A novel qPCR-based technique for identifying avian sex: An illustration within embryonic craniofacial bone.

Sex is a biological variable important to consider in all biomedical experiments. However, doing so in avian embryos can be challenging as sex can be morphologically indistinguishable. Unlike humans, female birds are the heterogametic sex with Z and W sex chromosomes. The female-specific W chromosome has previously been identified in chick using a species-specific polymerase chain reaction (PCR) technique. We developed a novel reverse transcription quantitative PCR (RT-qPCR) technique that amplifies the W chromosome gene histidine triad nucleotide-binding protein W (HINTW) in chick, quail, and duck. Accuracy of the HINTW RT-qPCR primer set was confirmed in all three species using species-specific PCR, including a novel quail-specific HINTW PCR primer set. Bone development-related gene expression was then analyzed by sex in embryonic lower jaws of duck and quail, as adult duck beak size is known to be sexually dimorphic while quail beak size is not. Trends toward sex differences were found in duck gene expression but not in quail, as expected. With these novel RT-qPCR and PCR embryo sexing methods, sex of chick, quail, and duck embryos can now be assessed by either/both RNA and DNA, which facilitates analysis of sex as a biological variable in studies using these model organisms.

Annotations of four high-quality indigenous chicken genomes identify more than one thousand missing genes in subtelomeric regions and micro-chromosomes with high G/C contents.

BACKGROUND: Although multiple chicken genomes have been assembled and annotated, the numbers of protein-coding genes in chicken genomes and their variation among breeds are still uncertain due to the low quality of these genome assemblies and limited resources used in their gene annotations. To fill these gaps, we recently assembled genomes of four indigenous chicken breeds with distinct traits at chromosome-level. In this study, we annotated genes in each of these assembled genomes using a combination of RNA-seq- and homology-based approaches. RESULTS: We identified varying numbers (17,497-17,718) of protein-coding genes in the four indigenous chicken genomes, while recovering 51 of the 274 "missing" genes in birds in general, and 36 of the 174 "missing" genes in chickens in particular. Intriguingly, based on deeply sequenced RNA-seq data collected in multiple tissues in the four breeds, we found 571 ~ 627 protein-coding genes in each genome, which were missing in the annotations of the reference chicken genomes (GRCg6a and GRCg7b/w). After removing redundancy, we ended up with a total of 1,420 newly annotated genes (NAGs). The NAGs tend to be found in subtelomeric regions of macro-chromosomes (chr1 to chr5, plus chrZ) and middle chromosomes (chr6 to chr13, plus chrW), as well as in micro-chromosomes (chr14 to chr39) and unplaced contigs, where G/C contents are high. Moreover, the NAGs have elevated quadruplexes G frequencies, while both G/C contents and quadruplexes G frequencies in their surrounding regions are also high. The NAGs showed tissue-specific expression, and we were able to verify 39 (92.9%) of 42 randomly selected ones in various tissues of the four chicken breeds using RT-qPCR experiments. Most of the NAGs were also encoded in the reference chicken genomes, thus, these genomes might harbor more genes than previously thought. CONCLUSION: The NAGs are widely distributed in wild, indigenous and commercial chickens, and they might play critical roles in chicken physiology. Counting these new genes, chicken genomes harbor more genes than originally thought.

ExonSurfer: a web-tool to design primers at exon-exon junctions.

BACKGROUND: Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design. RESULTS: ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets. CONCLUSION: ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.

RNA-seq validation: software for selection of reference and variable candidate genes for RT-qPCR.

BACKGROUND: Real-time quantitative PCR (RT-qPCR) is one of the most widely used gene expression analyses for validating RNA-seq data. This technique requires reference genes that are stable and highly expressed, at least across the different biological conditions present in the transcriptome. Reference and variable candidate gene selection is often neglected, leading to misinterpretation of the results. RESULTS: We developed a software named "Gene Selector for Validation" (GSV), which identifies the best reference and variable candidate genes for validation within a quantitative transcriptome. This tool also filters the candidate genes concerning the RT-qPCR assay detection limit. GSV was compared with other software using synthetic datasets and performed better, removing stable low-expression genes from the reference candidate list and creating the variable-expression validation list. GSV software was used on a real case, an Aedes aegypti transcriptome. The top GSV reference candidate genes were selected for RT-qPCR analysis, confirming that eiF1A and eiF3j were the most stable genes tested. The tool also confirmed that traditional mosquito reference genes were less stable in the analyzed samples, highlighting the possibility of inappropriate choices. A meta-transcriptome dataset with more than ninety thousand genes was also processed successfully. CONCLUSION: The GSV tool is a time and cost-effective tool that can be used to select reference and validation candidate genes from the biological conditions present in transcriptomic data.

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.

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.

Validation of Selected MicroRNA Transcriptome Data in the Bovine Corpus Luteum during Early Pregnancy by RT-qPCR.

In cattle, the corpus luteum (CL) is pivotal in maintaining early pregnancy by secreting progesterone. To establish pregnancy, the conceptus produces interferon-τ, preventing luteolysis and initiating the transformation of the CL spurium into a CL verum. Although this transformation is tightly regulated, limited data are available on the expression of microRNAs (miRNAs) during and after this process. To address this gap, we re-analyzed previously published RNA-Seq data of CL from pregnant cows and regressed CL from non-pregnant cows. This analysis identified 44 differentially expressed miRNAs. From this pool, three miRNAs-bta-miR-222-3p, bta-miR-29c, and bta-miR-2411-3p-were randomly selected for relative quantification. Using bovine ovaries (n = 14) obtained from an abattoir, total RNA (including miRNAs) was extracted and converted to cDNA for RT-qPCR. The results revealed that bta-miR-222-3p was downregulated (p = 0.016) in pregnant females compared to non-pregnant cows with regressed CL. However, no differences in miRNA expression were observed between CL of pregnant and non-pregnant cows for bta-miR-29c (p > 0.32) or bta-miR-2411-3p (p > 0.60). In silico prediction approaches indicated that these miRNAs are involved in pathways regulating pregnancy maintenance, such as the VEGF- and FoxO-signaling pathways. Additionally, their biogenesis is regulated by GABPA and E2F4 transcription factors. The validation of selected miRNA expression in the CL during pregnancy by RT-qPCR provides novel insights that could potentially lead to the identification of biomarkers related to CL physiology and pregnancy outcome.

Influence of storage solution, temperature, assay time and concentration on RT-qPCR nucleic acid detection for SARS-CoV-2 detection of SARS-CoV-2 by the RT-qPCR.

Real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is an important method for the early diagnosis of coronavirus disease 2019 (COVID-19). This study investigated the effects of storage solution, temperature and detection time on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection by RT-qPCR. Various concentrations of SARS-CoV-2 were added to inactive and non-inactive storage solution and the viral suspensions were stored at various temperatures (room temperature, 4, -20 and -80 °C). Then, at five different detection time points, the Ct values were determined by RT-qPCR. Active and inactive storage solutions and storage temperature have a great impact on the detection of N gene of SARS-CoV-2 at different concentration corridors but have little impact on the ORF gene. The storage time has a greater impact on the N gene and ORF gene at high concentrations but has no effect on the two genes at low concentrations. In conclusion, storage temperature, storage time and storage status (inactivated, non-inactivated) have no effect on the nucleic acid detection of SARS-CoV-2 at the same concentration. For different concentrations of SARS-CoV-2, the detection of N gene is mainly affected.

Transcriptome-based identification and validation of reference genes for corm growth stages, different tissues, and drought stress in Taro (Colocasia esculenta).

Taro is a widely utilized starch resource plant. It is essential to quantify the expression levels of functional genes associated with taro growth using real-time quantitative polymerase chain reaction (RT-qPCR). However, to obtain reliable RT-qPCR results, appropriate reference genes (RGs) are required for data normalization. In this study, we screened seven novel candidate RGs using transcriptome datasets from taro, encompassing data from growth corms and various tissues. The expression stability of these seven new RGs, along with the commonly used RGs Actin, EF1-α, and ß-tubulin, was assessed using Delta Ct, BestKeeper, geNorm, and NormFinder algorithms. Furthermore, we conducted a comprehensive analysis using the RefFinder program and validated the results using the target gene, CeAGPL1. The findings revealed that ACY-1 and PIA2 were the optimal multiple RGs for normalization during corm growth, while COX10 and Armc8 were suitable for samples including various types of tissues. Furthermore, we found three RGs, Armc8, COX10 and CCX4L, were the optimal RGs for drought stress. This study assessed the suitability of RGs in taro for the first time. The identified RGs provide valuable resources for studying corm growth, diverse tissues, and drought stress. This study contributes to the advancement of our understanding of the underlying mechanisms that govern the growth of taro.

Identification and validation of stable reference genes for RT-qPCR analyses of Kobresia littledalei seedlings.

BACKGROUND: Kobreisa littledalei, belonging to the Cyperaceae family is the first Kobresia species with a reference genome and the most dominant species in Qinghai-Tibet Plateau alpine meadows. It has several resistance genes which could be used to breed improved crop varieties. Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) is a popular and accurate gene expression analysis method. Its reliability depends on the expression levels of reference genes, which vary by species, tissues and environments. However, K.littledalei lacks a stable and normalized reference gene for RT-qPCR analysis. RESULTS: The stability of 13 potential reference genes was tested and the stable reference genes were selected for RT-qPCR normalization for the expression analysis in the different tissues of K. littledalei under two abiotic stresses (salt and drought) and two hormonal treatments (abscisic acid (ABA) and gibberellin (GA)). Five algorithms were used to assess the stability of putative reference genes. The results showed a variation amongst the methods, and the same reference genes showed tissue expression differences under the same conditions. The stability of combining two reference genes was better than a single one. The expression levels of ACTIN were stable in leaves and stems under normal conditions, in leaves under drought stress and in roots under ABA treatment. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was stable in the roots under the control conditions and salt stress and in stems exposed to drought stress. Expression levels of superoxide dismutase (SOD) were stable in stems of ABA-treated plants and in the roots under drought stress. Moreover, RPL6 expression was stable in the leaves and stems under salt stress and in the stems of the GA-treated plants. EF1-alpha expression was stable in leaves under ABA and GA treatments. The expression levels of 28 S were stable in the roots under GA treatment. In general, ACTIN and GAPDH could be employed as housekeeping genes for K. littledalei under different treatments. CONCLUSION: This study identified the best RT-qPCR reference genes for different K. littledalei tissues under five experimental conditions. ACTIN and GAPDH genes can be employed as the ideal housekeeping genes for expression analysis under different conditions. This is the first study to investigate the stable reference genes for normalized gene expression analysis of K. littledalei under different conditions. The results could aid molecular biology and gene function research on Kobresia and other related species.

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.

Genome-wide analysis of AP2/ERF transcription factors that regulate fruit development of Chinese prickly ash.

BACKGROUND: AP2/ERF is a large family of plant transcription factor proteins that play essential roles in signal transduction, plant growth and development, and responses to various stresses. The AP2/ERF family has been identified and verified by functional analysis in various plants, but so far there has been no comprehensive study of these factors in Chinese prickly ash. Phylogenetic, motif, and functional analyses combined with transcriptome analysis of Chinese prickly ash fruits at different developmental stages (30, 60, and 90 days after anthesis) were conducted in this study. RESULTS: The analysis identified 146 ZbAP2/ERF genes that could be classified into 15 subgroups. The motif analysis revealed the presence of different motifs or elements in each group that may explain the functional differences between the groups. ZbERF13.2, ZbRAP2-12, and ZbERF2.1 showed high levels of expression in the early stages of fruit development. ZbRAP2-4, and ZbERF3.1 were significantly expressed at the fruit coloring stage (R2 and G2). ZbERF16 were significantly expressed at fruit ripening and expression level increased as the fruit continued to develop. Relative gene expression levels of 6 representative ZbAP2/ERFs assessed by RT-qPCR agreed with transcriptome analysis results. CONCLUSIONS: These genes identified by screening can be used as candidate genes that affect fruit development. The results of the analysis can help guide future genetic improvement of Chinese prickly ash and enrich our understanding of AP2/ERF transcription factors and their regulatory functions in plants.

Genome-wide analysis of NAC transcription factors and exploration of candidate genes regulating selenium metabolism in Broussonetia papyrifera.

MAIN CONCLUSION: Genome-wide identification revealed 79 BpNAC genes belonging to 16 subfamilies, and their gene structures and evolutionary relationships were characterized. Expression analysis highlighted their importance in plant selenium stress responses. Paper mulberry (Broussonetia papyrifera), a deciduous arboreal plant of the Moraceae family, is distinguished by its leaves, which are abundant in proteins, polysaccharides, and flavonoids, positioning it as a novel feedstock. NAC transcription factors, exclusive to plant species, are crucial in regulating growth, development, and response to biotic and abiotic stress. However, extensive characterization of the NAC family within paper mulberry is lacking. In this study, 79 BpNAC genes were identified from the paper mulberry genome, with an uneven distribution across 13 chromosomes. A comprehensive, genome-wide analysis of BpNACs was performed, including investigating gene structures, promoter regions, and chromosomal locations. Phylogenetic tree analysis, alongside comparisons with Arabidopsis thaliana NACs, allowed for categorizing these genes into 16 subfamilies in alignment with gene structure and motif conservation. Collinearity analysis suggested a significant homologous relationship between the NAC genes of paper mulberry and those in Morus notabilis, Ficus hispida, Antiaris toxicaria, and Cannabis sativa. Integrating transcriptome data and Se content revealed that 12 BpNAC genes were associated with selenium biosynthesis. Subsequent RT-qPCR analysis corroborated the correlation between BpNAC59, BpNAC62 with sodium selenate, and BpNAC55 with sodium selenite. Subcellular localization experiments revealed the nuclear functions of BpNAC59 and BpNAC62. This study highlights the potential BpNAC transcription factors involved in selenium metabolism, providing a foundation for strategically breeding selenium-fortified paper mulberry.

In vitro regeneration, photomorphogenesis and light signaling gene expression in Hydrangea quercifolia cv. 'Harmony' under different LED environments.

MAIN CONCLUSION: Plant growth regulators, sucrose concentration, and light quality significantly impact in vitro regeneration of 'Harmony'. Blue light promotes photomorphogenesis by enhancing light energy utilization, adjusting transcription of light signal genes, and altering hormone levels. Hydrangea quercifolia cv. 'Harmony', celebrated for lush green foliage and clusters of white flowers, has been extensively researched for its regenerative properties. Regeneration in stem segments, leaves, and petioles is facilitated by exogenous auxin and cytokinins (CTKs), with the concentration of sucrose (SC) being a key determinant for shoot regeneration from leaves. The study also highlights the significant impact of light conditions on photomorphogenesis. With an increase in the proportion of red (R) light, there is an inhibitory effect, leading to a reduction in leaf area, a decrease in the quantum yield of PSII (ΦPSII), and an increase in non-photochemical quenching (ΦNPQ) and non-regulated energy dissipation in PSII (ΦNO). Conversely, blue (B) light enhances growth, characterized by an increase in leaf area, elevated ΦPSII, and stable ΦNPQ and ΦNO levels. Additionally, B light induces the upregulation of HqCRYs, HqHY5-like, HqXTH27-like, and HqPHYs genes, along with an increase in endogenous CTKs levels, which positively influence photomorphogenesis independent of HqHY5-like regulation. This light condition also suppresses the synthesis of endogenous gibberellins (GA) and brassinosteroids (BR), further facilitating photomorphogenesis. In essence, B light is fundamental in expediting photomorphogenesis in 'Harmony', demonstrating the vital role in plant growth and development.

Phenotypic and transcriptomics characterization uncovers genes underlying tuber yield traits and gene expression marker development in potato under aeroponics.

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.

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.

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.

Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in different tissues from mice infected by Ascaris suum.

Human ascariasis is the most prevalent helminth infection, affecting 445 million people worldwide. To better understand the impact of the immune system on the pathophysiology of individuals infected with Ascaris suum, mice have been used as experimental models. The RT-qPCR technique is a critical auxiliary tool of investigation used to quantify mRNA levels. However, proper normalization using reference genes is essential to ensure reliable outcomes to avoid analytical errors and false results. Despite the importance of reference genes for experimental A. suum infection studies, no specific reference genes have been identified yet. Therefore, we conducted a study to assess five potential reference genes (GAPDH, 18s, ACTB, B2M, and HPRT1) in different tissues (liver, lungs, small and large intestines) affected by A. suum larval migration in C57BL/6j mice. Tissue collection was carried out to analyze parasite burden and confirm the presence of larvae during the peak of migration in each tissue. Upon confirmation, we analyzed different genes in the tissues and found no common gene with stable expression. Our results highlight the importance of analyzing different genes and using different software programs to ensure reliable relative expression results. Based on our findings, B2M was ranked as the ideal reference gene for the liver, while 18S was the most stable gene in the lung and small intestine. ACTB, or a combination of ACTB with GAPDH, was deemed suitable as reference genes for the large intestine due to their stable expression and less variation between the control and infected groups. To further demonstrate the impact of using different reference genes, we normalized the expression of a chemokine gene (CXCL9) in all tissues. Significant differences in CXCL9 expression levels were observed between different groups in all tissues except for the large intestine. This underscores the importance of selecting appropriate reference genes to avoid overestimating target gene expression levels and encountering normalization-related issues that can lead to false results. In conclusion, our study highlights the significance of using reliable reference genes for accurate RT-qPCR analysis, especially in the context of A. suum infection studies in different tissues. Proper normalization is crucial to ensure the validity of gene expression data and avoid potential pitfalls in interpreting results.

Double infection of Nicotiana benthamiana with AMV and WCMV increases both virus concentrations and synergistically changes both host organelle ultrastructure and chlorophyll content.

To clarify the synergistic pathogenic mechanism of Nicotiana benthamiana double infection with alfalfa mosaic virus (AMV) and white clover mosaic virus (WCMV), AMV and WCMV co-inoculation of N. benthamiana as treatment and single inoculation of AMV or WCMV and phosphate buffer solution (pH 7.0, PBS) as control, respectively. The concentrations and the relative expression of AMV and WCMV coat proteins were determined by a double antibody sandwich enzyme-linked immune sorbent assay (DAS-ELISA) and real-time fluorescence quantitative PCR (RT-qPCR) in a double infection of N. benthamiana with AMV and WCMV. Meanwhile, virion morphology, ultrastructure morphology, and chlorophyll content were observed and determined by electron microscopy. The results showed that the diseased symptoms were more serious, and virus concentration and relative expression of AMV and WCMV coat proteins were also higher in N. benthamiana double infection with AMV and WCMV than in AMV or WCMV single infection. The main symptoms manifested as severe mottle mosaic, shrinkage, and chlorosis. The concentrations of AMV and WCMV were 182.23 pg/mL and 148.77 pg/mL of double infection with AMV and WCMV, which were 1.75-fold and 1.62-fold than AMV and WCMV single infection, respectively. The relative expression of AMV and WCMV coat proteins was 4.25-fold and 2.50-fold than the single virus infection, respectively. Electron microscopy also observed that chloroplast malformation, cell membrane deformation, contents dissolution, grana lamella disorder, fat granules increased and enlarged, starch granules enlarged, and mitochondria were seriously malformed in a double infection of N. benthamiana with AMV and WCMV. The chlorophyll content was significantly lower for double infection with AMV and WCMV than for AMV or WCMV single-infected and CK, reduced by 31.52 %, 22.83 %, and 76.09 %, respectively. This is the first report of a double infection of N. benthamiana with AMV and WCMV that increases both virus concentrations and synergistically changes both host organelle ultrastructure and chlorophyll content.

Recognizing the role of Epstein-Barr virus in gastric cancer: transcriptomic insights into malignancy modulation.

BACKGROUND: Studies show that Epstein-Barr virus (EBV) infection can play a role in malignancy and increase the risk of gastric cancer (GC). The objective of this research was to pinpoint genes whose expression may be influenced by EBV and play a role in the development of GC. METHODS: Candidate genes potentially susceptible to expression modulation in the presence of EBV were identified through the analysis of GSE185627 and GSE51575 datasets. The association of candidate genes with GC and the survival rate of patients was investigated based on the cancer genome atlas (TCGA) data. Also, pathways related to candidate genes were examined through the MsigDB database. The PPI network was used to identify Hub genes. To corroborate the obtained results, we utilized the RT-qPCR method, employing GC samples from both EBV + and EBV-cases, as well as adjacent normal samples. RESULTS: Our results showed that genes upregulated by the EBV in the GC cell line, as well as in EBV + samples, are significantly linked to pathways involving the immune response, inflammation, and the P53 pathway. Conversely, genes downregulated by EBV are closely linked to pathways involving cell proliferation and mTORC1. Examining the candidate genes revealed that a considerable portion of genes susceptible to downregulation under the influence of EBV exhibit oncogenic roles based on TCGA data. Moreover, some of these genes are associated with an unfavorable prognosis. Protein-protein interaction network analysis of candidate genes highlighted IFI44L and OAS2 as potential hub genes in the EBV-GC axis. Our RT-qPCR results further validated these findings, demonstrating that the expression levels of IFI44L and OAS2 were higher in EBV + samples compared to both healthy and EBV-samples. CONCLUSION: Our study underscores the capacity of EBV to exert regulatory control over genes associated with GC malignancy. In addition to its inflammatory effects, EBV elicits transcriptomic changes that appear to attenuate the progression of GC.