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.

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.

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.

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.

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.

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.

OBJECTIVE: The aim of this study was to evaluate the expression profiles of PIWI-like protein- 2 (PIWIL2), and HepPar1 and their immunohistochemical (IHC) characteristics in Hepatocellular Carcinoma (HCC), and determine their correlation with clinicopathological parameters of this type of cancer to determine their diagnostic value in combination.
METHODS: Seventy-five patients with HCC were assessed for the expression of PIWIL2 in serum and tissue using real-time polymerase chain reaction (RT-PCR) and IHC was performed for PIWIL2 and HepPar1 was performed on all patients.
RESULTS: A statistically significantly higher level of PIWIL2 was found in HCC compared to controls (p≤0.001). Both HepPar1 and PIWIL2 were detected in 84% of HCC cases, the diagnostic and prognostic factors for PIWIL2 were found to be significant in liver tumour tissue samples and non-tumorous sections p<0.001, and the same was observed for serum samples and results of healthy serum controls (p<0.001) when compared to AFP.
CONCLUSIONS: Our results affirm the hypothesis that reactivation of PIWI expression in various caner types is crucial for cancer development, and that a possible panel maybe used for these markers HCC diagnosis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus that undergoes rapid mutation. Based on viral whole genome sequencing analysis in Hebei Province, China, we identified several essential single nucleotide variants (SNVs) on primer-probe regions accumulating within some Omicron variants\' genomes. In this study, we focused on three SNVs, C28290T, T28297C, and C28311T emerging on 2019-nCoV-N1 (CDC-N1) primer-probe regions, recommended by CDC in 2020, and two SNVs, C26270T, A26275G emerging on E (Charité-E) primer-probe regions recommended by Charité, Germany. Our findings revealed that the presence of one or two SNVs in the primer or probe region affected the sensitivity of reverse transcription-quantitative polymerase chain reaction and droplet digital PCR to varying extents. This discovery underscores the importance of continuously monitoring the whole genome sequences of SARS-CoV-2 variants, especially the primer-probe targeting regions, and correspondingly updating commercial test kits or recommended primer-probe sequence sets.
OBJECTIVE: The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has resulted in a growing number of mutations in its genome, presenting new challenges for the diagnosis of SARS-CoV-2 using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and droplet digital PCR (RT-ddPCR) methods. There is an urgent need to develop refined methods for modifying primers and probes to improve the detection of these emerging variants. In this study, our focus was on the SNVs that have emerged in the CDC-N1 and Charité-E primer-probe regions. Our research has confirmed that the presence of these SNVs in the primer or probe region can significantly affect the results of coronavirus disease 2019 tests. we have developed and validated a modified detection method that can provide higher sensitivity and specificity. This study emphasizes the importance of refining the primer-probe sets to ensure the diagnostic accuracy of RT-qPCR and RT-ddPCR detection.

In 2020, there were numerous cases in Kazakhstan with clinical symptoms of COVID-19 but negative PCR results in nasopharyngeal and oropharyngeal swabs. The diagnosis was confirmed clinically and by CT scans (computed tomography). The problem with such negative PCR results for SARS-CoV-2 infection confirmation still exists and indicates the need to confirm the diagnosis in the bronchoalveolar lavage in such cases. There is also a lack of information about confirmation of SARS-CoV-2 infection in deceased patients. In this study, various tissue materials, including lungs, bronchi, and trachea, were examined from eight patients who died, presumably from SARS-CoV-2 infection, between 2020 and 2022. Naso/oropharyngeal swabs taken from these patients in hospitals tested PCR negative for SARS-CoV-2. This study presents a modified RNA isolation method based on a comparison of the most used methods for RNA isolation in laboratories: QIAamp Viral RNA Mini Kit and TRIzol-based method. This modified nucleic acid extraction protocol can be used to confirm SARS-CoV-2 infection by RT-qPCR in the tissues of deceased patients in disputed cases. RT-qPCR with RNA of SARS-CoV-2 re-extracted with such method from post-mortem tissues that were stored at -80 °C for more than 32 months still demonstrated high-yielding positive results.

The process of tumorigenesis is highly associated with the disruption of cell-cycle regulators and derangement of various signaling pathways, which end up with the inhibition of apoptosis and hyper-activation of survival pathways. The PI3K medicated AKT/mTOR pathway is the widely explained mechanism for cancer cell survival which causes the overexpression of MDM2 and downregulates the p53-BAX mediated apoptotic pathway. Curcumin (CUR), the phyto-compound, derived from Curcuma longa is currently being focused on for its anticancer activities against breast cancer cells, MDA-MB-231, not only because of its minimal cytotoxicity against healthy cells (HEK293) but also because it synergistically sensitizes the activity of Doxorubicin (DOXO) in lower doses, which can be a promising source for complementary drug development. This study aims to investigate the combinatorial effect of CUR and DOXO on PI3K/AKT/mTOR pathway proteins by sequential molecular docking analysis and MD simulation studies. The lower binding affinity of the sequentially docked protein-ligand complex proves the increasing binding affinity of CUR and DOXO in the combinatorial dose. The mRNA expressions of different genes of this pathway are observed and quantified using rt-qPCR, where the decreasing fold change (2-∆∆Ct) indicates the suppression of the AKT/mTOR pathway after co-treatment of CUR and DOXO against MDA-MB-231 cells. These in silico and in vitro findings can be a new horizon for further in vitro and clinical trials of breast cancer treatment.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s40203-024-00231-2.