National Institute of Animal Biotechnology

BACKGROUND:Spermatozoa undergo protein modifications due to oxidative stress as a result of freezing-thawing. With increasing information on oxidatively modified proteins of frozen-thawed spermatozoa, it is essential to investigate the effect of supplementing the semen extender with the repair enzyme protein L-isoaspartyl methyltransferase (PIMT) on the quality of frozen-thawed spermatozoa. OBJECTIVES:To investigate the expression of PIMT in male reproductive organs, including semen, and to evaluate the effect of recombinant PIMT (rPIMT) supplementation on the quality of frozen-thawed spermatozoa. MATERIALS AND METHODS:Expression of PIMT was studied using immunofluorescence assays, PCR, and Western blotting. Functionalities of spermatozoa were studied by HOST, viability, progressive motility, acrosome intactness, and zona-binding assays. RESULTS:The PIMT was detected all along the male reproductive tract, including spermatozoa, but could not be detected in the seminal plasma. The rPIMT was added to the extender at 0.5 (Group II), 1.0 (Group III), and 2.0 µg/80 million spermatozoa (Group IV), with the control (Group I) receiving no rPIMT. A significant effect was observed in Group IV, with an 8.9% increase in progressive motility, a 13.0% increase in viability, an 11.9% increase in HOST response, and an 11.8% improvement in acrosome integrity compared with the control. Further, motility and kinematic parameters (VCL, VAP, DSL, VSL, DAP, ALH, and BCF) of frozen-thawed spermatozoa were assessed using CASA. Most motion parameters were higher in group IV compared with the I, II, and III groups. Additionally, the in vitro fertilizability of Group IV spermatozoa was approximately 2.32 times higher compared with the control group. DISCUSSION AND CONCLUSION:The better functional attributes and a fold increase in fertilizability of frozen-thawed spermatozoa positively correlate with the quality of spermatozoa. It may be presumed that the rPIMT improved the cryopreserved semen quality through the repair of oxidatively damaged seminal plasma proteins.

Scrub typhus, caused by Orientia tsutsugamushi, is a clinically important vector-borne disease in the Asia-Pacific region. This study aimed to detect O. tsutsugamushi DNA in human serum samples collected during a scrub typhus outbreak in Vellore and adjacent districts of Tamil Nadu, India, and to genotype the pathogen based on the tsa56 gene sequence. A total of 40 serum samples were analyzed, including nine IgM ELISA-positive samples and 31 IgM-negative samples. Nested PCR targeting the 47-kDa htrA gene detected O. tsutsugamushi DNA in all nine IgM-positive samples and in 10 of the 31 IgM-negative samples, confirming the presence of O. tsutsugamushi DNA in serum. This study demonstrates the detection of Orientia tsutsugamushi DNA in human serum using a 47-kDa htrA gene-based nested PCR assay and highlights the value of integrating this molecular approach with IgM ELISA for early and accurate diagnosis of scrub typhus. From one serum sample, the 1571-bp tsa56 gene was amplified. The tsa56 gene based phylogenetic analysis revealed three distinct clusters within this geography and the isolate from this study was closely related to Cluster A. Amino acid sequence alignment of the TSA56 variable domains indicated distinct variations in VD-I, II and III, while VD-IV remained conserved in Clusters B and C. These findings highlight the importance of integrating molecular diagnostics with serology and emphasize the need for improved molecular surveillance and diagnostics to better characterize strain diversity, improve disease management and vaccine development.

Oocytes arrested at the dictyate stage of meiosis I must maintain genomic integrity for prolonged periods to preserve female fertility. During this extended arrest, DNA lesions arising from endogenous and exogenous sources threaten oocyte survival, yet the molecular mechanisms coordinating DNA repair in dormant oocytes remain poorly understood. Here, we identify cyclin-dependent kinase 1 (CDK1) as a critical regulator of the oocyte DNA damage response and homologous recombination (HR) repair under genotoxic stress. Using cisplatin-induced DNA damage models in fetal goat ovaries and neonatal mouse ovaries, we investigated repair mechanisms operating within the ovarian reserve. Label-free proteomic profiling revealed significant enrichment of DNA damage response pathways following cisplatin exposure, with CDK1 emerging as one of the most prominently upregulated kinases. Pharmacological inhibition of CDK1 had little effect on follicle survival under physiological conditions but aggravated oocyte and follicle loss following DNA damage, indicating a stress-dependent role for CDK1 in preserving ovarian follicle pool integrity. Mechanistically, DNA damage activated a Chk2-dependent signaling pathway that promoted p63 phosphorylation and altered the WEE1 -CDK1 regulatory axis, resulting in reduced inhibitory CDK1 phosphorylation (Thr14/Tyr15) and increased activating phosphorylation (Thr161). Activated CDK1 was associated with enhanced RAD51 phosphorylation and accumulation at DNA damage foci, supporting homologous recombination (HR)-mediated repair in dictyate-arrested oocytes. In contrast, CDK1 inhibition reduced phospho-RAD51 levels, impaired RAD51 localization, increased persistent γH2AX accumulation, and elevated oocyte apoptosis. Notably, suppression of CDK1 was accompanied by increased expression of the non-homologous end joining (NHEJ) marker Ku80 and the nucleotide excision repair (NER) factor XPA, suggesting increased engagement of alternative DNA repair pathways. Furthermore, inhibition of Chk2 abolished the DNA damage-associated CDK1 activation signature and restored WEE1 expression, supporting a model in which CDK1 functions downstream of Chk2 signaling during the oocyte DNA damage response. Collectively, our findings identify a previously unrecognized Chk2-CDK1-RAD51 signaling axis that coordinates homologous recombination repair in dormant oocytes and safeguards ovarian follicular pool integrity under genotoxic stress. These findings provide new mechanistic insight into how dictyate-arrested oocytes maintain genome stability during prolonged meiotic arrest.

Newcastle disease virus (NDV) is an avian paramyxovirus that has a significant impact on the global poultry industry. The accessory W protein (W) of NDV is generated by RNA editing of the phosphoprotein (P) gene; however, its precise function remains elusive. Consequently, we conducted an in-silico analysis of the W sequences from 1,011 NDV strains, utilizing the data from GenBank. Our investigation revealed 24 W-length variants ranging between 135 and 231 amino acids (aa), with variant 227 aa being the most prevalent. Further, the W-length variants were distinct for class I and class II NDV strains. We observed no apparent correlation between W lengths and genotype or pathogenicity of NDV. However, a nuclear localization signal (NLS) in the W seemed to be associated with virulence, with 55.9

Circadian rhythms coordinate daily fluctuations in physiology and behavior, yet their organization within primary sensory pathways remains poorly defined. Although somatosensory responsiveness varies across the day-night cycle, it is unclear whether peripheral sensory circuits possess molecular mechanisms for temporal regulation. Here, we demonstrate that the spinal-peripheral sensory axis harbors robust, tissue-autonomous circadian clocks. Using real-time bioluminescence imaging, we observed sustained oscillations of the core clock protein PER2 in the spinal dorsal horn and dorsal root ganglia (DRGs), indicating autonomous circadian timing within these tissues. To define the molecular scope of this regulation, we performed RNA sequencing across a 52-hour circadian time course in DRGs. Circadian analysis identified 626 rhythmic transcripts, representing 3.6% of expressed genes. These genes exhibited non-uniform phase distributions and segregated into discrete temporal clusters. Functional annotation revealed phase-specific enrichment of biological processes related to transport, neuronal structure, and proteostasis, suggesting coordinated temporal deployment of distinct molecular programs rather than uniform oscillations across the circadian cycle. Cross-referencing circadian genes with neuropathic pain-associated gene sets revealed limited overlap; however, overlapping genes aligned to specific baseline phase windows enriched for regenerative annotations. Potassium channel-related signaling components implicated in neuropathic pain also showed baseline circadian modulation. Together, these findings establish the spinal dorsal horn and DRGs as intrinsically circadian tissues and reveal a temporally structured molecular landscape in primary sensory neurons, providing a framework for understanding how peripheral sensory processing, plasticity, and homeostatic regulation are coordinated across the day-night cycle.