Modulation of lung cytoskeletal remodeling, RXR based metabolic cascades and inflammation to achieve redox homeostasis during extended exposures to lowered pO2.

Extended exposure to low pO2 has multiple effects on signaling cascades. Despite multiple exploratory studies, omics studies elucidating the signaling cascades essential for surviving extended low pO2 exposures are lacking. In this study, we simulated low pO2 (PB = 40 kPa; 7620 m) exposure in male Sprague-Dawley rats for 3, 7 and 14 days. Redox stress assays and proteomics based network biology were performed using lungs and plasma. We observed that redox homeostasis was achieved after day 3 of exposure. We investigated the causative events for this. Proteo-bioinformatics analysis revealed STAT3 to be upstream of lung cytoskeletal processes and systemic lipid metabolism (RXR) derived inflammatory processes, which were the key events. Thus, during prolonged low pO2 exposure, particularly those involving slowly decreasing pressures, redox homeostasis is achieved but energy metabolism is perturbed and this leads to an immune/inflammatory signaling impetus after third day of exposure. We found that an interplay of lung cytoskeletal elements, systemic energy metabolism and inflammatory proteins aid in achieving redox homeostasis and surviving extended low pO2 exposures. Qualitative perturbations to cytoskeletal stability and innate immunity/inflammation were also observed during extended low pO2 exposure in humans exposed to 14,000 ft for 7, 14 and 21 days.

Plasma protein(s)-based conceptual diagnostic tool for assessing high-altitude acclimation in humans.

Exposure to high altitude above 3000 m leads to two outcomes-acclimation or high-altitude maladies. To reach a particular outcome, the plasma proteome is modified differentially, either in context of an acclimation response or mal-acclimation response leading to disease. This ensures that hypoxia-responsive plasma protein trends reflect acclimation in acclimated individuals when compared with their levels prior to acclimation. Such protein trends could be used to assess acclimation in an individual and any significant deviation from this trend may indicate non-acclimation, thereby preventing high-altitude illnesses before they manifest. In this study, we investigate and statistically evaluate the trendlines of various hypoxia-responsive plasma protein levels, reported significantly perturbed in our previous studies, in individuals (male; n = 20) exposed to 3520 m at high-altitude day 1 (HAD1), HAD4, and HAD7L and to 4420 m at HAD7H, HAD30, and HAD120. We observe that thioredoxin (Trx), glutathione peroxidase 3 (GPx-3), and apolipoprotein AI (Apo-AI) are statistically robust markers to assess acclimation across the exposure duration while sulfotransferase 1A1 (ST1A1) is a capable negative control whose levels increase only in cases of HAPE. We also observe exposure day-specific and resident altitude-specific proteins capable of accurately assessing acclimation when compared with baseline levels or the lower altitude zone.

Saliva panel of protein candidates: A comprehensive study for assessing high altitude acclimatization.

Altitude acclimatization describes the processes whereby lowland humans respond to decreased partial pressure of oxygen. It refers to the changes seen as beneficial and involves a series of physiological adjustments that compensate for reduced ambient PO2, as opposed to changes that are pathological. Although numerous reports document the physiological effects of exposure to hypobaric hypoxia of varying durations but an interesting aspect overlooked by many researchers is that of acclimatization related studies. As proteome, a dynamic entity responds immediately to external stimuli, protein markers and their trends can be studied to assess acclimatization status of an individual. Compared to blood, the use of saliva is advantageous because sample collection and processing are easy, minimally invasive, low cost and better tolerated by individuals. In this study, we employed iTRAQ based LC-MS/MS technique for comparing saliva samples from humans exposed to hypobaric hypoxia from 7 to 120 days with normoxic controls followed by analysis using Ingenuity Pathway Analysis software and validation by immunoassays. Nearly 67 proteins were found to be differentially expressed in the exposed groups as compared to normoxia indicating modulated canonical pathways as lipid metabolism; acute phase response signalling and proteins as carbonic anhydrase 6, alpha-enolase, albumin, and prolactin inducible protein. Collectively, this study provides the proof of concept for the non-invasive assessment of high altitude acclimatization.

Intermittent normobaric hypoxia facilitates high altitude acclimatization by curtailing hypoxia-induced inflammation and dyslipidemia.

Intermittent hypoxic training (IHT) is a discrete cost-effective method for improving athletic performance and high altitude acclimatization. Unfortunately, IHT protocols widely vary in terms of hypoxia severity, duration, and number of cycles affecting physiological outcomes. In the present study, we evaluated the efficacy of a moderate normobaric IHT protocol (12% FiO2 for 4 h, 4 days) on acclimatization to high altitude (3250 m). Global plasma proteomics studies revealed that IHT elicited acute-phase response proteins like C-reactive protein (CRP), serum amyloid A-1 protein (SAA), and alpha-1-acid glycoprotein 2 (AGP 2) as well as altered levels of several apolipoproteins. On subsequent exposure to high altitude, the IH trained volunteers exhibited significant higher arterial oxygen saturation with concomitant lower incidences of acute mountain sickness (AMS) as compared to controls. Interestingly, IH trained subjects exhibited lower levels of positive acute-phase proteins like C-reactive protein (CRP), serum amyloid A-1 protein (SAA), and fibrinogen (FGA, FGB, and FGG) both after days 4 and 7 of high altitude ascent. High altitude exposure also decreased the levels of HDL, LDL, and associated proteins as well as key enzymes for assembly and maturation of lipoprotein particles like lecithin-cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), and phospholipid transfer protein (PLTP). In contrast, IHT curtailed hypoxia-induced alterations of HDL, LDL, Apo-AI, Apo-B, LCAT, CETP, and PLTP. Further validation of results also corroborated attenuation of hypoxia-induced inflammation and dyslipidemia by IHT. These results provide molecular evidences supporting the use of moderate IHT as a potential non-pharmacological strategy for high altitude acclimatization.

Post-translational modifications of eNOS augment nitric oxide availability and facilitates hypoxia adaptation in Ladakhi women.

The lower inhaled oxygen per volume at high altitude poses an intimidating challenge for humans to survive and reproduce. Indigenous populations of the Himalayas reportedly exhibit higher microcirculatory blood flow accompanied by higher orders of magnitude of nitric oxide (NO) products in lung, plasma and red blood cells as a vascular adaptation strategy for hypobaric hypoxia. The precise mechanism of such observed higher NO metabolites for hypoxia adaptation remains elusive. Studying high altitude native Ladakhi women, we observed significant higher eNOS mRNA and protein in blood/plasma as compared to lowland women. We also observed higher level of plasma l-citrulline and NOx (nitrates and nitrites) with concomitant lower levels of arginase mRNA and protein further suggesting higher eNOS activity and NO bioavailability. Interestingly, middle aged postmenopausal Ladakhi women exhibited significantly higher level of eNOS activity, NOx and cGMP as compared to age matched lowland women. Preferential phosphorylation of eNOS on stimulatory Ser1177 and Ser615 as well as dephosphorylation of inhibitory Thr495 site contributed to higher NO availability in Ladakhi women irrespective of age. We also observed higher levels of eNOS activating humoral factors like bradykinin and estrogen in both young and middle-aged Ladakhi women. These results suggest that an altered phosphorylation status, together with an enhanced expression of eNOS and potential humoral endothelial activators, are involved in enhanced activation of the eNOS-NO-cGMP pathway in Ladakhi women irrespective of age, reinforcing the hypothesis that NO metabolites play a major role in Himalayan pattern of hypoxia adaptation.

Revisiting cobalt chloride preconditioning to prevent hypobaric hypoxia-induced damage: identification of global proteomic alteration and key networks.

Several studies have supported the hypoxia mimetic roles and cytoprotective properties of cobalt chloride in vitro and in vivo. However, a clear understanding of biological process-based mechanism that integrates the available information remains unknown. This study was aimed to explore the potential mechanism of cobalt chloride deciphering its benefits and well-known physiological challenge caused by hypobaric hypoxia that reportedly affects nearly 24 % of the global population. In order to explore the mechanism of CoCl2, we used global proteomic and systems biology approach in rat model to provide a deeper insight into molecular mechanisms of preconditioning. Furthermore, key conclusions were drawn based on biological network analysis and their enrichment with ontological overlaps. The study was further strengthened by consistent identification of validation of proteins using immunoblotting. CoCl2-pretreated animals exposed to hypoxia showed two significant networks, one lipid metabolism and other cell cycle associated, with a total score of 23 and eight focus molecules. In this study, we delineated two primary routes: one, by direct modulation of reactive oxygen species metabolism and, second, by regulation of lipid metabolism which was not known until now. The previously known benefits of cobalt chloride during physiological challenge by hypobaric hypoxia are convincing and could be explained by some basic set of metabolic and molecular reorganization within the hypoxia model. Interestingly, we also observed some of the completely unknown roles of cobalt chloride such as regulation of lipid that could undulate the translational roles of cobalt chloride supplementation beyond hypoxia preconditioning.

Plasma kallikrein-bradykinin pathway promotes circulatory nitric oxide metabolite availability during hypoxia.

Nitric oxide (NO) is an indispensible signalling molecule under hypoxic environment for both ethnic high altitude natives as well as lowland residents at high altitude. Several studies have reported higher levels of NO and bioactive NO products for both high altitude natives as well as healthy high altitude sojourners. But the metabolic pathways regulating the formation of NO and associated metabolites during hypoxia still remain elusive. In the present study, we profiled plasma proteomes of Ladakhi natives (3520 m) and lowland residents (post 1, 4 and 7 days stay) at the same altitude. This has resulted in the identification of 208 hypoxia responsive proteins (p < 0.05) and kininogen-plasma kallikrein-bradykinin as a major pathway regulating eNOS activity during hypoxia. In corroboration, we have also observed significant higher levels of plasma biomarkers for NO production (l-citrulline, nitrite, nitrate) for Ladakhi natives as compared to both lowland individuals healthy high altitude sojourners indicating higher NO availability. Since hypoxia-induced free radicals reduce NO availability, we also measured plasma levels of 8-isoprostanes, protein carbonyls and protein oxidation products in both Ladakhi natives and high altitude sojourners. Interestingly Ladakhi natives had significant lower levels of oxidative stress in comparison to high altitude sojourners but higher than lowland controls. These results suggest that plasma kallikrein-bradykinin-eNOS pathway along with moderate oxidative stress contributes to high altitude adaptation of Ladakhi natives.

Hypobaric hypoxia induced arginase expression limits nitric oxide availability and signaling in rodent heart.

BACKGROUND: This study was aimed to evaluate regulation of cardiac arginase expression during hypobaric hypoxia and subsequent effect on nitric oxide availability and signaling. METHODS: Rats were exposed to hypobaric hypoxia (282mmHg for 3h) and ARG1 expression was monitored. The expression levels of eNOS and eNOS(Ser1177) were determined by Western blotting, cGMP levels were measured by ELISA and amino acid concentrations were measured by HPLC analysis. Transcription regulation of arginase was monitored by chromatin immunoprecipitation (ChIP) assay with anti-c-Jun antibody for AP-1 consensus binding site on ARG1 promoter. Arginase activity was inhibited by intra-venous dose of N-(ω)-hydroxy-nor-l-arginine (nor-NOHA) prior to hypoxia exposure and subsequent effect on NO availability and oxidative stress were evaluated. RESULTS: Hypobaric hypoxia induced cardiac arginase expression by recruiting c-Jun to AP-1 binding site on ARG1 promoter. This increased expression redirected l-arginine towards arginase and resulted in limited endothelial nitric oxide synthase (eNOS) activity, nitric oxide (NO) availability and cGMP mediated signaling. Inhibition of arginase restored the eNOS activity, promoted cardiac NO availability and ameliorated peroxynitrite formation during hypoxia. CONCLUSIONS: Hypoxic induced arginase under transcription control of AP-1 reciprocally regulates eNOS activity and NO availability in the heart. This also results in cardiac oxidative stress. GENERAL SIGNIFICANCE: This study provides understanding of hypoxia-mediated transcriptional regulation of arginase expression in the heart and its subsequent effect on eNOS activity, NO availability and signaling as well as cardiac oxidative stress. This information will support the use of arginase inhibitors as therapeutics for pathological hypoxia.

Electrochemical assay for the determination of nitric oxide metabolites using copper(II) chlorophyllin modified screen printed electrodes.

This work presents a novel electrochemical assay for the collective measurement of nitric oxide (NO) and its metabolites nitrite (NO2(-)) and nitrate (NO3(-)) in volume miniaturized sample at low cost using copper(II) chlorophyllin (CuCP) modified sensor electrode. Zinc oxide (ZnO) incorporated screen printed carbon electrode (SPCE) was used as a host matrix for the immobilization of CuCP. The morphological changes of the ZnO and CuCP modified electrodes were investigated using scanning electron microscopy. The electrochemical characterization of CuCP-ZnO-SPCE exhibited the characteristic quasi-reversible redox peaks at the potential +0.06 V versus Ag/AgCl. This biosensor electrode showed a wide linear range of response over NO concentrations from 200 nM to 500 µM with a detection limit of 100 nM and sensitivity of 85.4 nA µM(-1). Furthermore, NO2(-) measurement showed linearity of 100 nM to 1mM with a detection limit of 100 nM for NO2(-) and sensitivity of 96.4 nA µM(-1). Then, the concentration of NO3(-) was measured after its enzymatic conversion into NO2(-). Using this assay, the concentrations of NO, NO2(-), and NO3(-) present in human plasma samples before and after beetroot supplement were estimated using suitable membrane coated CuCP-ZnO-SPCE and validated with the standard Griess method.

Dietary nitrite attenuates oxidative stress and activates antioxidant genes in rat heart during hypobaric hypoxia.

The nitrite anion represents the circulatory and tissue storage form of nitric oxide (NO) and a signaling molecule, capable of conferring cardioprotection and many other health benefits. However, molecular mechanisms for observed cardioprotective properties of nitrite remain largely unknown. We have evaluated the NO-like bioactivity and cardioprotective efficacies of sodium nitrite supplemented in drinking water in rats exposed to short-term chronic hypobaric hypoxia. We observed that, nitrite significantly attenuates hypoxia-induced oxidative stress, modulates HIF-1α stability and promotes NO-cGMP signaling in hypoxic heart. To elucidate potential downstream targets of nitrite during hypoxia, we performed a microarray analysis of nitrite supplemented hypoxic hearts and compared with both hypoxic and nitrite supplemented normoxic hearts respectively. The analysis revealed a significant increase in the expression of many antioxidant genes, transcription factors and cardioprotective signaling pathways which was subsequently confirmed by qRT-PCR and Western blotting. Conversely, hypoxia exposure increased oxidative stress, activated inflammatory cytokines, downregulated ion channels and altered expression of both pro- and anti-oxidant genes. Our results illustrate the physiological function of nitrite as an eNOS-independent source of NO in heart profoundly modulating the oxidative status and cardiac transcriptome during hypoxia.

Altitude acclimatization via hypoxia-mediated oxidative eustress involves interplay of protein nitrosylation and carbonylation: A redoxomics perspective.

AIM: Hypoxia is an important feature of multiple diseases like cancer and obesity and also an environmental stressor to high altitude travelers. Emerging research suggests the importance of redox signaling in physiological responses transforming the notion of oxidative stress into eustress and distress. However, the behavior of redox protein post-translational modifications (PTMs), and their correlation with stress acclimatization in humans remains sketchy. Scant information exists about modifications in redoxome during physiological exposure to environmental hypoxia. In this study, we investigated redox PTMs, nitrosylation and carbonylation, in context of extended environmental hypoxia exposure. METHODS: The volunteers were confirmed to be free of any medical conditions and matched for age and weight. The human global redoxome and the affected networks were investigated using TMT-labeled quantitative proteo-bioinformatics and biochemical assays. The percolator PSM algorithm was used for peptide-spectrum match (PSM) validation in database searches. The FDR for peptide matches was set to 0.01. 1-way ANOVA and Tukey's Multiple Comparison test were used for biochemical assays. p-value<0.05 was considered statistically significant. Three independent experiments (biological replicates) were performed. Results were presented as Mean ± standard error of mean (SEM). KEY FINDINGS: This investigation revealed direct and indirect interplay between nitrosylation and carbonylation especially within coagulation and inflammation networks; interlinked redox signaling (via nitrosylation­carbonylation); and novel nitrosylation and carbonylation sites in individual proteins. SIGNIFICANCE: This study elucidates the role of redox PTMs in hypoxia signaling favoring tolerance and survival. Also, we demonstrated direct and indirect interplay between nitrosylation and carbonylation is crucial to extended hypoxia tolerance.

Upregulation of transcription factor NRF2-mediated oxidative stress response pathway in rat brain under short-term chronic hypobaric hypoxia.

Exposure to high altitude (and thus hypobaric hypoxia) induces electrophysiological, metabolic, and morphological modifications in the brain leading to several neurological clinical syndromes. Despite the known fact that hypoxia episodes in brain are a common factor for many neuropathologies, limited information is available on the underlying cellular and molecular mechanisms. In this study, we investigated the temporal effect of short-term (0-12 h) chronic hypobaric hypoxia on global gene expression of rat brain followed by detailed canonical pathway analysis and regulatory network identification. Our analysis revealed significant alteration of 33, 17, 53, 81, and 296 genes (p < 0.05, <1.5-fold) after 0.5, 1, 3, 6, and 12 h of hypoxia, respectively. Biological processes like regulation, metabolic, and transport pathways are temporally activated along with anti- and proinflammatory signaling networks like PI3K/AKT, NF-κB, ERK/MAPK, IL-6 and IL-8 signaling. Irrespective of exposure durations, nuclear factor (erythroid-derived 2)-like 2 (NRF2)-mediated oxidative stress response pathway and genes were detected at all time points suggesting activation of NRF2-ARE antioxidant defense system. The results were further validated by assessing the expression levels of selected genes in temporal as well as brain regions with quantitative RT-PCR and western blot. In conclusion, our whole brain approach with temporal monitoring of gene expression patterns during hypobaric hypoxia has resulted in (1) deciphering sequence of pathways and signaling networks activated during onset of hypoxia, and (2) elucidation of NRF2-orchestrated antioxidant response as a major intrinsic defense mechanism. The results of this study will aid in better understanding and management of hypoxia-induced brain pathologies.

Identification of haptoglobin and apolipoprotein A-I as biomarkers for high altitude pulmonary edema.

We have investigated the plasma proteome using 2D gel electrophoresis and matrix-assisted laser desorption/ionization tandem time of flight from patients with high altitude pulmonary edema (HAPE). A complete proteomic analysis was performed on 20 patients with HAPE and ten healthy sea level controls. In total, we have identified 25 protein spots in human plasma and found that 14 of them showed altered changes in HAPE patients, which mainly were acute phase proteins (APPs), compliment components, and apolipoproteins among others. Among the APPs, haptoglobin α2 chain, haptoglobin ß chain, transthyretin, and plasma retinol binding precursor showed overexpression in HAPE patients as compared to controls. To validate the result of proteomic analysis, two proteins were selected for enzyme-linked immunosorbent assay and Western blotting analysis. Our data conclusively shows that two proteins, haptoglobin and apolipoprotein A-I are upregulated in plasma of HAPE patients. These proteins may provide a fast and effective control of inflammatory damage until the subsequent mechanisms can begin to operate. Taken together, our findings further support the hypothesis that inflammatory response system is linked to the pathophysiology of HAPE.

D4F prophylaxis enables redox and energy homeostasis while preventing inflammation during hypoxia exposure.

Apo-A1 is correlated with conditions like hyperlipidemia, cardiovascular diseases, high altitude pulmonary edema and etc. where hypoxia constitutes an important facet.Hypoxia causes oxidative stress, vaso-destructive and inflammatory outcomes.Apo-A1 is reported to have vasoprotective, anti-oxidative, anti-apoptotic, and anti-inflammatory effects. However, effects of Apo-A1 augmentation during hypoxia exposure are unknown.In this study, we investigated the effects of exogenously supplementing Apo-A1-mimetic peptide on SD rats during hypoxia exposure. For easing the processes of delivery, absorption and bio-availability, Apo-A1 mimetic peptide D4F was used. The rats were given 10 mg/kg BW dose (i.p.) of D4F for 7 days and then exposed to hypoxia. D4F was observed to attenuate both oxidative stress and inflammation during hypoxic exposure. D4F improved energy homeostasis during hypoxic exposure. D4F did not affect HIF-1a levels during hypoxia but increased MnSOD levels while decreasing CRP and Apo-B levels. D4F showed promise as a prophylactic against hypoxia exposure.

Intermittent hypoxia modulates redox homeostasis, lipid metabolism associated inflammatory processes and redox post-translational modifications: Benefits at high altitude.

Intermittent hypoxia, initially associated with adverse effects of sleep apnea, has now metamorphosed into a module for improved sports performance. The regimen followed for improved sports performance is milder intermittent hypoxic training (IHT) as compared to chronic and severe intermittent hypoxia observed in sleep apnea. Although several studies have indicated the mechanism and enough data on physiological parameters altered by IH is available, proteome perturbations remain largely unknown. Altitude induced hypobaric hypoxia is known to require acclimatization as it causes systemic redox stress and inflammation in humans. In the present study, a short IHT regimen consisting of previously reported physiologically beneficial FIO2 levels of 13.5% and 12% was administered to human subjects. These subjects were then airlifted to altitude of 3500 m and their plasma proteome along with associated redox parameters were analyzed on days 4 and 7 of high altitude stay. We observed that redox stress and associated post-translational modifications, perturbed lipid metabolism and inflammatory signaling were induced by IHT exposure at Baseline. However, this caused activation of antioxidants, energy homeostasis mechanisms and anti-inflammatory responses during subsequent high-altitude exposure. Thus, we propose IHT as a beneficial non-pharmacological intervention that benefits individuals venturing to high altitude areas.

Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features of mitochondrial translation elongation.

The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.

Reverse translating SULT1A1, a potential biomarker in roentgenographically tested rat model of rapid HAPE induction.

AIMS: HAPE remains the most common lethal high-altitude disease. Although its pathophysiology and other associated causal factors have been partially uncovered along with some potential biomarker proteins, it has not been completely elucidated. A major hindrance to improving the understanding of HAPE pathophysiology and associated molecular events has been the absence of a quick, reliable and definitive animal model of HAPE. This study is aimed at development of a rapid and reliable SD rat model of high altitude pulmonary edema (HAPE) that can be roentgenographically confirmed and be used to study protein markers of HAPE. MAIN METHODS: In this study, we detail the process of rapidly inducing HAPE in male SD rats within 18 h of simulated high-altitude exposure without causing high rates of mortality. Thereafter, we confirmed HAPE using roentgenography. We assessed Sulfotransferase 1A1 (SULT1A1), IL-1 beta, TNF- alpha and IFN-gamma using ELISA. Finally, H&E staining of lung tissues was also performed. KEY FINDINGS: A roentgenographically confirmed HAPE model was demonstrated. SULT 1A1 levels are found to be highest in rats suffering HAPE, as previously confirmed in human patients. Inflammation was also assessed based on levels of inflammatory proteins like IL-1b, TNF-a, and IFN-g in addition to H&E staining of lung tissues. Inflammation and HAPE were observed to be synergistic events and not cause and effect of each other. SIGNIFICANCE: This rat model of HAPE will help researchers and clinicians in evaluating performance of therapies, potential biomarker and also further elucidate underlying molecular processes causing HAPE.

Salivary proteome patterns of individuals exposed to High Altitude.

OBJECTIVE: Identification of molecular signatures having key roles in hypobaric hypoxia by analysing the salivary proteome. Saliva holds a promising future in the search for new clinical biomarkers that are easily accessible, less complex, accurate, and cost effective as well as being non-invasive. METHODOLOGY: We employed qualitative proteomics approach to develop discriminatory biomarker signatures from human saliva exposed to hypobaric hypoxia. Salivary proteins were analyzed and compared between age-matched healthy subjects exposed to high altitude (∼13700 ft) for seven days (HAD7) with control subjects at sea level (Normoxia) by using 2-Dimensional gel electrophoresis/Mass Spectrometry approach. RESULTS: Several proteins with significant differential expression were found. The up-regulated proteins were apoptosis inducing factor-2, cystatin S, cystatin SN and carbonic anhydrase 6. The down regulated proteins were polymeric immunoglobulin receptor, alpha-enolase and prolactin-inducible protein. Further confirmation of the altered proteins such as alpha enolase, carbonic anhydrase 6, prolactin-inducible protein, apoptosis inducing factor 2, cystatin S and cystatin SN were performed using immunoblotting. The expression patterns of the selected proteins observed by immunoblot were in concurrence with 2-Dimesional gel electrophoresis results, therefore affirming the authenticity of the proteomic investigation. CONCLUSION: This study provides the proof of concept of salivary biomarkers for the non-invasive detection of hypobaric hypoxia induced effects. It is highly feasible to turn these biomarkers into an applicable clinical test after large scale validation.

STAT3-RXR-Nrf2 activates systemic redox and energy homeostasis upon steep decline in pO2 gradient.

Hypobaric hypoxia elicits several patho-physiological manifestations, some of which are known to be lethal. Among various molecular mechanisms proposed so far, perturbation in redox state due to imbalance between radical generation and antioxidant defence is promising. These molecular events are also related to hypoxic status of cancer cells and therefore its understanding has extended clinical advantage beyond high altitude hypoxia. In present study, however, the focus was to understand and propose a model for rapid acclimatization of high altitude visitors to enhance their performance based on molecular changes. We considered using simulated hypobaric hypoxia at some established thresholds of high altitude stratification based on known physiological effects. Previous studies have focused on the temporal aspect while overlooking the effects of varying pO2 levels during exposure to hypobaric hypoxia. The pO2 levels, indicative of altitude, are crucial to redox homeostasis and can be the limiting factor during acclimatization to hypobaric hypoxia. In this study we present the effects of acute (24h) exposure to high (3049m; pO2: 71kPa), very high (4573m; pO2: 59kPa) and extreme altitude (7620m; pO2: 40kPa) zones on lung and plasma using semi-quantitative redox specific transcripts and quantitative proteo-bioinformatics workflow in conjunction with redox stress assays. It was observed that direct exposure to extreme altitude caused 100% mortality, which turned into high survival rate after pre-exposure to 59kPa, for which molecular explanation were also found. The pO2 of 59kPa (very high altitude zone) elicits systemic energy and redox homeostatic processes by modulating the STAT3-RXR-Nrf2 trio. Finally we posit the various processes downstream of STAT3-RXR-Nrf2 and the plasma proteins that can be used to ascertain the redox status of an individual.

Competing trends of ROS and RNS-mediated protein modifications during hypoxia as an alternate mechanism of NO benefits.

Hypoxia, especially altitude associated hypoxia is known to cause severe physiological alterations and life-threatening conditions. Impaired redox balance along with oxidative stress, protein carbonylation and instigation of apoptotic events are common sub-cellular events that follow the hypoxic insult. The role of nitric oxide (NO) is very dynamic and versatile in preventing the ill effects of hypoxia vis-a-vis reacting with oxidative species and causing protein nitrosylation. Although several mechanisms of NO-mediated cytoprotection are known during hypoxic insult, limited pieces of evidence are available to support the relationship between two downstream events of oxidative stress, protein carbonylation (caused by carbonyl; CO radical) and protein nitrosylation/nitration (caused by NO/peroxynitrite; ONOO radical). In this study, we investigated an entirely new aspect of NO protection in hypoxia involving crosstalk between carbonylation and nitrosylation. Using standard NO inhibitor l-NAME and simulated hypoxic conditions in hypoxia-sensitive cell line H9c2, we evaluated the levels of radicals, cell death, mitochondrial membrane potential, levels of protein nitrosylation, protein nitration and carbonylation and glutathione content. The results were then carefully analyzed in light of NO bioavailability. Our study shows that reducing NO during hypoxia caused cell death via the increased degree of carbonylation in proteins. This provides a new aspect of NO benefits which furthers opens new possibilities to explore potential mechanisms and effects of cross-talk between nitrosylation, protein nitration and carbonylation, especially through some common antioxidant mediators such as glutathione and thioredoxin.