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Salivary microRNAs (miRNAs) have been recently revealed as the next generation of non-invasive biomarkers for the diagnostics of diverse diseases. However, their short and highly homologous sequences make their quantification by RT-qPCR technique highly heterogeneous and study dependent, thus limiting their implementation for clinical applications. In this study, we evaluated the use of a widely used commercial RT-qPCR kit for quantification of salivary miRNAs for clinical diagnostics. Saliva from ten healthy volunteers were sampled four times within a three month time course and submitted for small RNA extraction followed by RT-qPCR analysed. Six miRNAs with different sequence homologies were analysed. Sensitivity and specificity of the tested miRNA assays were corroborated using synthetic miRNAs to evaluate the reliability of all tested assays. Significant variabilities in expression profiles of six miRNAs from ten healthy participants were revealed, yet the poor specificity of the assays offered insufficient performance to associate these differences to biological context. Indeed, as the limit of quantification (LOQ) concentrations are from 2-4 logs higher than that of the limit of detection (LOD) ones, the majority of the analysis for salivary miRNAs felt outside the quantification region. Most importantly, a remarkable number of crosstalk reactions exhibiting considerable OFF target signal intensities was detected, indicating their poor specificity and limited reliability. However, the spike-in of synthetic target miRNA increased the capacity to discriminate endogenous salivary miRNA at the LOQ concentrations from those that were significantly lower. Our results demonstrate that comparative analyses for salivary miRNA expression profiles by this commercial RT-qPCR kit are most likely associated to technical limitations rather than to biological differences. While further technological breakthroughs are still required to overcome discrepancies, standardization of rigorous sample handling and experimental design according to technical parameters of each assay plays a crucial role in reducing data inconsistencies across studies.

Background: Zinc deficiency poses significant health risks, particularly in low-income settings. This study aims to evaluate the impact of agronomically zinc biofortified (fermented and non-fermented) and post-harvest wheat flour flatbread on zinc status and metabolic health in adolescents and adult women in rural Pakistan., Methods: A four-arm triple-blind randomized controlled trial will be conducted in a rural district of Pakistan. Participants (adolescents aged 10-19 and adult women aged 20-40) will be assigned to receive fermented or unfermented high zinc agronomically biofortified wheat flour flatbread, post-harvest zinc-fortified wheat flour flatbread, or low zinc conventional whole wheat flour flatbread. The meal would be served once a day, six days a week for six months. The study aims to enroll 1000 participants and will be analyzed based on the intention-to-treat principle. The trial is registered with number NCT06092515., Outcomes: Primary outcomes will include serum zinc concentration and metabolic markers, while secondary outcomes include anthropometric measurements, blood pressure, and dietary intake., Conclusion: This trial will provide valuable insights into the efficacy of agronomically zinc biofortified wheat flour in improving zinc status and metabolic health. Findings may inform public health strategies to combat zinc deficiency in resource-limited settings., Competing Interests: The authors have declared that no competing interests., (Copyright: © 2024 Das et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Tuberculosis (TB) is the largest infectious disease with 10 million new active-TB patients and1.7 million deaths per year. Active-TB is an inflammatory disease and is increasingly viewed as an imbalance of immune responses to M. tb. infection. The mechanisms of a switch from latent infection to active disease is not well worked out but a shift in the immune responses is thought to be responsible. Increasingly, the role of gut microbiota has been described as a major influencer of the immune system. And because the gut is the largest immune organ, we aimed to analyze the gut microbiome in active-TB patients in a TB-endemic country, Pakistan. The study revealed that Ruminococcacea, Enetrobactericeae, Erysipelotrichaceae, Bifidobacterium, etc. were the major genera associated with active-TB, also associated with chronic inflammatory disease. Plasma antibody profiles against several M. tb. antigens, as specific biomarkers for active-TB, correlated closely with the patient gut microbial profiles. Besides, bcoA gene copy number, indicative of the level of butyrate production by the gut microbiome was five-fold lower in TB patients compared to healthy individuals. These findings suggest that gut health in TB patients is compromised, with implications for disease morbidity (e.g., severe weight loss) as well as immune impairment., Competing Interests: The authors have declared that no competing interests exist.