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Objective: Cholesterol efflux capacity (CEC), the ability of extracellular acceptors to pick-up cholesterol from macrophages, is a clinically relevant cardiovascular biomarker. CEC is inversely associated with incident atherosclerotic cardiovascular disease events. However, CEC is only modestly associated with HDL-C (high-density lipoprotein cholesterol) levels, which may explain the failure of HDL-C raising therapies to improve atherosclerotic cardiovascular disease outcomes. Determinants of variation in CEC are not well understood. Thus, we sought to establish whether extreme high and low CEC is a robust persistent phenotype and to characterize associations with cholesterol, protein, and phospholipids across the particle size distribution. Approach and Results: CEC was previously measured in 2924 participants enrolled in the Dallas Heart Study, a multi-ethnic population-based study from 2000 to 2002. We prospectively recruited those who were below the 10th and above 90th percentile of CEC. Our study revealed that extreme low and high CEC are persistent, robust phenotypes after 15 years of follow-up. Using size exclusion chromatography, CEC to fractionated plasma depleted of apolipoprotein B (fraction-specific CEC) demonstrated significant differences in CEC patterns between persistent high and low efflux groups. Fraction-specific CEC was correlated with fraction-specific total phospholipid but not apolipoprotein A-I, cholesterol, or total protein. These correlations varied across the size distribution and differed among persistent high versus low efflux groups. Conclusions: Extreme high and low CEC are persistent and robust phenotypes. CEC patterns in fractionated plasma reveal marked variation across the size distribution. Future studies are warranted to determine specific molecular species linked to CEC in a size-specific manner.

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.

Background: Haptoglobin has three common genotypes: Hp 1-1, Hp 2-1, and Hp 2-2. The Hp 2-2 genotype, known to have poorer antioxidizing capacity, is associated with worse cardiovascular outcomes in select patients with diabetes. In this study, we explore the association between various cardiometabolic biomarker profiles and the three Hp genotypes in a multiethnic population. Methods: A cross-sectional analysis was performed for all participants with diabetes in the Dallas Heart Study and Multi-Ethnic Study of Atherosclerosis cohorts. Hp genotype was measured in 1,158 participants (399 from DHS and 759 from MESA) using ELISA technique (Savyon Diagnostics, Ltd). Associations between Hp genotype and 26 cardiometabolic circulating and imaging biomarkers were assessed by ANOVA and stratified by ethnicity. Results: The study included 51% women, 44% Black (513 of 1,158: Hp 1-1=45%, Hp 2-1=35%, Hp 2-2=20%), 26% Hispanic (300 of 1,158: Hp 1-1=25%, Hp 2-1=49%, Hp 2-2=26%), 20% White (237 of 1,158: Hp 1-1=16%, Hp 2-1=56%, Hp 2-2=28%), and 9.2% Asian/Pacific Islander/East Indian (107 of 1,158: Hp 1-1=15%, Hp 2-1=36%, Hp 2-2=49%) participants. Significant differences were seen in lipid metabolism as well as atherosclerosis, inflammatory, structural, and endothelial markers with no significant differences seen in imaging biomarkers. In White participants, Hp 1-1 was associated with elevated total cholesterol (p=0.016), LDL-p (p=0.001), and LDL-C (p=0.004). In Asian/Pacific Islander/East Indian participants, Hp 2-2 was associated with elevated systolic blood pressure (p=0.029), BMI (p=0.029), and inflammatory markers such as IL-6 (p=0.018) and D-dimer (p=0.027). In Black participants, Hp 2-2 was associated with elevated ICAM (p=0.048) and BMI (p=0.007). Lastly, in Hispanic participants, no significant associations were seen. In exploratory analysis, when comparing Hp 1-1 and Hp 2-1 with Hp 2-2, there was greater magnitude effect size of certain biomarkers in Black and Asian participants, suggesting potential differential effects by ethnicity. Conclusion: Associations between Hp genotype and various cardiometabolic biomarkers are varied and ethnicity dependent with further studies needed to investigate the underlying physiologic mechanisms.

Reverse cholesterol transport is a main anti-atherosclerotic function of high-density lipoprotein (HDL), and cholesterol efflux capacity (CEC) is a key first step of this function. Inflammation and oxidation are metabolically linked to cholesterol transport but the relationships between these functions have not been well characterized. The objective of this study was to determine if extreme high or low CEC also had differences in inflammation and anti-oxidative functions. We hypothesized that inflammation and anti-oxidative functions of HDL are worse in those with extreme low vs. high CEC. Cholesterol efflux capacity was previously measured in 2,924 participants in the Dallas Heart Study, a multi-ethnic population-based cohort. Those below the 10 th and above the 90 th percentile were recruited 15-17 years later, and those with persistent extreme high and low CEC were studied (N=36). Several inflammatory markers such as interleukin-1(IL-1), IL-6, IL-10, tumor necrosis factor α (TNF- α), vascular cell adhesion protein 1(VCAM-1), and serum amyloid A (SAA) were measured using MAGPIX instrument on whole EDTA plasma. The antioxidant levels of paraoxonase-1 (PON1) were measured based on arylesterase activity using ELISA kit on whole citrated plasma. We analyzed data in whole group and stratified by sex, race/ethnicity, and diabetes status. Despite a two-fold difference in CEC, there were no significant differences in inflammatory markers between extreme high vs. low CEC. In those with diabetes, SAA levels were significantly higher in those with low vs. high efflux (P=0.02). High CEC group showed the inverse association with inflammatory marker (IL-1) while direct association with anti-inflammatory marker (IL-10) performing Pearson Correlation Coefficient, R=1, P

Cholesterol efflux (CE) is a major athero-protective function of high density lipoproteins (HDL). HDL is a heterogeneous mixture of lipoproteins that contain apoA1, the prime protein of HDL, and may contain one or more of >200 other proteins. These other proteins cluster on subpopulations of HDL particles to form HDL subspecies. It is not entirely clear by simply measuring individual protein abundances what drives HDL function. We aimed to study the differential association of 7 HDL subspecies defined by presence or absence of a functional protein with extreme and persistent cholesterol efflux capacity (CEC). From ~3000 individuals in the multi-ethnic population-based DHS (Dallas Heart Study) cohort, people at the bottom 10% and top 90% of CEC were reassessed after 15 years resulting in 36 participants with extreme persistent high or low CEC for the present study. HDL with and without 7 individual proteins potentially relevant to HDL function were quantified using ELISA. Differences in association with CEC between HDL subspecies that contain or lack a defining protein were examined by heterogeneity test. In the low CEC group, HDL lacking apolipoprotein C3 (apoC3), complement C3, apoE (apolipoprotein E) and PLMG (plasminogen) were each associated positively with CEC while HDL subspecies containing these proteins were not positively associated with CEC. (Pearson correlation coefficient range 0.55369 to 0.66562 vs -0.08432 to -0.25198; p

Hypoxia, particularly hypobaric hypoxia, is a multifaceted entity which includes certain molecular, patho-physiological and biochemical aspects. Any single aspect in itself cannot help us elucidate hypobaric hypoxia in its entirety. We observed three crucial lacunae within the existing literature. These include a lack of high-throughput investigations into redox PTMs, particularly NO-based PTMs; a prophylactic supplement with proven efficacy and safety which doesn’t involve medical supervision and is not contraindicated in hepatic, renal and cardiac insufficiencies; and a clinically validated rodent model of HAPE without any genetic/pharmacological manipulations. In the present study, we present an antagonistic interplay between nitrosylation and carbonylation which shows an additional NO-based network that is active in acclimatised individuals. Then we present a micronised aqueous suspension of silymarin which is efficacious at low doses in providing antioxidant, anti-inflammatory and hypoxia-adaptive vascular responses in addition to being a free radical quencher itself. Silymarin has an excellent safety and efficacy profile in humans. Finally, we create a SD rat model of HAPE which was used to reverse-translate a previously known HAPE marker in humans (SULT1A1) and elucidate the synergistic occurrence of HAPE and inflammation cascades. This is the first radiologically validated rodent HAPE model. In conclusion, we were able to elucidate the molecular, biochemical and patho-physiological aspects of hypobaric hypoxia which were left out by previous studies.

Hypobaric hypoxia (HH), a pathophysiological condition caused by an ascent to a high altitude. It occurs due to a deficiency of oxygen at the tissue level. Saliva is an advantageous biological sample because of easy, low cost and minimally invasive sample collection and processing. Although few reports have documented the effect of long term exposure to hypobaric hypoxia on salivary proteome but short term proteome based study remains uninvestigated. In this study, age-matched male SD rats were exposed to simulated hypobaric hypoxia (~ 25,000 ft, 7620 m) for 1 day (HD1), 3 days (HD3) and 7 days (HD7) followed by iTRAQ based LCMS/MS comparison with normoxic controls. Oxidative stress being the main event of hypobaric hypoxia suggested an initial increase in levels of oxidative stress parameters such as reactive oxygen species (ROS) and lipid peroxidation; and decrease in antioxidant enzymes such as the activity of catalase and reduced glutathione. Based on iTRAQ, out of 67 differentially expressed proteins, 36 (22 up-regulated and 14 down-regulated), 45 (24 up-regulated and 21 down-regulated), and 45 (19 up-regulated and 26 down-regulated) were modulated in HD1, HD3, and HD7 group respectively. Few proteins such as BPI fold-containing family A member 2, cystatin, and carbonic anhydrase 1 showed many folds differential expression in exposed groups as compared to normoxia. Through Ingenuity Pathways analysis, we have observed glucocorticoid receptor signaling and MSP-RON pathway to be most significantly affected. Glucocorticoid receptor signaling and MSP-RON signalling being the most significant pathways involved modulated proteins such as annexin 1, hsp90, keratin, actin-beta, and kallikrein 1. These proteins were found to aid in acclimatization and survival during hypobaric hypoxia. Taken together, this study provided the proof of concept for footprints of hypobaric hypoxia-mediated events during short term exposure through rat salivary proteome.

Hypobaric hypoxia (HH) is the hypoxia caused by decreased ambient atmospheric pressure (partial pressure of oxygen) as one ascends beyond 2500 m. The entire spectrum of molecular and biochemical studies regarding the effects of HH on an organism heavily depends on biological fluids during exposure to HH. The biological fluids are important sample types. This is because they are easiest to collect using minimally invasive collection procedures, easy to handle and store and low quantities are required for tests/assays. Although many reviews on hypobaric hypoxia in the recent past have been dedicated to the various advances made possible due to the study of biological fluids, biological fluids themselves have never been categorized according to studies done using them. This review concisely outlines the myriad results (particularly translational) in context of hypobaric hypoxia that have been observed in biological fluids, their collection strategies, storage strategies and ethical guidelines. Biological fluidscategorized here are blood (plasma and serum), saliva, tears and urine in context of research on HH and diseases like cancer where hypoxia is an essential condition. This review shall be of help to young investigators for choice of biological samples to be used in their experiments concerning hypoxia related studies and to clinicians involved in translational research providing them a ready reckoner for improving or modifying their approaches.

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.

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.

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.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-value0.05 was considered statistically significant. Three independent experiments (biological replicates) were performed. Results were presented as Mean ± standard error of mean (SEM).This investigation revealed direct and indirect interplay between nitrosylation and carbonylation especially within coagulation and inflammation networks; interlinked redox signaling (via nitrosylation‑carbonylation); and novel nitrosylation and carbonylation sites in individual proteins.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.

Extended exposure to low pO

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.

Lack of zero side-effect, prescription-less prophylactics and diagnostic markers of acclimatization status lead to many suffering from high altitude illnesses. Although not fully translated to the clinical setting, many strategies and interventions are being developed that are aimed at providing an objective and tangible answer regarding the acclimatization status of an individual as well as zero side-effect prophylaxis that is cost-effective and does not require medical supervision. This short review brings together the twin problems associated with high-altitude acclimatization, i.e. acclimatization status and zero side-effect, easy-to-use prophylaxis, for the reader to comprehend as cogs of the same phenomenon. We describe current research aimed at preventing all the high-altitude illnesses by considering them an assault on redox and energy homeostasis at the molecular level. This review also entails some proteins capable of diagnosing either acclimatization or high-altitude illnesses. The future strategies based on bioinformatics and systems biology is also discussed.

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.

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 (24 h) exposure to high (3049 m; pO2: 71 kPa), very high (4573 m; pO2: 59 kPa) and extreme altitude (7620 m; pO2: 40 kPa) 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 59 kPa, for which molecular explanation were also found. The pO2 of 59 kPa (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., Graphical abstract fx1, Highlights • A comparison of effects on redox and energy homeostasis between three altitude zones. • Exposure at 59 kPa (10 h) followed by normoxia (1 h) rescues homeostasis at 40 kPa. • Redox and energy homeostasis dependent on STAT-3-RXR-Nrf interplay. • Lung redox and energy homeostasis fails at 15,000 ft. • Systemic redox and energy homeostasis activated on failure of lung homeostatic mechanisms.

Padhy, Gayatri, Anamika Gangwar, Manish Sharma, Kalpana Bhargava, and Niroj Kumar Sethy. Plasma proteomics of Ladakhi natives reveal functional regulation between renin-angiotensin system and eNOS-cGMP pathway. High Alt Med Biol. 18:27-36, 2017.-Humans have been living in high altitudes for more than 25,000 years but the molecular pathways promoting survival and performance in these extreme environments are not well elucidated. In an attempt to understand human adaptation to high altitudes, we used two-dimensional gel electrophoresis combined with MALDI-TOF/TOF to identify plasma proteins and associated pathways of ethnic Ladakhi natives residing at 3520 m. This resulted in the identification of 36 differential proteins compared with sea-level individuals. Proteins belonging to coagulation cascade and complement activation were found to be less abundant in Ladakhi natives. Interestingly, we observed lower abundance of angiotensinogen (ANGT) and subsequent analysis also revealed lower levels of both ANGT and angiotensin II (Ang II) in Ladakhi natives. Concomitantly, we observed elevated levels of eNOS, phosphorylated eNOS (Ser1177), and plasma biomarkers for nitric oxide (NO) production (nitrate and nitrite) and availability (cGMP). These results suggest that functional interplay between renin-angiotensin system and NO-cGMP pathway contributes to the hypoxia adaptation in Ladakhi natives. These findings will augment the present understanding of higher NO and NO-derived metabolite availability during human adaptation to 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.

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.

Acute exposure to high dose of ultraviolet (UV) radiations is known to cause significant harm to skin, primarily due to the generation of free radicals and damage to DNA, which often culminate in rapid aging of the skin, or cancers. Keratinocytes being the most abundant skin’s cells are affected most by UV. Although a degree of endogenous protection is present, the vulnerability of UV-induced damaged can be minimized using protective agents. A few UV filters (organic and inorganic) have been successfully commercialized, yet, due to prevailing disadvantages such as low solubility, photostability, and aesthetic sense, suitable and more efficient UV filters continue to be explored as potential ingredients of cosmaceutical agents. A recently studied antioxidant enzyme mimetic cerium oxide nanoparticles showed emerging piece of evidence on benefits under environmental stress. However, its protective abilities as potential UV filter and therefore applicability in cosmaceutical has not yet been completely explored. This study provides a piece of evidence in support of beneficial effects of this new class of UV filters, polyethyleneglycol functionalized nanoceria (PEG-CNP) against UV - induced damage in vitro and in vivo. The nanomolar concentration of PEG-CNPs in the cell culture showed significant protection from UV exposure, by direct ROS scavenging, the rescue of cells from cell cycle arrest and DNA damage. Further, a proof of the concept study in dehaired rat skin showed that the topical application of 50 μM PEG-CNPs prevented the initial signs of UV induced damage. Unlike conventional UV filters, PEG-CNPs confer protection by internalizing the cells, and scavenging the radicals.

Biosensors are amazing tools for analysis of biological or nonbiological systems using fundamental biomolecules such as enzymes, antibodies, proteins, or small metabolites. Biosensors have emerged as prominent tool for clinicians as well as researchers and remain essential components of several point-of-care devices, dipstick portable tests, and even research kits. Animal biotechnology, which deals with improving or using the animals and their parts such as cells or tissues for use of human betterment, and sometimes involves genetic manipulation, has great dependence on biosensors. Besides some of the very popular and successfully commercialized biosensors such as glucose biosensor, potentially new classes of biosensors are rapidly emerging, which are severalfold advanced in terms of their specificity, portability, and cost. Use of biosensors in animal biotechnology mainly involves detection of metabolites, biochemical assays, in particular related to cell death, cancer research, aging, etc. In this chapter, we will discuss basic details of biosensors, principle, types of existing biosensors, and emerging concepts with future scope of biosensors in animal biotechnology.

Oxidative stress is associated with the generation of reactive oxygen/nitrogen species (RNOS) which non-enzymatically modify active functional groups in proteins mostly turning them into protein carbonyls or nitrosyls. These changes render the protein molecules non-functional and drive them to degradation or formation of cross linking aggregates. Limited methods are available for detections of protein modifications. Enzyme linked immunoassays as quantitative method and Immunoblotting based qualitative methods are most common. Visual examination of Immunoblots containing a characteristic pattern is difficult for a fair comparison if the differences are trifling or the modifications are of varying degree across the complete range. This necessitates the use of image processing tools for a fair comparison. We, report here a computational approach using ImageJ, to process and obtain significant inter group comparison. Also, this method provides, software developers and programmers an opportunity to augment the gel processing tools with such plugins and features.

Background Exposure to high-altitude leads to two outcomes: acclimation and mal-acclimation leading to high-altitude illnesses (HAIs). Acclimation to altitude actively negates HAIs like acute mountain sickness, high-altitude pulmonary and cerebral edemas. Although HAIs have been scrutinized, acclimation has remained only empirically understood with minimal knowledge of it at molecular level in an organism. Methods In this study, we investigated (using ELISA) and statistically evaluated the trendlines of various hypoxia-responsive plasma proteins' levels, reported significantly perturbed in multiple 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. Findings We observed 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 HAPE cases. We observed day and resident altitude specific proteins capable of accurately assessing acclimation when compared to baseline levels or the lower altitude zone. Interpretation The trendlines of Trx, GPx-3, Apo-AI and ST1A1 shown in this study are of acclimated individuals. These proteins have been shown to be statistically most accurate in indicating acclimation. Thus, they can serve as indicators of acclimation. A proof-of-concept protein panel is shown here which can be used to assess acclimation objectively. Funding: The study was funded by Ministry of Defence, Govt of India under Project DIP-263. Subhojit Paul is a recipient of CSIR fellowship. Anamika Gangwar is a recipient of DST-INSPIRE fellowship. Declaration of Interest: Authors declare no conflict of interest. Ethical Approval: All study protocols were cross-checked and approved by the institutional ethics committee (IEC/DIPAS/B2/1) in accordance with Helsinki declaration. Informed written consent was obtained prior to the study from all participants.

Oxidative stress is associated with a variety of disorders, diseases as well as the natural aging process thus making antioxidant discovery of medical relevance. In the present study, we report the simultaneous antioxidant as well as bio-imaging activities of a fluorescent extract (fluorophore) obtained from the tasar silk cocoons of the silk moth Antheraea mylitta. Using a nanocarrier-based strategy, we were able to localize the fluorophore in the cytosol of cardiomyoblast cell line H9c2 without any alterations to cellular morphology. In search of additional uses of the fluorophore beyond bio-imaging, we evaluated the antioxidant efficacies of the fluorophore against hydrogen peroxide-induced oxidative stress. Using microscopic, flow-cytometry and ELISA based studies; we observed that the silk fluorophore ameliorated hydrogen peroxide-induced reactive oxygen species (ROS) levels and oxidative stress. Moreover, enhanced levels of endogenous antioxidant enzymes were also observed in fluorophore pre-treated cells during oxidative stress. This resulted in significant reduction of oxidative stress-induced cell death. Our cumulative results suggest concomitant antioxidant and bio-imaging activities of the naturally occurring silk fluorophore. Given that a tasar silk fluorophore has never been isolated from cocoons in the silk textile industry, further commercial exploration will provide economic support to silk farmers.

Oxidative stress is proposed as leading event in the deterioration of health and basic biological processes. Ever since the Harman’s theory of aging was proposed based on the ill effects of oxidative in the body, the pace of oxidative stress research became rapid. The antioxidants were proposed as putative therapeutic and prophylactic agents for the prevention of oxidative damage and its aftermath. Despite the escalating research publications in the domain of oxidative stress and antioxidant therapy, apparent clinical transitions are fairly low. Perhaps, this should not be looked as question on the studies which were performed on the antioxidants, rather our poor understanding of cross talk of antioxidants and oxidants in the cells and its downstream effects. It seems that decision of considering antioxidants as miracle drugs for aging and similar condition was too early. There is lot more to be explored in this domain, and as we move deeper, we realize that oxidative stress and antioxidant interplay is one of the most complicated biological events that has several fold more complexity than basic cellular processes and metabolism. The scientific questions such as how much antioxidant dose is optimal and which antioxidant is most suitable can only be answered in a context-specific manner. The several anomalies and unfruitful clinical translations of antioxidants have led to the continuation and intensification of antioxidant research. With the advent of a new domain of science named nanotechnology, few exciting possibilities have emerged in the antioxidant researches which are likely to answer some of the issues of conventional antioxidants. This chapter is aimed to discuss the emerging trends in nano-antioxidants with a special focus on much-studied antioxidant nanoceria.

Aditya Arya,1 Anamika Gangwar,1 Sushil Kumar Singh,2 Manas Roy,3,4 Mainak Das,3 Niroj Kumar Sethy,1 Kalpana Bhargava1 1Peptide and Proteomics Division, Defense Institute of Physiology and Allied Sciences, 2Functional Materials Division, Solid State Physics Laboratory, Defense Research and Development Organization, Timarpur, Delhi, 3Biological Science and Bioengineering, Indian Institute of Technology, Kanpur, 4Department of Chemistry, Indian Institute of Engineering Science and Technology, Howrah, India Abstract: Structural and functional integrity of the brain is adversely affected by reduced oxygen saturation, especially during chronic hypoxia exposure and often encountered by altitude travelers or dwellers. Hypoxia-induced generation of reactive nitrogen and oxygen species reportedly affects the cortex and hippocampus regions of the brain, promoting memory impairment and cognitive dysfunction. Cerium oxide nanoparticles (CNPs), also known as nanoceria, switch between+3 and +4 oxidation states and reportedly scavenge superoxide anions, hydrogen peroxide, and peroxynitrite in vivo. In the present study, we evaluated the neuroprotective as well as the cognition-enhancing activities of nanoceria during hypobaric hypoxia. Using polyethylene glycol-coated 3 nm nanoceria (PEG-CNPs), we have demonstrated efficient localization of PEG-CNPs in rodent brain. This resulted in significant reduction of oxidative stress and associated damage during hypoxia exposure. Morris water maze-based memory function tests revealed that PEG-CNPs ameliorated hypoxia-induced memory impairment. Using microscopic, flow cytometric, and histological studies, we also provide evidences that PEG-CNPs augmented hippocampus neuronal survival and promoted neurogenesis. Molecular studies revealed that PEG-CNPs promoted neurogenesis through the 5'-adenine monophosphate-activated protein kinase–protein kinase C–cyclic adenosine monophosphate response element-binding protein binding (AMPK-PKC-CBP) protein pathway. Our present study results suggest that nanoceria can be translated as promising therapeutic molecules for neurodegenerative diseases. Keywords: cerium oxide nanoparticles, oxidative stress, memory, hypoxia, neuroprotection

The advent of high throughput technologies and development of mathematical models of systems biology has led to finer and clearer observations in various domains of biology, especially proteomics, metabolomics, and genomics. Redox biology is certainly a prospective domain to include these approaches. Some initial attempts have been successfully made by researchers at the development of redox protein database, systems biology of thiol redox systems. However, the development of an integrated database of redox parameters is highly required to pace up the redox biology research. The challenges are primarily a collection of a large dataset of various redox parameters which remain scattered across several thousands of research articles and their curation. We developed a community-based approach to gather the data and develop a composite database. We used some existing models of community-based data collection systems used in tuberculosis research and prepared an organized hierarchical system for data collection, validation, and manual curation. We have gathered more than 20 different parameters of redox systems in more than 200 datasets, which is likely to increase to >20,000 datasets by the second phase of the project. Use Google forms and Google live sheets were used for the collection of data and this was followed by data curation by specific team leaders. We observed that there was a high degree of heterogeneity in the data in terms of the units used, prevailing experimental conditions, which were normalized and segregated by curators, that can be beneficial in the next level integration with the systems biology tools. The dataset will be transformed into a highly interactive web-portal with machine learning and artificial intelligence tools, to provide live simulations of redox biology in various model systems.

Despite its extraordinary antioxidant capacity, the clinical usage of silymarin has remained restricted to amelioration of hepatic pathology. Perhaps its low bioavailability and uneven bio-distribution, owing to its poor aqueous solubility, are two main causes that have dampened the clinical applicability and scope of this preparation. We took these two challenges and suggested an unexplored application of silymarin. Apart from liver, two of the most susceptible vital organs at the highest risk of oxidative stress are brain and lung, especially during reduced oxygen saturation (hypoxia) at anatomical level. Hypoxia causes excess generation of radicals primarily in the lungs as it is the first organ at the interphase of atmosphere and organism making it the most prone and vulnerable to oxidative stress and the first responder against hypobaric hypoxia. As our first objective, we improved the silymarin formulation by restricting its size to the lower threshold and then successfully tested the prophylactic and therapeutic action in rat lung challenged with simulated hypobaric hypoxia. After dose optimization, we observed that 50mg/kg BW silymarin as size restricted and homogenous aqueous suspension successfully minimized the reactive oxygen species and augmented the antioxidant defense by significant upregulation of catalase and superoxide dismutase and reduced glutathione. Moreover, the well-established hypoxia markers and proteins related to hypoxia adaptability, hif1a and VEGF were differentially regulated conferring significant reduction in the inflammation caused by hypobaric hypoxia. We therefore report,the unexplored potential benefits of silymarin for preventing high altitude associated pathophysiology further paving its road to clinical trials.

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

Generation of reactive oxygen species (ROS) is well accepted phenomenon in the skin exposed to Ultraviolet radiations. Ultraviolet radiations initiate an acute cascade of photochemical reactions culminating in the generation of reactive oxygen species such as superoxides. The downstream effects of these acute changes are directly involved in various cutaneous changes such as sunburn erythema, phytocarcionoma and photoaging. The risk and exposure of UV radiations varies dramatically over time, space and topology across the globe suggesting the clinical relevance of identifying efficient UV protective agents. Based on the prior understanding from our lab and other studies cerium oxide has emerged as one of the promising antioxidants due to its superoxide dismutase and catalase mimetic activity. In this study we evaluated the UV protective efficiency of custom synthesized cerium oxide nanoparticles tagged with polyethyleneglycol (PEG-CNPs). The internalization of nanoparticles was successfully evaluated using DiD florescent tags. Preliminary studies were conducted on primary keratinocyte culture (HAEK cell line) using standard flow cytometry and microscopy. Significant reduction in ROS was observed in UV irradiated cells pretreated with 25 μM PEG-CNPs for 6 hours with concomitant reduction in protein modification such as carbonylation and nitrosylation and eventually the cell death. Moreover, on gaining confidence in cell culture model the hypothesis was tested in rat skin exposed to known doses of UV the PEG-CNPs showed significant reduction in clinical cutaneous parameters such as erythema. Histopatholical examination and basic biochemical indicators of oxidative stress including 8-OHdG, protein carbonyl, nitrosylation was significantly reduced on topical application prior to UV exposure. Apart from evaluating the minimum effective dose we also evaluated basic toxicological assessment and developed a topical formulation with hydrophobic solvents. In conclusion, the PEG-CNPs were highly efficient in protein skin from UV damage therefore providing a promising cosmaceutical application of this novel class of antioxidants.

High altitude pulmonary edema (HAPE) is a serious pathological condition associated with rapid ascent to high altitude occurring in non-acclimatized but otherwise healthy individuals. Decades of scientific studies on HAPE have unraveled the disease pathology, diagnosis and therapeutic interventions yet, the etiology is still unknown. A vast scientific literature is available on HAPE for a quick reference of clinicians, researchers and academicians. Perhaps, the view of mountain travelers is different and their anticipation of HAPE susceptibility comprises of personal experience. Ever-increasing number of visitors to high altitude demands the possibility of HAPE susceptibility screening, however, scientific community is yet to find a staunch solution. This review is an update of recent information on HAPE susceptibility indicators from genomics, proteomics and metabolomics as well as information pertaining to treatment/prognosis of HAPE.

'Exercise' is a double-edged sword for the skeletal muscle. Small amount of ROS generated during mild exercise, is essential for normal force generation; whereas large quantity of ROS generated during intense exercise, may cause contractile dysfunction, resulting in muscle weakness and fatigue. One of the key question in skeletal muscle physiology is 'could antioxidant therapy improve the skeletal muscle endurance? A question, which has resulted in contradictory experimental findings till this date. This work has addressed this 'very question' using a synthetic, inorganic, antioxidant nano-material viz., 'cerium oxide nanozyme' (CON). It has been introduced in the rat by intramuscular injection, and the skeletal muscle endurance has been evaluated. Intramuscular injections of CON, concurrent with exercise, enhanced muscle mass, glycogen and ATP content, type I fiber ratio, thus resulting in significantly higher muscle endurance. Electron microscope studies confirmed the presence of CON in the vicinity of muscle mitochondria. There was an increase in the number and size of the muscle mitochondria in the CON treated muscle, following exercise, as compared to the untreated group with only exercised muscle. Quantitative proteomics data and subsequent biological network analysis studies, identified higher levels of oxidative phosphorylation, TCA cycle output and glycolysis in CON supplemented exercised muscle over only exercised muscle. This was further associated with significant increase in the mitochondrial respiratory capacity and muscle contraction, primarily due to higher levels of electron transport chain proteins like NDUFA9, SDHA, ATP5B and ATP5D, which were validated by real-time PCR and western blotting. Along with this, persistence of CON in muscle was evaluated with ICP-MS analysis, which revealed clearance of the particles after 90 d, without exhibiting any inflammation or adverse affects on the health of the experimental animals. Thus a higher physiological endurance of the CON supplemented exercised muscle' opens new avenues in skeletal muscle therapeutic, space and sports medicine.