BATF sustains homeostasis and functionality of bone marrow Treg cells to preserve homeostatic regulation of hematopoiesis and development of B cells.

Bone marrow Treg cells (BM Tregs) orchestrate stem cell niches crucial for hematopoiesis. Yet little is known about the molecular mechanisms governing BM Treg homeostasis and function. Here we report that the transcription factor BATF maintains homeostasis and functionality of BM Tregs to facilitate homeostatic regulation of hematopoiesis and B cell development. Treg-specific ablation of BATF profoundly compromised proportions of BM Tregs associated with reduced expression of Treg effector molecules, including CD44, ICOS, KLRG1, and TIGIT. Moreover, BATF deficiency in Tregs led to increased numbers of hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), and granulocyte-macrophage progenitors (GMPs), while reducing the functionality of myeloid progenitors and the generation of common lymphoid progenitors. Furthermore, Tregs lacking BATF failed to support the development of B cells in the BM. Mechanistically, BATF mediated IL-7 signaling to promote expression of effector molecules on BM Tregs and their homeostasis. Our studies reveal a previously unappreciated role for BATF in sustaining BM Treg homeostasis and function to ensure hematopoiesis.

Arsenic-induced autophagic alterations and mitochondrial impairments in HPG-S axis of mature male mice offspring (F1-generation): A persistent toxicity study.

Arsenic (As) has been implicated in causing reproductive toxicity, but the precise cellular pathway through which the As toxicity in mature F1- male mice hypothalamic-pituitary- gonadal- sperm (HPG-S) axis is induced has not well been documented. Hence, parental mice were treated to As2O3 (0, 0.2, 2, and 20 ppm in deionized water) from five weeks before mating until weaning, and the male pups from weaning to maturity. Afterward, the markers of oxidative stress, mitochondrial impairment, and autophagy as fundamental mechanisms of cytotoxicity and organ injury were evaluated. Higher As2O3 doses (2 and 20 ppm) were a potent inducer of oxidative stress, mitochondrial dysfunction, and autophagy in HPG-S axis. Concomitant with a dose-dependent increase in the number of MDC-labeled autophagic vacuoles in the HPG axis, an adverse dose-dependent effect was observed on the mean body weight, litter size, organ coefficient, and spermatogenesis. Transmission electron microscopy also revealed more autophagosomes at high As2O3 dosage. Concomitant with a dose-dependent increment in gene expression of PI3K, Atg5, Atg12, as well as protein expression of Beclin1, LC3- I, II, P62 in HPG axis tissues and Atg12 in the pituitary; a dose-dependent decrease in mTOR gene expression was recorded in the HPG tissues of mature F1-males. These observations provide direct evidence that oxidative stress-induced mitochondrial impairments and autophagic cell death, through AMPK/TSC/mTOR and LC3 related pathways, are fundamental mechanisms for As2O3- induced toxicity on the reproductive system in mature male mice offspring.

Immune disruption occurs through altered gut microbiome and NOD2 in arsenic induced mice: Correlation with colon cancer markers.

The gut microbial compositions are easily affected by the environmental chemicals like arsenic (As) leading to dysbiosis. The dysbiosis of gut microbiome has associated with numerous diseases; among which cancer is one of the major diseases. The meticulous mechanism underlying As- altered gut microbiome, Nucleotide domine containing protein 2 (NOD2) and how altered gut microbiome disturbs the intestinal homeostasis to regulate colon cancer markers remains unclear. For this, one hundred twenty 8-week old age male mice were divided into two exposure periods (3 and 6 months), and each exposure group animals were further divided into four groups as control (received only distilled H2O), low (0.15 mg As2O3/L), medium (1.5 mg As2O3/L) and high (15 mg As2O3/L) dose (each group containing 15 mice) administrated for 3 and 6 months. The results showed that As exposure highly altered gut microbiome with a significant depletion in NOD2 in contrast to control groups. Moreover, the dendritic cells (CD11a, CD103, CX3CR1) and macrophages (F4/80) were significantly increased by As exposure. Interestingly, increased trend of inflammatory cytokines (TNF-α, IFN-γ, IL-17) and depleted anti-inflammatory cytokines (IL-10) was observed in As exposed mice. Furthermore, the colon cancer markers ß-catenin has increased while APC was arrested by As both in 3 and 6 months treated animals. Many studies reported that As altered gut microbial compositions, in this study, our results suggested that altered gut microbiome indirectly regulates colon cancer marker through immune system destruction mediated by inflammatory cytokines.

Arsenic influences spermatogenesis by disorganizing the elongation of spermatids in adult male mice.

Arsenic (As) has become a major problem in maintaining the environment and human health due to its wide application in the production of agriculture and industry. Many studies indicate that As can affect spermatogenesis process and lower sperm quality. However, the undergoing molecular mechanism is unclear. For this, forty-eight 8-week old adult male mice were divided into four groups of twelve each, which were administrated to 0, 0.2, 2, 20 ppm As2O3 in their drinking water respectively for six months. The results showed that As treatment reduced sperm counts and increased the sperm malformation ratio of mice. Interestingly, both the amounts of round and elongated spermatids, and the ratios of spermatids elongation were decreased significantly by As exposure. Furthermore, the structure of Chromatoid Body (CB) which presents a typical nebulous shape in round spermatids after spermatogenesis arrested, and the mRNA expression levels of gene TDRD1, TDRD6 and TDRD7 related to CB were changed by arsenic. Again, the mRNA and protein expression levels of the markers DDX25 and CRM1 in haploid periods of spermatogenesis and the associated proteins HMG2, PGK2, and H4 with DDX25 regulation were declined significantly with As treatment. Taken together; it reveals that As interferes with spermatogenesis by disorganizing the elongation of spermatids. H4, HMG2 and PGK2 are regulated by DDX25 which interacts with CRM1 and may play a vital role in spermatogenesis disorder induced by As exposure, which maybe provides one of the underlying mechanisms for As-induced male reproductive toxicity.

Fluoride induced mitochondrial impairment and PINK1-mediated mitophagy in Leydig cells of mice: In vivo and in vitro studies.

It is very important to explore the potential harm and underlying mechanism of fluoride due to the extensive distribution and the significant health risks of fluoride in environment. The objective of this study to investigate whether fluoride can induce mitochondrial impairment and mitophagy in testicular cells. For this, 40 male mice were randomly divided into four groups treated with 0, 0.6, 1.2, 2.4 mM NaF deionized water, respectively, for 90 days continuously. The results showed that mitophagy was triggered by F in testicular tissues, especially in the Leydig cells by transmission electron microscopy and mitophagy receptor PHB2 locations by immunofluorescence. Furthermore, TM3 Leydig cells line was employed and treated with 0, 0.125, 0.25, and 0.5 mM NaF for 24 h. The mitochondrial function indicators and mitophagy maker PHB2, COX IV and regulator PINK1 in transcript and protein levels in Leydig cells were examined by the methods of qRT-PCR, western blotting, and immunofluorescence co-localization. The results showed that fluoride decreased the mitochondrial membrane potential with a concomitant increase in the number of lysosomes. Meanwhile, fluoride exposure also increased the expressions of PINK1 and PHB2 in TM3 Leydig cells. These results revealed that fluoride could induce mitochondrial impairment and excessive PINK1/Parkin-mediated mitophagy in testicular cells, especially in Leydig cells, which could contribute to the elucidation of the mechanisms of F-induced male reproductive toxicity.

Paternal exposure to arsenic resulted in oxidative stress, autophagy, and mitochondrial impairments in the HPG axis of pubertal male offspring.

Despite the knowledge of AS-induced reprotoxicity, the literature concerning arsenic trioxide (As2O3)-induced oxidative stress and consequent intracellular events, like autophagy process, in the hypothalamic-pituitary- gonadal (HPG) axis of F1- pubertal male mice is sparse to date. Hence, we made an attempt to study the reproductive toxicities and the underlying mechanisms induced by As2O3 in the HPG axis of pubertal F1- male mice in correlation with oxidative stress-induced autophagy. Parental mice were challenged with As2O3 (0, 0.2, 2, and 20 ppm) from five weeks before mating, and continued till puberty age for the male pups. It was recorded that higher As2O3 doses (2 and 20 ppm) were a potent inducer of oxidative stress and autophagy in the HPG axis. Concomitant with a decrease on mean body weight, total antioxidant capacity, and stereology indices, an increase in the number of MDC-labeled autophagic vacuoles, and MDA/GSH ratio in HPG axis of pubertal F1- male mice which were exposed to higher As2O3 doses was observed. Meanwhile, concomitant with a dose-dependent increment in the gene expression of ATG3, ATG5, Beclin, as well as protein expression of P62, ATG12, and Beclin in HPG axis tissues; a dose-dependent decrease in PI3K and mTOR gene expression was recorded in the HPG tissues of pubertal F1-males. Altogether, our observations suggest that higher doses of As2O3 have detrimental effects on the functionality of HPG axis in pubertal male mice offspring by increasing MDA/GSH ratio and autophagic cell death-related genes and proteins, as well as by reducing total antioxidant capacity.

Combination of Fluoride and SO2 Induce DNA Damage and Morphological Alterations in Male Rat Kidney.

BACKGROUND/AIMS: We investigated the combined toxic effect of sodium fluoride (NaF) and sulfur dioxide (SO2) on kidney morphological changes and DNA damage in male Wistar rats. METHODS: In this study we selected totally 96 male Wistar rats (12-week-old) then randomly group-housed them into four cages, treated with deionized water, NaF, SO2 and co-treatment of NaF and SO2 respectively. Morphological changes of kidney were detected by hematoxylin and eosin (H&E) staining at 2, 4, 6 and 8 weeks. Correspondingly, tailing ratio and comet length were measured by BAB Bs Comet Assay System, including DNA damage special unit were calculated to evaluate the grades of kidney DNA damage at the same time. RESULTS: Treated groups showed a body weight decrease when compared to control group. However, no significant difference in the relative weight of kidney was found in all four groups. It is noteworthy that at 2, 4, 6 and 8 weeks after exposure, the morphological alteration of renal tubules were observed in all treated groups, especially in group-IV. Also, at 4 and 6 weeks, notable DNA damage was found in all treated groups, as assessed by significantly increasing trend of comet length tailing ratio. CONCLUSION: The study manifests that presence of NaF and SO2 will not only induce renal tissue lesions but also impact DNA integrity. In addition, this combined exposure exhibits a synergistic effect, characterizing a dose-dependence and time correlation. These findings may provide novel insights regarding perturbations of DNA damage and its functions as a potential new mechanism, by which cautious interpretation of NaF and SO2 co-exposure evolved in both animals and human beings is necessary.

Arsenic induces autophagy in developmental mouse cerebral cortex and hippocampus by inhibiting PI3K/Akt/mTOR signaling pathway: involvement of blood-brain barrier's tight junction proteins.

For the past decade, there has been an increased concern about the health risks from arsenic (As) exposure, because of its neurotoxic effects on the developing brain. The exact mechanism underlying As-induced neurotoxicity during sensitive periods of brain development remains unclear, especially the role of blood-brain barrier's (BBB) tight junction (TJ) proteins during As-induced neurotoxicity. Here, we highlight the involvement of TJ proteins in As-induced autophagy in cerebral cortex and hippocampus during developmental periods [postnatal day (PND) 21, 28, 35 and 42]. Here, the administration of arsenic trioxide (As2O3) at doses of 0.15 mg or 1.5 mg or 15 mg As2O3/L in drinking water from gestational to lactational and continued to the pups till PND42 resulted in a significant decrease in the mRNA expression levels of TJ proteins (Occludin, Claudin, ZO-1 and ZO-2) and Occludin protein expression level. In addition, As exposure significantly decreased PI3K, Akt, mTOR, and p62 with a concomitant increase in Beclin1, LC3I, LC3II, Atg5 and Atg12. Moreover, As exposure also significantly downregulated the protein expression levels of mTOR with a concomitant upregulation of Beclin 1, LC3 and Atg12 in all the developmental age points. However, no significant alterations were observed in low and medium dose-exposed groups of PND42. Histopathological analysis in As-exposed mice revealed decreased number of pyramidal neurons in hippocampus; and neurons with degenerating axons, shrinkage of cells, remarkable vacuolar degeneration in cytoplasm, karyolysis and pyknosis in cerebral cortex. Ultrastructural analysis by transmission electron microscopy revealed the occurrence of autophagosomes and vacuolated axons in the cerebral cortex and hippocampus of the mice exposed to high dose As at PND21 and 42. The severities of changes were found to more persist in the cerebral cortex than in the hippocampus of As-exposed mice. Finally, we conclude that the leaky BBB in cerebral cortex and hippocampus may facilitate the transfer of As and induces autophagy by inhibiting PI3K/Akt/mTOR signaling pathway in an age-dependent manner, i.e., among the four different developmental age points, PND21 animals were found to be more vulnerable to the As-induced neurotoxicity than the other three age points.

Arsenic-Induced Autophagy in the Developing Mouse Cerebellum: Involvement of the Blood-Brain Barrier's Tight-Junction Proteins and the PI3K-Akt-mTOR Signaling Pathway.

This study was designed to determine whether the tight-junction (TJ) proteins of the blood-brain barrier (BBB) and the PI3K-Akt-mTOR signaling pathway are involved during arsenic (As)-induced autophagy in developing mouse cerebella after exposure to different As concentrations (0, 0.15, 1.5, and 15 mg/L As(III)) during gestational and lactational periods. The dosage was continually given to the pups until postnatal day (PND) 42. Studies conducted at different developmental age points, like PND21, 28, 35, and 42, showed that exposure to As led to a significant decrease in the mRNA-expression levels of TJ proteins (occludin, claudin, ZO-1, and ZO-2), PI3K, Akt, mTOR, and p62, with concomitant increases in Beclin1, LC3I, LC3II, Atg5, and Atg12. Also, As significantly downregulated occludin and mTOR protein-expression levels with concomitant upregulation of Beclin1, LC3, and Atg12 at all the developmental age points. However, no significant alterations were observed in low- and medium-dose-exposed groups at PND42. Histopathological analysis revealed the irregular arrangement of the Purkinje cell layer in the As-exposed mice. Ultrastructural analysis by transmission electron microscopy (TEM) revealed the occurrence of autophagosomes and vacuolated axons in the cerebella of the mice exposed to high doses of As at PND21 and 42, respectively. Finally, we conclude that developmental As exposure significantly alters TJ proteins, resulting an increase in BBB permeability, facilitating the ability of As to cross the BBB and induce autophagy, which might be partly the result of inhibition of the PI3K-Akt-mTOR signaling pathway, in an age-dependent manner (i.e., PND21 mice were found to be more vulnerable to As-induced neurotoxicity), which could be due to the immature BBB allowing As to cross through it. However, the effect was not significant in PND42, which could be due to the developed BBB.

Isolation and characterization of ethanol tolerant yeast strains.

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains.