Milk miRNA expression in buffaloes as a potential biomarker for mastitis.

BACKGROUND: Buffaloes have the highest potential for production due to a promising gene pool that is being enhanced and upgraded. Mastitis is a significant health impediment that greatly diminishes milk yield and quality, affecting rural farmers' livelihoods. The traditional gold standard used for diagnosing mastitis or subclinical mastitis is CMT, but it has the drawback of false positive or negative results. Subclinical mastitis, if not treated promptly, can lead to mammary tumors. To address the gap in early diagnosis of subclinical mastitis in CMT-negative milk of buffaloes, we performed a retrospective analysis and evaluated the milk miRNA expression profiles as potential biomarkers. RESULTS: Thirty buffalo milk samples based on clinical signs and CMT were divided into normal, subclinical, and clinical mastitis. SCC evaluation showed significant differences between the groups. The data analysis demonstrated that the elevation of miR-146a and miR-383 differed substantially between normal, subclinical, and clinical mastitis milk of buffaloes with 100% sensitivity and specificity. The relationship of SCC with miR-146a and miR-383 in normal/healthy and subclinical mastitis was positively correlated. CONCLUSION: The overexpression of miR-146a and miR-383 is associated with inflammation. It can be a valuable prognostic and most sensitive biomarker for early mastitis detection in buffaloes with SCC below 2 lakhs and CMT-ve, enhancing the accuracy of subclinical mastitis diagnosis.

Delineation of altered brain proteins associated with furious rabies virus infection in dogs by quantitative proteomics.

Rabies is a fatal zoonotic disease caused by rabies virus (RABV). Despite the existence of control measures, dog-transmitted human rabies accounts for ˃95% reported cases due to unavailability of sensitive diagnostic methods, inadequate understanding of disease progression and absence of therapeutics. In addition, host factors and their role in RABV infection are poorly understood. In this study, we used 8-plex iTRAQ coupled with HRMS approach to identify differentially abundant proteins (DAPs) of dog brain associated with furious rabies virus infection. Total 40 DAPs including 26 down-regulated and 14 up-regulated proteins were statistically significant in infected samples. GO annotation and IPA showed that calcium signaling and calcium transport, efficient neuronal function, metabolic pathway associated proteins were mostly altered during this infection. Total 34 proteins including 10 down-regulated proteins pertaining to calcium signaling and calcium transport pathways were successfully verified by qRT-PCR and two proteins were verified by western blot, thereby suggesting these pathways may play an important role in this infection. This study provides the map of altered brain proteins and some insights into the molecular pathophysiology associated with furious rabies virus infection. However, further investigations are required to understand their role in disease mechanism. SIGNIFICANCE: Transmission of rabies by dogs poses the greatest hazard world-wide and the rare survival of post-symptomatic patients as well as severe neurological and immunological problems pose a question to understand the molecular mechanism involved in rabies pathogenesis. However, information regarding host factors and their function in RABV infection is still inadequate. Our study has used an advanced quantitative proteomics approach i.e. 8-plex iTRAQ coupled with HRMS and identified 40 DAPs in furious rabies infected dog brain tissues compared to the controls. Further analysis showed that calcium signaling and transport pathway, efficient neuronal functions and metabolic pathway associated brain proteins were most altered during furious rabies virus infection. This data provides a map of altered brain proteins which may have role in furious rabies virus infection. Hence, this will improve our understanding of the molecular pathogenesis of RABV infection.

Evolutionary analysis of rabies virus using the partial Nucleoprotein and Glycoprotein gene in Mumbai region of India.

Nearly 1.7 million cases of dog bites are reported every year in India and many cases of animal rabies are left unattended and undiagnosed. Therefore, a mere diagnosis of rabies is not sufficient to understand the epidemiology and the spread of the rabies virus (RV) in animals. There is a paucity of information about the evolutionary dynamics of RV in dogs and its biodiversity patterns in India. In total, 50 dog-brain samples suspected of rabies were screened by the nucleoprotein- (N) and glycoprotein- (G) gene PCR. The N and G genes were subsequently sequenced to understand the molecular evolution in these genes. The phylogenetic analysis of the N gene revealed that six isolates in the Mumbai region belonged to a single Arctic lineage. Time-scaled phylogeny by Bayesian coalescent analysis of the partial N gene revealed that the time to the most recent common ancestor (TMRCA) for the sequences belonged to the cluster from 2006.68 with a highest posterior density of 95 % betweeen 2005-2008, which is assigned to Indian lineage I. Migration pattern revealed a strong Bayes factor between Mumbai to Delhi, Panji to Hyderabad, Delhi to Chennai, and Chennai to Chandigarh. Phylogenetic analysis of the G gene revealed that the RVs circulating in the Mumbai region are divided into three lineages. Time-scaled phylogeny by the Bayesian coalescent analysis method estimated that the TMRCA for sequences under study was from 1993 and Indian clusters was from 1962. In conclusion, the phylogenetic analysis of the N gene revealed that six isolates belonged to single Arctic lineages along with other Indian isolates and they were clustered into a single lineage but divided into three clades based on the G-gene sequences. The present study highlights and enhances the current molecular epidemiology and evolution of RV and revealed strong location bias and geographical clustering within Indian isolates on the basis of N and G genes.

Inhalable liposomes of Glycyrrhiza glabra extract for use in tuberculosis: formulation, in vitro characterization, in vivo lung deposition, and in vivo pharmacodynamic studies.

OBJECTIVE: The current study involves the development of liposomal dry powder for inhalation (LDPI) containing licorice extract (LE) for use in tuberculosis. SIGNIFICANCE: The current epidemiology of tuberculosis along with the increasing emergence of resistant forms of tuberculosis necessitates the need for developing alternative efficacious medicines for treatment. Licorice is a medicinal herb with reported activity against Mycobacterium tuberculosis. METHODS: Liposomes with LE were prepared by thin film hydration technique and freeze dried to obtain LDPI. The comprehensive in vitro and in vivo characterization of the LDPI formulation was carried out. RESULTS: The particle size of liposomes was around 210 nm with drug entrapment of almost 75%. Transmission electron microscopy revealed spherical shape of liposome vesicles. The flow properties of the LDPI were within acceptable limits. Anderson Cascade Impactor studies showed the mean median aerodynamic diameter, geometric standard deviation and fine particle fraction of the LDPI to be 4.29 µm, 1.23, and 54.68%, respectively. In vivo lung deposition studies of LDPI in mice showed that almost 46% of the drug administered reaches the lungs and 16% of administered drug is retained in the lungs after 24 hours of administration. The in vivo pharmacodynamic evaluation of the LDPI showed significant reduction in bacterial counts in lungs as well as spleen of TB-infected mice. CONCLUSIONS: LE LDPI thus has a promising potential to be explored as an effective anti-tubercular medicine or as an adjunct to existing anti-tubercular drugs.