% of sera were found positive, confounding even more the definitive number of cases, and emphasizing the need of several distinct diagnostic tools for accurate diagnostics. Molecular characterization of the prM and E genes from isolated viruses revealed that the American/Asian genotype of DENV-2 and the Asian lineage of ZIKV were circulating during this outbreak. Two different clades of DENV-2 American/Asian genotype were identified to co-circulate in the same region and a difference in the platelet and leukocyte count was noted between people infected with each clade, suggesting a putative distinct virulence. Our study sheds light on the necessity for healthcare strategies in managing arbovirus outbreaks, emphasizing the importance of comprehensive molecular and serological diagnostic approaches, as well as molecular characterization. This approach aids in enhancing our understanding of the clinical and epidemiological aspects of arboviral diseases during outbreaks. Our research highlights the need to strengthen training programs for health professionals and the need to increase research-based on laboratory evidence for diagnostic accuracy, guidance, development and implementation of public health interventions and epidemiological surveillance.

Screening of reliable reference genes for the normalization of RT-qPCR in chicken oviduct tract.

Utilizing publicly available RNA-seq data to screen for ideal reference genes is more efficient and accurate than traditional methods. Previous studies have identified optimal reference genes in various chicken tissues, but none have specifically focused on the oviduct (including the infundibulum, magnum, isthmus, uterus, and vagina), which is crucial for egg production. Identifying stable reference genes in the oviduct is essential for improving research on gene expression levels. This study investigated genes with consistent expression patterns in the chicken oviduct, encompassing both individual oviduct tract tissues and the entire oviduct, by utilizing multiple RNA-seq datasets. The screening results revealed the discovery of 100 novel reference genes in each segment of oviduct tissues, primarily associated with cell cycle regulation and RNA binding. Moreover, the majority of housekeeping genes (HKGs) showed inconsistent expression levels across distinct samples, suggesting their lack of stability under varying conditions. The stability of the newly identified reference genes was assessed in comparison to previously validated stable reference genes in chicken oviduct and commonly utilized HKGs, employing traditional reference gene screening methods. HERPUD2, CSDE1, VPS35, PBRM1, LSM14A, and YWHAB were identified to be suitable novel reference gene for different parts of the oviduct. HERPUD2 and YWHAB were reliable for gene expression normalization throughout the oviduct tract. Furthermore, overexpression and interference assays in DF1 cells showed LSM14A and YWHAB play a crucial role in cell proliferation, highlighting the importance of these newly reference genes for further research. Overall, this study has expanded the options for reference genes in RT-qPCR experiments in different segments of the chicken oviduct and the entire oviduct.

Studying the impact of titanium dioxide nanoparticles on the expression of pivotal genes related to menthol biosynthesis and certain biochemical parameters in peppermint plants (Mentha Piperita L.).

BACKGROUND: This study examines the impact of titanium dioxide nanoparticles (TiO2NPs) on gene expression associated with menthol biosynthesis and selected biochemical parameters in peppermint plants (Mentha piperita L.). Menthol, the active ingredient in peppermint, is synthesized through various pathways involving key genes like geranyl diphosphate synthase, menthone reductase, and menthofuran synthase. Seedlings were treated with different concentrations of TiO2NPs (50, 100, 200, and 300 ppm) via foliar spray. After three weeks of treatment, leaf samples were gathered and kept at -70 °C for analysis. RESULTS: According to our findings, there was a significant elevation (P ≤ 0.05) in proline content at concentrations of 200 and 300 ppm in comparison with the control. Specifically, the highest proline level was registered at 200 ppm, reaching 259.64 ± 33.33 µg/g FW. Additionally, hydrogen peroxide and malondialdehyde content exhibited a decreasing trend following nanoparticle treatments. Catalase activity was notably affected by varying TiO2NP concentrations, with a significant decrease observed at 200 and 300 ppm compared to the control (P ≤ 0.05). Conversely, at 100 ppm, catalase activity significantly increased (11.035 ± 1.12 units/mg of protein/min). Guaiacol peroxidase activity decreased across all nanoparticle concentrations. Furthermore, RT-qPCR analysis indicated increased expression of the studied genes at 300 ppm concentration. CONCLUSIONS: Hence, it can be inferred that at the transcript level, this nanoparticle exhibited efficacy in influencing the biosynthetic pathway of menthol.

Selection and application of small non-coding RNAs for normalizing RT-qPCR data of bovine preimplantation embryo conditioned medium.

Small non-coding RNAs (sncRNAs) present in the conditioned medium (CM) of bovine preimplantation embryos are potential noninvasive biomarkers for assessing embryo quality. Accurate quantification of sncRNA levels in the spent CM is of utmost importance in this regard. RT-qPCR is considered as the gold standard for quantifying RNA. In order to standardize RT-qPCR data in the sample type under investigation, the use of suitable stable sncRNAs is essential. Here, we selected 10 sncRNAs from small RNA sequencing of CM samples derived from both bovine blastocysts and degenerate embryos, and evaluated their expression stability together with that of cel-miR-39 as a spike and the often-used U6 small nuclear RNA at different embryo developmental stages. In CM of 2-cell embryos, rsRNA-1044 showed the most stable expression, while tDR-1:32-Gly-CCC-1 was the most stable expressed sncRNA in CM of the stages beyond the 2-cell stage. Next, tDR-1:32-Gly-CCC-1 was used for normalizing the RT-qPCR data from the CM of blastocysts and degenerate embryos. Bta-miR-155 and tDR-39:75-Arg-CCG-2 were found to be significantly up-regulated in the CM of blastocysts compared to that of the degenerated embryos (P = 0.028 and P = 0.017, respectively), suggesting their expression levels are related to embryo development stage. In conclusion, tDR-1:32-Gly-CCC-1 can serve as a suitable reference sncRNA for normalization of RT-qPCR data of the CM from bovine blastocysts.

Integrated mRNA-miRNA transcriptome profiling of blood immune responses potentially related to pulmonary fibrosis in forest musk deer.

Background: Forest musk deer (FMD, Moschus Berezovskii) is a critically endangered species world-widely, the death of which can be caused by pulmonary disease in the farm. Pulmonary fibrosis (PF) was a huge threat to the health and survival of captive FMD. MicroRNAs (miRNAs) and messenger RNAs (mRNAs) have been involved in the regulation of immune genes and disease development. However, the regulatory profiles of mRNAs and miRNAs involved in immune regulation of FMD are unclear. Methods: In this study, mRNA-seq and miRNA-seq in blood were performed to constructed coexpression regulatory networks between PF and healthy groups of FMD. The hub immune- and apoptosis-related genes in the PF blood of FMD were explored through Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Further, protein-protein interaction (PPI) network of immune-associated and apoptosis-associated key signaling pathways were constructed based on mRNA-miRNA in the PF blood of the FMD. Immune hub DEGs and immune hub DEmiRNAs were selected for experimental verification using RT-qPCR. Results: A total of 2744 differentially expressed genes (DEGs) and 356 differentially expressed miRNAs (DEmiRNAs) were identified in the PF blood group compared to the healthy blood group. Among them, 42 DEmiRNAs were negatively correlated with 20 immune DEGs from a total of 57 correlations. The DEGs were significantly associated with pathways related to CD molecules, immune disease, immune system, cytokine receptors, T cell receptor signaling pathway, Th1 and Th2 cell differentiation, cytokine-cytokine receptor interaction, intestinal immune network for IgA production, and NOD-like receptor signaling pathway. There were 240 immune-related DEGs, in which 186 immune-related DEGs were up-regulated and 54 immune-related DEGs were down-regulated. In the protein-protein interaction (PPI) analysis of immune-related signaling pathway, TYK2, TLR2, TLR4, IL18, CSF1, CXCL13, LCK, ITGB2, PIK3CB, HCK, CD40, CD86, CCL3, CCR7, IL2RA, TLR3, and IL4R were identified as the hub immune genes. The mRNA-miRNA coregulation analysis showed that let-7d, miR-324-3p, miR-760, miR-185, miR-149, miR-149-5p, and miR-1842-5p are key miRNAs that target DEGs involved in immune disease, immune system and immunoregulation. Conclusion: The development and occurrence of PF were significantly influenced by the immune-related and apoptosis-related genes present in PF blood. mRNAs and miRNAs associated with the development and occurrence of PF in the FMD.

Selection and Multiplexing of Reverse Transcription-Quantitative PCR Tests Targeting Relevant Honeybee Viral Pathogens.

Verifying the inclusivity of molecular detection methods gives indications about the reliability of viral infection diagnosis because of the tendency of viral pathogens to undergo sequence variation. This study was aimed at selecting inclusive probes based on reverse transcription-quantitative PCR (RT-qPCR) assays for the diagnosis of the most widespread and detrimental viruses infecting honeybees, namely the acute bee paralysis virus (ABPV), the black queen cell virus (BQCV), the chronic paralysis bee virus (CBPV), the deformed wing virus variants A (DWVA) and B (DWVB), and the sacbrood virus (SBV). Therefore, previously described detection methods were re-evaluated in silico for their specificity and inclusivity. Based on this evaluation, selected methods were modified, or new ones were designed and tested in duplex RT-qPCR reactions. The limits of detection (LODs), effect of multiplexing on sensitivity and the viral RNA quantification potential in bees and hive debris were assessed. This study made available diagnostic assays able to detect an increased number of virus variants compared with previously described tests and two viral pathogens in a single PCR reaction.

The Spread of Southern Rice Black-Streaked Dwarf Virus Was Not Caused by Biological Changes in Vector Sogatella furcifera.

The pandemic of Southern rice black-streaked dwarf virus (SRBSDV) in and after the late 2000s caused serious yield losses in rice in Southeast and East Asia. This virus was first recorded in China in 2001, but its exclusive vector insect, Sogatella furcifera, occurred there before then. To clarify the evolutionary origin of SRBSDV as the first plant virus transmitted by S. furcifera, we tested virus transmission using three chronological strains of S. furcifera, two of which were established before the first report of SRBSDV. When the strains fed on SRBSDV-infected rice plants were transferred to healthy rice plants, those established in 1989 and 1999 transmitted the virus to rice similarly to the strain established in 2010. SRBSDV quantification by RT-qPCR confirmed virus accumulation in the salivary glands of all three strains. Therefore, SRBSDV transmission by S. furcifera was not caused by biological changes in the vector, but probably by the genetic change of the virus from a closely related Fijivirus, Rice black-streaked dwarf virus, as suggested by ecological and molecular biological comparisons between the two viruses. This result will help us to better understand the evolutionary relationship between plant viruses and their vector insects and to better manage viral disease in rice cropping in Asia.

Development of Multiplex RT qPCR Assays for Simultaneous Detection and Quantification of Faecal Indicator Bacteria in Bathing Recreational Waters.

In this study, we designed and validated in silico and experimentally a rapid, sensitive, and specific multiplex RT qPCR for the detection and quantification of faecal indicator bacteria (FIB) used as microbiological references in marine bathing water regulations (Escherichia coli and intestinal enterococci). The 16S rRNA gene was used to quantify group-specific enterococci and Escherichia/Shigella and species-specific such as Enterococcus faecalis and E. faecium. Additionally, a ybbW gene encoding allantoin transporter protein was used to detect E. coli. An assessment of marine coastal systems (i.e., marine water and sediment) revealed that intestinal enterococci were the predominant group compared to Escherichia/Shigella. The low contribution of E. faecalis to the intestinal enterococci group was reported. As E. faecalis and E. faecium were reported at low concentrations, it is assumed that other enterococci of faecal origin are contributing to the high gene copy number of this group-specific enterococci. Moreover, low 16S rRNA gene copy numbers with respect to E. faecalis and E. faecium were reported in seawater compared to marine sediment. We conclude that marine sediments can affect the quantification of FIBs included in bathing water regulations. Valuing the quality of the marine coastal system through sediment monitoring is recommended.

Capsid Integrity Detection of Enteric Viruses in Reclaimed Waters.

Climate change, unpredictable weather patterns, and droughts are depleting water resources in some parts of the globe, where recycling and reusing wastewater is a strategy for different purposes. To counteract this, the EU regulation for water reuse sets minimum requirements for the use of reclaimed water for agricultural irrigation, including a reduction in human enteric viruses. In the present study, the occurrence of several human enteric viruses, including the human norovirus genogroup I (HuNoV GI), HuNoV GII, and rotavirus (RV), along with viral fecal contamination indicator crAssphage was monitored by using (RT)-qPCR methods on influent wastewater and reclaimed water samples. Moreover, the level of somatic coliphages was also determined as a culturable viral indicator. To assess the potential viral infectivity, an optimization of a capsid integrity PMAxx-RT-qPCR method was performed on sewage samples. Somatic coliphages were present in 60% of the reclaimed water samples, indicating inefficient virus inactivation. Following PMAxx-RT-qPCR optimization, 66% of the samples tested positive for at least one of the analyzed enteric viruses, with concentrations ranging from 2.79 to 7.30 Log10 genome copies (gc)/L. Overall, most of the analyzed reclaimed water samples did not comply with current EU legislation and contained potential infectious viral particles.

Selection of Reference Genes for Expression Normalization by RT-qPCR in Dracocephalum moldavica L.

Dracocephalum moldavica is widely used as an ornamental, medicine, and perfume in industry. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) is widely and accurately utilized for gene expression evaluations. Selecting optimal reference genes is essential for normalizing RT-qPCR results. However, the identification of suitable reference genes in D. moldavica has not been documented. A total of 12 reference genes in D. moldavica were identified by PEG6000 (15%) treatment under hypertonia conditions in different tissues (roots, stem, leaves, flower, seeds and sepal) and during three stages of flower development, then used to validate the expression stability. There were four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper) used to analyze the stability. Finally, the RefFinder program was employed to evaluate the candidate reference genes' stability. The results showed that ACTIN, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and EF1α (elongation factor-1α) were stable reference genes under the PEG6000 treatment. Heat shock protein 70 (HSP70) was the most stable gene across different flower development stages. ADP-ribosylation factor (ARF) was the most stable gene in different tissues and total samples. This study provides reliable gene expression studies for future research in D. moldavica.

Oropouche Virus Exposure in Febrile Patients during Chikungunya Virus Introduction in the State of Amapá, Amazon Region, Brazil.

Oropouche orthobunyavirus (OROV) is an arbovirus transmitted by midges that has been involved in outbreaks throughout Central and South America. In Brazil, human cases have been historically concentrated in the northern region of the country. Oropouche fever in humans range from mild clinical signs to rare neurological events, and is considered a neglected tropical disease in Brazil. Due to the clinical similarities to other arboviruses, such as chikungunya and dengue viruses, OROV infections are likely to be underreported. Chikungunya virus (CHIKV) cases in Brazil were first recognized in 2014 in the states of Amapá and Bahia in the north and northeast regions, respectively. Both OROV and CHIKV cause nonspecific symptoms, making clinical diagnosis difficult in a scenario of arbovirus cocirculation. Aiming to investigate OROV transmission during the CHIKV introduction in the state of Amapá located in the Brazilian Amazon, we conducted a retrospective molecular (RT-qPCR) and serological investigation in febrile cases (N = 166) collected between August 2014 and May 2015. All acute serum samples were negative for OROV RNA using RT-qPCR. However, neutralizing antibodies for OROV were detected using a plaque reduction neutralization test (PRNT90) in 10.24% (17/166) of the patients, with neutralizing antibody titers ranging from 20 to ≥640, suggesting the previous exposure of patients to OROV. Regarding CHIKV, recent exposure was confirmed by the detection of CHIKV RNA in 20.25% (33/163) of the patients and by the detection of anti-CHIKV IgM in 28.57% (44/154) of the patients. The additional detection of anti-CHIKV IgG in 12.58% (19/151) of the febrile patients suggests that some individuals had been previously exposed to CHIKV. Whether the OROV exposure reported here occurred prior or during the CHIKV circulation in Amapá, is unknown, but because those arboviral infections share similar clinical signs and symptoms, a silent circulation of enzootic arboviruses during the introduction of exotic arboviruses may occur, and highlights the importance of syndromic cases' surveillance to arboviruses in Brazil.