Proteomic Analysis of Mouse Hypothalamus under Simulated Microgravity

Exposure to altered microgravity during space travel induces changes in the brain and these are reflected in many of the physical behavior seen in the astronauts. The vulnerability of the brain to microgravity stress has been reviewed and reported. Identifying microgravity-induced changes in the brain proteome may aid in understanding the impact of the microgravity environment on brain function. In our previous study we have reported changes in specific proteins under simulated microgravity in the hippocampus using proteomics approach. In the present study the profiling of the hypothalamus region in the brain was studied as a step towards exploring the effect of microgravity in this region of the brain. Hypothalamus is the critical region in the brain that strictly controls the pituitary gland that in turn is responsible for the secretion of important hormones. Here we report a 2-dimensional gel electrophoretic analysis of the mouse hypothalamus in response to simulated microgravity. Lowered glutathione and differences in abundance expression of seven proteins were detected in the hypothalamus of mice exposed to microgravity. These changes included decreased superoxide dismutase-2 (SOD-2) and increased malate dehydrogenase and peroxiredoxin-6, reflecting reduction of the antioxidant system in the hypothalamus. Taken together the results reported here indicate that oxidative imbalance occurred in the hypothalamus in response to simulated microgravity.     

Synthesis and antidyslipidemic activity of chalcone fibrates

A series of chalcone based PPAR-α agonists were synthesized and evaluated for their antidyslipidemic activity in high fructose high fat fed dyslipidemic Syrian golden hamsters. Most of the compounds exhibited antidyslipidemic activity. The compounds 4c and 4f have been identified as most potent antidyslipidemics. A definite structure-activity relationship was observed while varying the nature as well as the position of the substituent.     

Inhibition of Syk activity by R788 in platelets prevents remote lung tissue damage after mesenteric ischemia-reperfusion injury

Tissue injury following ischemia-reperfusion (I/R) occurs as a consequence of actions of soluble factors and immune cells. Growing evidence supports a role for platelets in the manifestation of tissue damage following I/R. Spleen tyrosine kinase has been well documented to be important in lymphocyte activation and more recently in platelet activation. We performed experiments to evaluate whether inhibition of platelet activation through inhibition of spleen tyrosine kinase prevents tissue damage after mesenteric I/R injury. Platelets isolated from C57BL/6J mice fed with R788 for 10 days were transfused into C57BL/6J mice depleted of platelets 2 days before mesenteric I/R injury. Platelet-depleted mice transfused with platelets from R788-treated mice before mesenteric I/R displayed a significant reduction in the degree of remote lung damage, but with little change in the degree of local intestinal damage compared with control I/R mice. Transfusion of R788-treated platelets also decreased platelet sequestration, C3 deposition, and immunoglobulin deposition in lung, but not in the intestine, compared with control groups. These findings demonstrate that platelet activation is a requisite for sequestration in the pulmonary vasculature to mediate remote tissue injury after mesenteric I/R. The use of small-molecule inhibitors may be valuable to prevent tissue damage in remote organs following I/R injury.     

Lactobacillus protected bone damage and maintained the antioxidant status of liver and kidney homogenates in female wistar rats

The aim of the study was to evaluate protective property of Lactobacillus casei and Lactobacillus acidophilus in minimizing oxidative stress associated with arthritis from liver and kidney. Subsequently, protective property of Lactobacillus against the bone damage was also taken into consideration. Arthritis was induced by injecting freund's complete adjuvant (100 μl) into sub plantar surface of hind paw. Oral administration of culture, vehicle, and drug started after induction of arthritis (i.e. on day 9th). Indomethacin was used as a standard drug. Radiographic analysis of the hind paw knee joint was also done at the end of the 21st day. Oxidative stress parameters were studied from liver and kidney homogenate. Histopathology of liver and kidney was also performed. Lactobacillus treatment significantly rescued the enzymatic level of catalase, superoxide dismutase, reduced glutathione, and glutathione peroxidase in both liver and kidney homogenates, whereas it has decreased the malonaldehyde accumulation. Oral administration of Lactobacillus also significantly decreased the serum ceruloplasmin level. Radiographic analysis also corroborated these findings. Lactobacillus treatment maintained the normal histopathology of liver and kidney. Results of this study clearly suggest that L. casei and L. acidophilus, alone or in combination, decreased the bone damaged and effectively restored antioxidant status of liver and kidney. Lactobacillus has a significant antiarthritic and antioxidant activity against freund's complete adjuvant induced arthritis in rats.     

Abnormal mitochondrial autophagy in nephropathic cystinosis

Cystinosis, which is characterized by lysosomal accumulation of cystine in many tissues, was the first known storage disorder caused by defective metabolite export from the lysosome. The molecular and cellular mechanisms underlying nephropathic cystinosis, the most severe form, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. We used renal proximal tubular epithelial (RPTE) cells and fibroblasts from patients with three clinical variants of cystinosis: nephropathic, intermediate and ocular to explore the specific injury mechanism in nephropathic cystinosis. We demonstrate enhanced autophagy of mitochondria, increase in apoptosis and mitochondrial dysfunction in the nephropathic cystinosis phenotype. Furthermore, specific inhibition of autophagy results in significant attenuation of cell death in nephropathic cystinosis. This study provides ultrastructural and functional evidence of abnormal mitochondrial autophagy in nephropathic cystinosis, which may contribute to renal Fanconi syndrome and progressive renal injury.Key words: cystinosis, autophagy, mitochondria, kidney, lysosome, apoptosis, cell death, mitophagyCystinosis is an autosomal recessive metabolic disorder caused by mutations in the CTNS gene, which encodes a 7-transmembrane domain protein, cystinosin, a lysosomal cystine transporter. Cystinosis belongs to the family of lysosomal storage disorders (LSDs) characterized by the tissue accumulation of cystine crystals leading to multiple organ dysfunction. The three types of cystinosis, i.e., nephropathic (classic renal and systemic disease), intermediate (a late-onset variant of nephropathic cystinosis) and non-nephropathic (clinically affecting only the cornea) are allelic disorders caused by CTNS mutations. Children affected with nephropathic cystinosis present with the Fanconi syndrome and usually develop progressive renal failure within the first decade of life. The mechanism linking lysosomal cystine storage to pathological manifestations, in particular to the prominent proximal tubular defect and renal injury, remains unclear. Renal injury in nephropathic cystinosis may not simply be caused just by cystine accumulation, as disruption of the ctns gene in mice induces cystine storage in many tissues but does not result in signs of tubulopathy or renal failure; renal injury is not seen in other human forms of cystinosis and progressive renal injury occurs despite cystine depletion therapy.The purpose of our study was to investigate the specific mechanism leading to tubulopathy and end stage renal injury in nephropathic cystinosis. We used primary fibroblast and renal proximal tubular epithelial (RPTE) cells derived from patients with three clinical phenotypes of cystinosis. Our data show an abnormal increase in macroautophagy (hereafter referred to as autophagy), specific to the nephropathic variant of cystinosis. We also demonstrate that specific inhibition of autophagy rescues cell death in nephropathic cystinotic RPTE cells. Our results indicate that mitochondrial autophagy may be a critical mechanism contributing to renal Fanconi syndrome and progressive renal injury in nephropathic cystinosis.Abnormal autophagy was also recently observed in other types of lysosomal storage diseases (LSD). However, our study provides the first evidence supporting the extensive involvement of autophagy in nephropathic cystinosis pathogenesis. Abundant vacuolization and abnormal mitochondria are detected by electron microscopy (EM) in nephropathic cystinotic cells. Additionally, elevated levels of LC3-II and Beclin 1 are also observed in nephropathic cystinotic RPTE cells, indicating a role of Beclin 1-mediated autophagy in cystinosis. These results altogether establish an abnormal increase in autophagy in nephropathic cystinotic cells.Renal biopsies from patients with nephropathic cystinosis can reveal abnormally large mitochondria, but the relevance of this finding and other ultrastructural abnormalities is unclear. Our study further demonstrates a significant decrease in mitochondrial ATP generation with an increase in reactive oxygen species (ROS) in cystinotic cells. To further dissect the association of abnormal mitochondria with increased autophagy in cystinosis, we carefully examined the electron micrographs at higher magnifications. We discovered various stages of degradation of mitochondria by autophagy (hereafter referred to as mitophagy). To further validate mitophagy in cystinosis, we used an immunofluorescence (IF) approach to capture colocalization images of LC3, LAMP-2 (lysosomal marker) and ATP5H (mitochondrial marker). Intriguingly, an increase in LAMP-2 perinuclear staining is detected by IF assay in cystinotic cells. This observation may also denote enhanced active autophagy as LAMP-2 is involved in lysosomal biogenesis and/or the fusion between autophagosomes and lysosomes. Alternatively, LAMP-2 accumulation could be a manifestation of retarded autophagic flux in cystinotic cells. A decreased ability of lysosomes to fuse with autophagosomes has been reported in various LSDs. However, the colocalization of LC3 and LAMP-2 in nephropathic cystinotic RPTE cells argues against this possibility. Nevertheless, the possibility of autophagic flux blockade after autophagosome-lysosome fusion leading to detrimental effects is yet to be investigated. Interestingly, previously published EM reports of the renal biopsies of patients with nephropathic cystinosis show only the nucleus and a thin rim of cytoplasm as remnants in a proximal tubular cell, while mitochondria and lysosomes completely disappear.Conventionally, autophagy has been suggested as a cytoprotective mechanism to ensure cell survival during starvation. In contrast, several forms of cell death have been associated with the appearance of autophagic vesicles. To gain insight into the role of autophagy as regards to cell death or cell survival in nephropathic cystinosis, we used 3-methyladenine (3MA), a specific inhibitor of autophagy and assayed cell viability and apoptosis in cystinotic cells. Increased apoptosis has been previously reported in cultured cystinotic fibroblasts and RPTE cells. Treatment with 3MA in cystinotic cells significantly rescues cell death, thus suggesting a synergistic role of apoptosis and autophagy in cystinosis.In conclusion, as illustrated in Figure 1, we speculate that there is a multifaceted impact of autophagy in nephropathic cystinosis as follows: (1) the mechanism linking autophagy to lysosomal cystine or apoptosis in cystinotic cells could potentially be related to lysosomal membrane permeabilization (LMP), proposed as an early step in apoptosis in cystinosis. We hypothesize that abnormal induction of autophagy besides providing more cargo to be digested in the lysosomes, leads to increased fusion of autophagosomes with cystine-laden lysosomes, rendering them more sensitive to membrane destabilization, and thus making them readily enter the apoptotic pathway; (2) the second most important question is the link between abnormal mitochondria and mitophagy in cystinosis. A decreased level of cytosolic glutathione in cystinotic cells is one of the known factors responsible for generating damaged mitochondria. Our data also indicate an impairment of complex I activity, an increase in ROS and a decrease in mitochondrial ATP generation in cystinotic cells. We hypothesize that the abnormal induction of autophagy leads to depletion of mitochondria, forcing cells to enter the ‘starvation mode,’ thereby leading to an uncontrolled autophagy and cell death; (3) the third key question yet to be answered is the link between autophagy and renal injury in nephropathic cystinosis. Skeletal muscles and neuronal tissues are the primary organs where autophagy is physiologically enhanced. Recently, it has been shown that mouse kidneys exert a high level of autophagy under basal conditions, influencing the susceptibility to glomerular disease and renal failure. Thus, we postulate an organ- and tissue-specific effect of abnormally induced autophagy in nephropathic cystinosis, causing severe injury to kidneys leading to loss of renal function, ultimately culminating in end-stage renal disease.Open in a separate windowFigure 1A schematic view of the interplay between autophagy, abnormal mitochondria and cell death in cystinosis. Abnormal induction of autophagy, typically mitophagy, forces cells into a starvation mode leading to cell death; and renders cystine-laden lysosomes sensitive to lysosomal membrane permeabilization (LMP) making it readily enter the apoptosis pathway. A potential block in autophagic flux, after autophagosome-lysosome fusion, remains to be elucidated. Preferential severe kidney damage in nephropathic cystinosis may be due to the tissue- and organ-specific injury effect of autophagy.The recent progress in autophagy research has increased the need for additional studies so that we can fully understand the underlying pathological mechanisms and the significance of the lysosomal cell death axis in lysosomal storage disorders.     

A heuristic method towards deadline-aware energy-efficient mapreduce scheduling problem in Hadoop YARN

Cluster Computing - The MapReduce (MR) scheduling is a prominent area of research to minimize energy consumption in the Hadoop framework in the era of green computing. Very few scheduling...