Biology subjects

Bhushan, S.

Publications and source records attributed to Bhushan, S..

7 recordsLinked to original sources

bioRxiv2025

Depletion of CX3CR1+ macrophages results in disrupted functionality and immune surveillance within epididymis and testis

A finely tuned immune regulation within the epididymis and testis is essential for male reproductive health. This balance is especially critical in the epididymis, where sperm mature and ascending infections frequently disrupt homeostasis, resulting in regionally different immune responses and potential long-term fertility impairments. We previously demonstrated that the epididymis harbors a region-specific immunological scaffold, with CX3CR1+ macrophages as the most prominent epithelium-associated immune cell population. Here, we established a transgenic mouse model to selectively deplete these intraepithelial CX3CR1+ macrophages within the epididymis, resulting in focal epithelial damage and impaired sperm maturation processes essential for proper sperm functionality. Additionally, a mild reduction of the testicular macrophage pool resulted in transient disruptions in spermatogenesis and steroidogenesis. Although the macrophage niche was repopulated after depletion, the newly recruited cells displayed altered phenotypes consistent with persistent sperm alterations. Following infection with uropathogenic Escherichia coli (UPEC), macrophage-depleted mice exhibited exacerbated immune responses - particularly in normally protected proximal epididymal regions - with earlier onset and more severe tissue damage. Transcriptomic analysis revealed a failure to restrain inflammatory responses, especially in genes involved in immune regulation and antibacterial defense, accompanied by elevated immune cell infiltration in infected macrophage-depleted mice. Overall, our findings confirm a crucial role for CX3CR1 macrophages in preserving epithelial integrity and modulating immune responses, supporting a stable tissue environment necessary for efficient organ function of both epididymis and testis. Significance statementMaintaining immune balance in the epididymis is essential for tissue health and protection against ascending infections. Using a transgenic mouse model that allows for selective depletion of CX3CR1 macrophages, this study examines their role in both the epididymis and testis under normal and infectious conditions. The results show that the removal of these macrophages causes localized epithelial damage, changes in immune cell make-up, and increased inflammation in the epididymis after bacterial infection, while also causing mild, reversible problems with spermatogenesis and steroid production in the testis. These findings support the idea that CX3CR1 macrophages contribute to region-specific immune regulation and epithelial stability--key features for keeping the tissue environment suitable for proper sperm development.

immunology↗

bioRxiv2023

Iron regulatory proteins 1 and 2 have opposing roles in regulating inflammation in bacterial orchitis

Acute bacterial orchitis (AO) is a prevalent cause of intra-scrotal inflammation, often resulting in sub-or infertility. A frequent cause eliciting AO is uropathogenic Escherichia coli (UPEC), a gram negative pathovar, characterized by the expression of various iron acquisition systems to survive in a low-iron environment. On the host side, iron is tightly regulated by iron regulatory proteins (IRP) 1 and 2 and these factors have been reported to play a role in testicular and immune cell function, however, their precise role remains unclear. Here, we show in a mouse model of UPEC-induced orchitis that the absence of IRP1 results in reduced immune response and testicular damage. Compared to infected wild-type (WT)-mice, testis of UPEC-infected Irp1-/- mice showed impaired ERK signalling. Conversely, IRP2 deletion led to a stronger inflammatory response. Notably, differences in immune cell infiltrations were observed among the different genotypes. In contrast to WT and Irp2-/- mice, no increase in monocytes and neutrophils was detected in testis of Irp1-/- mice upon UPEC-infection. Interestingly, in Irp1-/- UPEC-infected testis, we observed an increase in a subpopulation of macrophages (F4/80+ CD206+) associated with anti-inflammatory and wound-healing activities compared to WT. These findings suggest that IRP1 deletion may protect against UPEC-induced inflammation by modulating ERK signalling and dampening the immune response.

immunology↗

bioRxiv2022

Streptomycin and Pyridomycin drug molecules provide the most potential inhibitors against Rv3871 to cure Tuberculosis

The ESX-1 secretion system of Mycobacterium tuberculosis delivers bacterial virulence factors to host cells during infection. The most abundant factor, the ESAT-6/CFP-10 dimer, is targeted for secretion via a C-terminal signal sequence on CFP-10 that is recognized by the cytosolic ATPase, Rv3871. ATPase that is a component of the ESAT-6/CFP-10 secretion system. ESX locus contains genes encoding conserved secretion machinery components termed EccCb1. these core components are required for ESAT-6/CFP-10 secretion [70, 71]. Rv3871 is a cytoplasmic protein connected with the Rv3870 and encoded by the EccCb1 gene. These proteins supply energy for the secretion process. Each of these ATPases is involved in targeting protein for ESX-1 secretion. EccCb1 binds a seven amino acid C-terminal signal peptide of CFT-10, which is required for secretion of the ESAT-6/CFP-10 complex. In this we target the Rv3871 seven amino acid c-terminal region and block it through multiple drugs so, it cannot be activated by the ATPases and not to supply energy for the secretion protein during infection (Tuberculosis). In the present study we have comparatively studied different Rv3871 inhibitory antagonist drug molecule which could be a potential drug molecule to inhibit ATPases on the Rv3871 molecule that is responsible for the energy supply. We have 16 antagonistic drug molecules as shortlisted based on previous studies, that block the target site on the Rv3871 molecule.

microbiology↗

bioRxiv2022

Raltitrexed and Edatrexate drug molecules against human Proton Coupled Folate Transporter to cure cancer

Proton pump inhibitors (PPIs) (e.g.: - rabeprazole, pantoprazole, omeprazole, lansoprazole, and esomeprazole) are widely used to treat gastroesophageal reflux disease and other acid-related disorders. Folic acid transport is important for proper cell proliferation. In human, folic acid is not synthesized in the body, it is obtained externally. Therefore, specific transporters are involved in absorption of folic acid, which is concentrated in intestine. Malignant cancer cells require this folic acid very frequently in large quantities for the rapid reproduction of cancer cell. In the current study, we compared different types of proton pump inhibitor drug molecules that may be potential candidates for preventing the absorption of folate by (hPCFT), its responsible for unusually large and frequent folate production. Each of these anti-drug candidates has 27 finalists based on previous studies. Among these competitors, leukovorin and noletraxid molecules were found to be particularly associated with the hPCFT transporters key active site loop(G155XXG158).

bioinformatics↗

bioRxiv2022

The regional distribution of resident immune cells shapes distinct immunological environments along the murine epididymis

Summary/ AbstractThe epididymis constitutes an important transition zone for post-testicular sperm maturation and storage. As the organ consists of a single convoluted duct, inflammation-associated tissue damage has a severe impact on fertility. In order to clarify the reasons for region-specific differences in the intensity of immune responses observed in a mouse model of acute bacterial epididymitis, we investigated the heterogeneity of resident immune cell populations within the epididymis under physiological conditions by scRNASeq analysis of extravascular CD45+ cells. 12 distinct immune cell subsets were identified, displaying substantial differences in distribution along the epididymis. Several distinct subsets of macrophages constituted the majority of these cells. Crucially, the proximal and distal regions showed striking differences in their immunological landscapes. These findings indicate that resident immune cells are strategically positioned along the epididymal duct, potentially providing different immunological environments required for sperm maturations and elimination of pathogens ascending the urogenital tract.

immunology↗

bioRxiv2022

Persistence of parental age effect on somatic mutation rates across generations in Arabidopsis.

In the model plant Arabidopsis thaliana, parental age is known to affect somatic mutation rates in their immediate progeny and here we show that this age dependent effect persists across successive generations. Using a set of detector lines carrying the mutated uidA gene, we examined if a particular parental age maintained across five consecutive generations affected the rates of base substitution (BSR), intrachromosomal recombination (ICR), frameshift mutation (FS), and transposition. The frequency of functional GUS reversions were assessed in seedlings as a function of identical/different parental ages across generations. In the context of a fixed parental age, BCR/ICR rates were unaffected in the first three generations, then dropped significantly in the 4th and increased in most instances in the 5th generation. On the other hand, with advancing parental ages, BSR/ICR rates remained high in the first two/three generations, with a striking resemblance in the pattern of mutation rates. We adopted a novel approach of identifying and tagging flowers pollinated on a particular day, thereby avoiding biases due to potential emasculation induced stress responses. Our results suggest a time component in counting the number of generations a plant has passed through self-fertilization at a particular age in determining the somatic mutation rates.

developmental biology↗

bioRxiv2019

Pyrazinamide triggers degradation of its target aspartate decarboxylase

The introduction of pyrazinamide (PZA) in the tuberculosis drug regimen shortened treatment from 12 to 6 months 1. PZA is a prodrug that is activated by a Mycobacterium tuberculosis (Mtb) amidase to release its bioactive component pyrazinoic acid (POA) 2. Aspartate decarboxylase PanD, a proenzyme activated by autocatalytic cleavage (Supplementary Fig. 1A, 3) and required for Coenzyme A (CoA) biosynthesis, emerged as a target of POA 4-7. In vitro and in vivo screening to isolate spontaneous POA-resistant Mtb mutants identified missense mutations in either panD or the unfoldase clpC1, encoding a component of the caseinolytic protease ClpC1-ClpP 4,6-9. Overexpression and binding studies of PanD or ClpC1 pointed to PanD as the direct target of POA whereas clpC1 mutations appeared to indirectly cause resistance 4,5,7,9,10. Indeed, supplementing growth media with CoA precursors downstream of the PanD catalyzed step conferred POA resistance 4,7,11. Metabolomic analyses and biophysical studies using recombinant proteins confirmed targeting of PanD by POA 5. However, the exact molecular mechanism of PanD inhibition by POA remained unknown. While most drugs act by inhibiting protein function upon target binding, we show here that POA is not a bona fide enzyme inhibitor. Rather, POA binding to PanD triggers degradation of the protein by ClpC1-ClpP. Thus, the old tuberculosis drug PZA promotes degradation of its target. While novel for an antibacterial, drug-induced target degradation has recently emerged as a strategy in drug discovery across disease indications. Our findings provide the basis for the rational discovery of next generation PZA.

microbiology↗