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Sharma, Vandana, Rajeev Varshney, and Niroj Kumar Sethy. Identification of suitable reference genes for lowlanders exposed to high altitude and Ladakhi highlanders.

Objectives: The present study was planned to delineate the blood pressure (BP) response of the lowlanders during initial 6 days of acclimatisation to high altitude. Materials and Methods: 398 normotensive sojourners at sea level ascended to HA (3,500 m). Blood pressure response of these subjects were analysed for 6 consecutive days at HA. Based on the BP response of these subjects on the 6th day at HA they were divided into two groups: HBP-high BP group and NBP- normal BP group. Results: It showed that on 6th day at HA, 51 out of 398 subjects presented with BP≥140/90 mmHg (HBP) while others remained normotensive (NBP). Comparative systolic BP of HBP and NBP groups during successive days at HA were as follows: HA day 1 (146.8 ± 13.7 mmHg vs 146.3 ± 8.8 mmHg, p>0.05), HA day 4 (144.5 ± 8.7 mmHg vs 135.0 ± 7.0 mmHg, p0.05), HA day 4 (88.9 ± 6.0 mmHg vs 85.3 ± 5.1 mmHg, p Conclusion: It was found that NBP had initially raised BP which returned to normal levels after acclimatisation period of 6 days, but in HBP, it started rising after 4th day. It can be concluded that those individuals whose BP on 6th day at HA does not return to normal after acclimatisation period needs regular follow up as these individuals can have a delayed acclimatisation normalising BP later on or can become hypertensive subsequently.

Higher levels of circulatory nitric oxide (NO) and NO metabolites reportedly facilitate high altitude acclimatization. But the underlying factors and molecular pathways promoting NO production at high altitude has been poorly characterized. Studying healthy lowlanders at sea level (C, lowlander) and high altitude (3500 m, after day 1, 4 and 7 of ascent), we report higher protein levels of eNOS and eNOSSer1177, higher plasma levels of BH4, NOx (nitrate and nitrites), cGMP and lower levels of endogenous eNOS inhibitor ADMA during healthy high altitude acclimatization. Our qRT-PCR-based gene expression studies identified higher levels of eNOS/NOS3 mRNA along with several other eNOS pathway genes like CALM1, SLC7A1 and DNM2. In addition, we observed higher mRNA levels of estrogen (E2) receptors ERα/ESR1 and ERβ/ESR2 at high altitude that transcriptionally activates NOS3. We also observed higher mRNA level of membrane receptor ERBB2 that phosphorylates eNOS at Ser1177 and thus augments NO availability. Evaluating E2 biosynthesis at high altitude, we report higher plasma levels of CYP11A1, CYP19A1, E2, lower levels of testosterone (T) and T/E2 ratio as compared to sea level. Correlation studies revealed moderate positive correlation between E2 and NOx (R = 0.68, p = 0.02) after day 4 and cGMP (R = 0.69, p = 0.02) after day 7 at high altitude. These findings suggest a causative role of E2 and its receptors ESR1 and ESR2 in augmenting eNOS activity and NO availability during healthy high altitude ascent. These results will aid in better understanding of NO production during hypobaric hypoxia and help in designing better high altitude acclimatization protocols.

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.

Pooja, Vandana Sharma, Manish Sharma, Rajeev Varshney, Bhuvnesh Kumar, and Niroj Kumar Sethy. Association between 17β-estradiol receptors and nitric oxide signaling augments high-altitude adaptation of Ladakhi highlanders.

Superoxide dismutases (SODs), a family of ubiquitous enzymes, provide essential protection to biological systems against uncontrolled reactions with oxygen- and nitrogen- based radical species. We review first the role of SODs in oxidative stress and the other biological functions such as peroxidase, nitrite oxidase, thiol oxidase activities etc., implicating its role in neurodegenerative, cardiovascular diseases, and ageing. Also, this review focuses on the development of electrochemical label-free immunosensor for SOD1 and the recent advances in biosensing assay methods based on their catalytic and biological functions with various substrates including reactive oxygen species (superoxide anion radical, hydrogen peroxide), nitric oxide metabolites (nitrite, nitrate) and thiols using thiol oxidase activity. Furthermore, we emphasize the progress made in improving the detection performance through incorporation of the SOD into conducting polymers and nanocomposite matrices. In addition, we address the potential opportunities, challenges, advances in electrochemical-sensing platforms and development of portable analyzer for point-of-care applications.

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.

Nitrite (NO2-) supplementation limits hypoxia-induced oxidative stress and activates the alternate NO pathway which may partially account for the nitrite-mediated cardioprotection. So, sensitive and selective biosensors with point-of-care devices need to be explored to detect the physiological nitrite level due to its important role in human pathophysiology. In this work, cytochrome c reductase (CcR) biofunctionalized self assembled monolayer (SAM) functionalized on gold nanoparticles (GNPs) in polypyrrole (PPy) nanocomposite onto the screen printed carbon electrode (SPCE) was investigated as a biosensor for the detection of nitrite based on its electrochemical and catalytic properties. CcR was covalently coupled with SAM layers on GNPs by using EDC and NHS. Direct electrochemical response of CcR biofunctionalized electrodes showed a couple of well-defined and nearly reversible cyclic voltammetric peaks at -0.34 and -0.45 vs. Ag/AgCl. Under optimal conditions, the biosensor could be used for the determination of NO2- with a linear range from 0.1-1600µm and a detection limit of 60nM with a sensitivity of 0.172µAµM-1cm-2. Further, we have designed and developed a novel and cost effective portable electrochemical analyzer for the measurement of NO2- in hypoxia induced H9c2 cardiac cells using ARM microcontroller. The results obtained here using the developed portable electrochemical nitrite analyzer were also compared with the standard cyclic voltammetry instrument and found in agreement with each other.

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.

Brain is well known for its disproportionate oxygen consumption and high energy-budget for optimal functioning. The decrease in oxygen supply to brain, thus, necessitates rapid activation of adaptive pathways - the absence of which manifest into vivid pathological conditions. Amongst these, oxygen sensing in glio-vascular milieu and H

High energy laser, particularly 532 nm, is widely used in defense and medical applications and there is need to address its occupational safety. Thermal and non-thermal effects of 532 nm high energy laser on skin are cause of concern. This study indicates impact of 532 nm laser on rat skin and first of its kind of attempt to understand transcriptional activation of genes as an early response following laser exposure. Skin of experimental rats were exposed to 532 nm radiance at 0.1, 0.25 and 0.50 W/cm2 for 10 sec. Thermographic changes of skin exposed to 532 nm laser exhibited increased Tmax temperature in radiance dependent manner. After thermal imaging, skin of experimental rats was collected 1 h post laser exposure for studying differential gene expression. The skin exposed to lower power density (0.1 W/cm2) did not show significant changes in expression of gene pathways studied. At moderate radiance (0.25 W/cm2), predominantly canonical wnt/B-catenin pathway genes notch1, axin2, ccdn1, wnt5a and redox homeostasis genes; txn1, nqo1 and txnrd1 were expressed. At higher radiance (0.5 W/cm2), significant repression of genes related to wound healing process particularly notch/wnt pathway viz. hes5, wnt1, wn3b with higher expression of dab2 was recorded. The data obtained from these studies would help in drawing safety limits for skin exposure to 532 nm laser. Further, genes expressed at moderate and high level of radiance exposure to skin were distinct and differential and provide new avenue to configure pathway to counteract laser induced delay in tissue injury and hair follicular damage.

In this work, we have fabricated a label-free electrochemical immunosensor for the detection of Cu,Zn superoxide dismutase (SOD1) which is clinically important to a wide variety of neurodegenerative, cardiovascular, and chronic immune diseases. The immunosensor is comprised of a screen printed carbon electrode (SPCE) modified with self-assembled monolayers (SAMs) of gold nanoparticles (GNPs) in electropolymerized polypyrrole (PPy) and biofunctionalized with monoclonal anti-SOD1 antibody. The morphological changes of each electrode modification step were analyzed by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) while electrochemical changes were monitored via cyclic voltammetry (CV) and impedance spectroscopy (EIS) by using ferri-ferrocyanide as electrochemical redox probe. The GNP nanostructured immunosensor indirectly monitors the SOD1 levels via electrocatalytic oxidation of nitrite, due to inherent nitrite oxidase activity of SOD1, with a wide linear sensing range (0.5 nM to 5 μM), low detection limit (0.5 nM), and high sensitivity (46.6 ± 3.5 nA nM −1 ). SOD1 concentration levels were also measured in real biological samples ( i.e. , cultured human epidermal keratinocytes) and the results correlated well with a western blot densitometry assay. Such rapid detection of SOD1 concentration levels in real biological samples is well-suited for point-of-care (POC) diagnostics.

Cordyceps sinensis (C. sinensis) is well established as a traditional Chinese medicine (TCM) that has been valued as a health food for centuries. It is an entomopathogenic fungus in Ascomycetes that naturally occurs at high altitude in Himalayan region and has received considerable attention due to the abundance of various biologically active compounds. Despite having reported health benefits and economic importance, qualitative phytochemical analysis, proximate composition and proteome study of Indian isolates of C. sinensis grown at high altitude remains untapped. In the present study, qualitative phytochemical analysis was carried on powdered whole body of C. sinensis (CSWb) and its aqueous extract (CSAq) prepared by accelerated solvent extraction technique which indicated the presence of several bioactive constituents such as alkaloids, amino acids and proteins, carbohydrates, flavonoids and phenols, gums, mucilages and saponins. We evaluated chemical composition of the Indian Himalayan medicinal mushroom C. sinensis in terms of its carbohydrate (55.68%) content, crude fiber (6.40%), fat (1.80%), moisture (7.18%), protein (21.46%) and total ash (7.48%). Furthermore, soluble protein identification of both CSWb and CSAq by SDS-PAGE followed by MALDI-TOF-TOF analysis revealed the presence of various types of most abundant proteins such as P-type II A ATPase, TE1b [Blumeriagraminis f. sp. hordei], Chitin synthase Chs [Penicilliummarneffei ATCC 18224], Serine/threonine-protein kinase CLA4, DEHA2C06820p [Debaryomyceshansenii CBS767], YALI0E29887p [Yarrowialipolytica] etc. In conclusion, the present study provides a comprehensive qualitative phytochemical analysis, proximate composition and proteome study on Indian isolate of C. sinensis which could endorse its use as a functional food.

Brain is well known for its disproportionate oxygen consumption and high energy-budget for optimal functioning. The decrease in oxygen supply to brain, thus, necessitates rapid activation of adaptive pathways - the absence of which manifest into vivid pathological conditions. Amongst these, oxygen sensing in glio-vascular milieu and H2S-dependent compensatory increase in cerebral blood flow (CBF) is a major adaptive response. We had recently demonstrated that the levels of H2S were significantly decreased during chronic hypobaric hypoxia (HH)-induced neuro-pathological effects. The mechanistic basis of this phenomenon, however, remained to be deciphered. We, here, describe experimental evidence for marked limitation of cysteine during HH - both in animal model as well as human volunteers ascending to high altitude. We show that the preservation of brain cysteine level, employing cysteine pro-drug (N-acetyl-L-cysteine, NAC), markedly curtailed effects of HH - not only on endogenous H2S levels but also, impairment of spatial reference memory in our animal model. We, further, present multiple lines of experimental evidence that the limitation of cysteine was causally governed by physiological propensity of brain to utilize cysteine, in cystathionine beta synthase (CBS)-dependent manner, past its endogenous replenishment potential. Notably, decrease in the levels of brain cysteine manifested despite positive effect (up-regulation) of HH on endogenous cysteine maintenance pathways and thus, qualifying cysteine as a conditionally essential nutrient (CEN) during HH. In brief, our data supports an adaptive, physiological role of CBS-mediated cysteine-utilization pathway - activated to increase endogenous levels of H2S - for optimal responses of brain to hypobaric hypoxia.

A brief seed pretreatment of 12 hours, in an aqueous suspension of synthesized nano iron pyrite (FeS2), significantly increases the yield of spinach and other crops. The effector mechanism is not clear. An aqueous suspension of FeS2, produces very trace amounts of H2O2, Fe2O3, FeS, FeSO4, Fe(SO4)3, SO2, S and H+ ionic species. Thus for 12 hours, seeds are exposed to this complex aqueous suspension. Among these trace species, H2O2 and Fe2O3 are known seed stimulants and plant growth promoters respectively. In this work, an attempt has been made to quench one of these trace compounds generated in the aqueous suspension of FeS2, viz., H2O2; and the long-term effect on the mature plant was monitored. To test this, along with FeS2, an agriculturally relevant inorganic peroxide scavenger viz., nano cerium oxide (CeO2) was introduced into this system. Four seed pretreatment regimens were followed for the spinach viz., (i) control (water), (ii) FeS2 + water, (iii) CeO2 + water, (iv) FeS2 + CeO2 + water; and growth was monitored for the next 80 days. It was found that, at maturity, CeO2 and FeS2 + CeO2, resulted in significantly smaller leaves, as compared to the control and FeS2; furthermore, FeS2 resulted in leaves with increased chlorophyll and carbohydrate. Thus, the data indicates that by quenching the H2O2, the seed-stimulant effect of FeS2 is compromised. So, while the FeS2 + water suspension functions as a seed vigor enhancer, CeO2 + water on the contrary, functions as a ‘seed vigor reducer’. It is noteworthy, that CeO2 is used by Chinese farmers, as a micro-nutrient to increase crop production. Current data indicates that, it delays germination of seeds, whereas FeS2 hastens germination. Thus such an approach could be used for hastening or delaying germination, manipulating weed population, seed storage in critical conditions, timing the life-cycle of a plant and developing more energy-efficient plants, especially in regions, where there is limited sunlight during significant parts of the year.

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.

Doping of graphene with nitrogen is of much interest, since it improves the overall conductivity and supercapacitive properties. Besides conductivity, nitrogen doping also enhances the pseudo-capacitance due to fast and reversible surface redox processes. In this work, we have developed a cheap and easy process for synthesizing heavily nitrogen doped graphene (15% nitrogen) from non-mulberry silk cocoon membrane (Tassar, Antheraea mylitta) by pyrolyzing the cocoon at 400 °C in argon atmosphere. Further we have investigated the performance of this heavily ‘nitrogen doped graphene’ (NDG) in a supercapacitor device. Our results suggest that NDG obtained from cocoon has improved supercapacitor performance. The improved performance is due to the high electronegativity of nitrogen that forms dipoles on the graphene surface. These dipoles consequently enhance the tendency of graphene to attract charged species to its surface. This is a green and clean synthesis approach for developing electronic materials for energy applications.

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.

In this paper, a portable, cost-effective electrochemical assay is presented for rapid, sensitive, and quantitative detection of cytochrome c (cyt c ) release. The developed cyt c assay consists of two parts: (i) a miniaturized electrochemical biosensor based on cytochrome c reductase (CcR) functionalized screen printed electrodes (SPE); (ii) a microcontroller based data acquisition unit integrated with potentiostat circuit capable of performing cyclic voltammetry technique for the analysis. The working electrode surface of SPE was integrated with polypyrrole (PPy)-carbon nanotubes (CNT) nanocomposite for an enhanced immobilization of the enzyme, CcR. The acquired biosensor data are processed into digital form by the microcontroller and further transferred to a PC through USB port for analysis. GUI based system implemented here makes the analyzer easy to operate. Under optimal conditions, the electroanalytical behavior of the CcR-CNT-PPy-SPE biosensor linearly responds to the cyt c concentration range from 10 nM to 500 μM with a detection limit of 10 nM and a sensitivity of 0.102 ± 0.005 μA μM −1 cm −2 . The performance of the volume miniaturized SPE based biosensor coupled with the portable microcontroller based instrument was further evaluated by applying it for the measurement of mitochondrial cyt c release during cardiomyocytes apoptosis; the results are validated well with the commercial electrochemical analyzer and standard ELISA.

NAP (davunetide) is a clinical octapeptide and reportedly possesses neuroprotective, neurotrophic and cognitive protective properties. The information for NAP-mediated neuroproteome changes and associated signaling pathways during hypoxia will help in drug development programmes across the world. In the present study, we have evaluated the antioxidant activities of NAP in rat hippocampus exposed to hypobaric hypoxia (25,000 ft, 282 mm Hg) for 3, 6 and 12 h respectively. Using 2D-gel electrophoresis (2D-GE) with matrix-assisted laser desorption ionization time of flight (MALDI-TOF/TOF) mass spectrometry, we have identified altered expression of 80 proteins in NAP-supplemented hippocampus after hypoxia. Pathway analysis revealed that NAP supplementation significantly regulated oxidative stress response, oxidoreductase activity and cellular response to stress pathways during hypoxia. Additionally, NAP supplementation also regulated energy production pathways along with AMP-activated protein kinase (AMPK) signaling and signaling by Rho family GTPases pathways. We observed higher expression of antioxidant Sod1, Eno1, Prdx2 and Prdx5 proteins that were subsequently validated by Western blotting. A higher level of Prdx2 was also observed by immunohistochemistry in NAP-supplemented hippocampus during hypoxia. In corroboration, we are able to detect significant lower level of protein carbonyls in NAP-supplemented hypoxic hippocampus suggesting amelioration of oxidant molecules by NAP supplementation. These results emphasize the antioxidant and signaling properties of NAP in rodent hippocampus during hypobaric hypoxia.

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 (282 mm Hg for 3 h) 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.

Cerium oxide nanoparticles (CNPs) of spherical shape have unique antioxidant capacity primarily due to alternating + 3 and + 4 oxidation states and crystal defects. Several studies revealed the protective efficacies of CNPs in cells and tissues against the oxidative damage. However, its effect on mitochondrial functioning, downstream effectors of radical burst and apoptosis remains unknown. In this study, we investigated whether CNPs treatment could protect the primary cortical cells from loss of mitochondrial membrane potential (Δψm) and Δψm-dependent cell death. CNPs with spherical morphology and size range 7-10 nm were synthesized and utilized at a concentration of 25 nM on primary neuronal culture challenged with 50 μM of hydrogen peroxide (H2O2). We showed that optimal dose of CNPs minimized ROS content of the cells and also curbed related surge in cellular calcium flux. Importantly, CNPs treatment prevented apoptotic loss of cell viability. Reduction in the apoptosis could be successfully attributed to the maintenance of Δψm and restoration of major redox equivalents NADH/NAD(+) ratio and cellular ATP. These findings, therefore, suggest possible route of CNPs protective efficacies in primary cortical culture.

This work presents a novel bienzymatic biosensor for the simultaneous determination of nitrite (NO2(-)) and nitrate (NO3(-)) ions using copper, zinc superoxide dismutase (SOD1) and nitrate reductase (NaR) coimmobilized on carbon nanotubes (CNT)-polypyrrole (PPy) nanocomposite modified platinum electrode. Morphological changes of the PPy and CNT modified electrodes were investigated using scanning electron microscopy. The electrochemical behavior of the bienzymatic electrode (NaR-SOD1-CNT-PPy-Pt) was characterized by cyclic voltammetry exhibiting quasi-reversible redox peak at +0.06 V and reversible redox peaks at -0.76 and -0.62V vs. Ag/AgCl, for the immobilized SOD1 and NaR respectively. The electrocatalytic activity of SOD1 towards NO2(-) oxidation observed at +0.8 V was linear from 100 nM to 1mM with a detection limit of 50 nM and sensitivity of 98.5 ± 1.7 nA µM(-1)cm(-2). Similarly, the coimmobilized NaR showed its electrocatalytic activity towards NO3(-) reduction at -0.76 V exhibiting linear response from 500 nM to 10mM NO3(-) with a detection limit of 200 nM and sensitivity of 84.5 ± 1.56 nA µM(-1)cm(-2). Further, the present bienzymatic biosensor coated with cellulose acetate membrane for the removal of non-specific proteins was used for the sensitive and selective determinations of NO2(-) and NO3(-) present in human plasma, whole blood and saliva samples.

This work addresses an interesting, interdisciplinary problem of nature- “the mechanism by which animal senses the earth's magnetic field and navigate”. Currently there are two existing theories trying to explain, “How the animal senses the magnetic field of earth”. One theory is based on the presence of biogenic magnetic materials in the body of the animals. Such magnetic materials which are present inside the body, orients itself according to the earth's weak magnetic field and convey the information to the nervous system to develop the navigational map. The second theory is based on a light dependent photochemical reaction. A photochemical reaction leads to the generation of radical pairs, which helps in sensing the weak magnetic field of the earth. In this work, we are proposing a new model of magnetoreception. Unlike the existing radical pair system of magnetoreception, where a light-dependent reaction is essential to generate free radicals, here we show the presence of a large pool of stable carbon-centric free radicals in the nano-domains of the antennae and the wings of silk moth. This stable pool of carbon-centric free radicals is intrinsic in the nano-domains of these anatomical structures and responds to weak magnetic fields similar to that of Earth's (50 μT) even in the absence of light. Hence we are proposing that nocturnal animals in their navigation could utilize such a light independent mechanism. We further observed the presence of ferromagnetic elements (Fe, Ni, Co, Mn) in these structures. In conclusion, we have discussed how carbon centric free radicals along with other ferromagnetic components present in the antennae and the wings of the nocturnal silk moth, might help them to avoid the bats.

Hypoxia is one of the major stressors at high altitude. Exposure to hypobaric hypoxia induces several adverse consequences to the structural and functional integrity of brain. In an attempt to understand the proteome modulation, we used 2-DE coupled with MALDI-TOF/TOF for cortex and hippocampus exposed to short-term temporal (0, 3, 6, 12 and 24h) hypobaric hypoxia. This enabled us in the identification of 88 and 73 hypoxia responsive proteins in cortex and hippocampus respectively. We further compared the proteomes of both the regions and identified 37 common proteins along with 49 and 32 specific proteins for cortex and hippocampus respectively. We observed significant up-regulation of glycolytic enzymes like Gapdh, Pgam1, Eno1 and malate-aspartate shuttle enzymes Mdh1 and Got1in cortex as compared to hippocampus deciphering efficient use of energy producing substrates. This was coupled with concomitant increase in expression of antioxidant enzymes like Sod1, Sod2 and Pebp1 in cortex to neutralize the hypoxia-induced reactive oxygen species (ROS) generation. Our comparative proteomics studies demonstrate that efficient use of energy generating pathways in conjugation with abundance of antioxidant enzymes makes cortex less vulnerable to hypoxia than hippocampus.

Silk cocoon membrane (SCM), a solid matrix of protein fiber, responds to light, heat and moisture and converts these energies to electrical signals. Essentially it exhibits photo-electric and thermo-electric properties; making it a natural electro-magnetic sensor, which may influence the pupal development. This raises the question: ‘is it only electricity?’, or ‘it also posses some kind of magnetic memory?’ This work attempted to explore the magnetic memory of SCM and confirm its soft magnetism. Fe, Co, Ni, Mn, Gd were found in SCM, in traces, through energy dispersive X-ray analysis (EDX), X-ray photoelectron spectroscopy (XPS) and inductively coupled plasma mass spectrometry (ICP-MS). Presence of iron was ascertained by electron paramagnetic resonance (EPR). In addition, EPR-spectra showed the presence of a stable pool of carbon-centric free radical in the cocoon structure. Carbon-centric free radicals behaves as a soft magnet inherently. Magnetic-Hysteresis (M-H) of SCM confirmed its soft magnetism. It can be concluded that the soft bio-magnetic feature of SCM is due to the entrapment of ferromagnetic elements in a stable pool of carbon centric radicals occurring on the super-coiled protein structure. Natural soft magnets like SCM provide us with models for developing eco-friendly, protein-based biological soft magnets.

Continuous agricultural innovations are required to feed the exploding human population through natural or artificial resources. Though light is ample on earth, two-third of unavailable ocean and one-third of available soil are major limiting factors to free growth. Excessive fertilizer usage is irreversibly altering the chemical ecology of soil, further reducing the available area. Seed metabolism might be a potential answer to this resource crunch. Without genetic modification and thus maintaining the existing biodiversity, manipulation of seed metabolism at the very onset of germination is a sustainable alternative. The current work presents seed priming with iron pyrite (FeS2) prior to sowing as one such sustainable and innovative intervention to reduce fertilizer consumption in vegetable (beetroot, carrot), spice (fenugreek), fodder (alfalfa), and oilseed (mustard, sesamum) crops. A 12-h seed pretreatment in an aqueous suspension of nano-iron disulfide/pyrite (FeS2) resulted in significant yield increase in the above crops. While agriculturists aim to restore the natural genomic diversity of different domesticated crops, environmental engineers require technologies to reduce fertilizer consumption without compromising agricultural yields, thereby making the planet more sustainable. This nanoscale seed pretreatment approach using FeS2, otherwise a benign earth abundant mineral, suggests the sustainable opportunity to translate this technology to other crops thereby enhancing the global agricultural production.

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

Silkworm metamorphosis is governed by the intrinsic and extrinsic factors. One key intrinsic factor is the temporal electrical firing of the neuro-secretory cells of the dormant pupae residing inside the silk cocoon membrane (SCM). Extrinsic factors are environmental like temperature, humidity and light. The firing pattern of the cells is a function of the environmental factors that eventually controls the pupal development. How does the nervous organization of the dormant pupae sense the environment even while enclosed inside the cocoon shell? We propose that the SCM does this by capturing the incident light and converting it to electricity in addition to translating the variation in temperature and humidity as an electrical signal. The light to electricity conversion is more pronounced with ultraviolet (UV) frequency. We discovered that a UV sensitive fluorescent quercetin derivative that is present on the SCM and pupal body surface is responsible for generating the observed photo current. Based on these results, we propose an equivalent circuit model of the SCM where an overall electrical output transfers the weather information to pupae, directing its growth. We further discuss the implication of this electrical energy conversion and its utility for consumable electricity.

High altitude exposure results in decreased partial pressure of oxygen and increased formation of reactive oxygen and nitrogen species, which causes oxidative damage to lipids, proteins and DNA. Exposure to high altitude appears to decrease the activity and effectiveness of antioxidant enzyme system. The antioxidant system is limited in brain tissue and is very much susceptible to hypoxic stress. High metabolic rate along with a rich store of polyunsaturated fatty acids makes brain tissues a vulnerable target to oxidative damage. NAP peptide in particular has been reported to cross the blood–brain barrier and it improves cognitive functions and neuronal bioenergetics mechanisms. However, the therapeutic potential of NAP in hypoxic stress and the precise mechanism involved still remains unexplored. Our in vivo results show that the significant changes (antioxidant status of brain) were observed at 14 days hypoxic exposure. In order to monitor the proteins of rat brain cortex, adult Sprague Dwaly male rats were exposed to stimulated condition of high altitude (25000ft, 28±2°C) for 14 days alongside intranasal administration of NAP peptide (2µg/kg body weight/day). In this study, we identified 25 differentially expressed proteins which were antioxidant in nature and associated with energy metabolism.

An electrochemical highly sensitive nitrite (NO2 and superoxide anion radical (O •− 2 sensor was fabricated and developed based on an electrochemical deposition of manganese(III) tetrakis(1methyl-4-pyridyl)porphyrin (MnTMPyP) in polypyrrole (PPy) matrix on Pt electrode. The surface morphological image of MnTMPyP-PPy and PPy matrix on Pt electrode was obtained by scanning electron microscopy exhibiting microporous structure. The electrochemical behavior of the MnTMPyP-PPy-Pt electrode as sensor investigated by cyclic voltammetry revealed that the characteristic of MnTMPyP reversible redox peaks obtained at −0.12 V and −0.38 V versus Ag/AgCl respectively. The sensor electrode showed an excellent electrocatalytic nitrite oxidase and superoxide dismutase (SOD) activities. This electrochemical sensor exhibited a linear current response over the concentration range from 0.8 to 1000 M, with a detection limit of 0 8±0 03 M for NO2 and a corresponding linear range from 0.6 to 1000 M, with a detection limit of 0 6± 0 02 M for O•− 2 . In addition, the sensor displayed high sensitivity, good reproducibility and retained stability over a period of ∼4 weeks. This SOD-mimetic electrode was proved to be effective not only in detecting NO2 and O •− 2 independently but also in determining the concentrations of NO − 2 and O •− 2 simultaneously in in vitro systems.

Cordyceps sinensis is a traditional Chinese medicine used for promotion of health, longevity and athletic power. However, the molecular mechanism for anti-fatigue activity and physical fitness has not yet been reported.The present study was conducted to evaluate the exercise endurance promoting activities of fungal traditional Chinese medicine (FTCM) Cordyceps sinensis cultured whole mycelium (CS) and the underlying mechanisms.CS was orally supplemented (200mg/kg body weight/day) to rats for 15days with or without swimming exercise along with exercise and placebo groups.Both CS supplementation and supplementation concurrent with exercise improved exercise endurance by 1.79- (P0.05) and 2.9-fold (P0.01) respectively as compared to placebo rats. CS supplementation concurrent with exercise also increased the swimming endurance by 1.32-fold (P0.05) over the exercise group. To study the molecular mechanism of the observed effect, we measured the expression levels of endurance responsive skeletal muscle metabolic regulators AMPK, PGC-1α and PPAR-δ as well as endurance promoting and antioxidant genes like MCT1, MCT4, GLUT4, VEGF, NRF-2, SOD1 and TRX in red gastrocnemius muscle. Our results indicate that CS supplementation significantly upregulates the skeletal muscle metabolic regulators, angiogenesis, better glucose and lactate uptake both in exercised and non-exercised rats. We have also observed increased expression of oxidative stress responsive transcription factor NRF-2 and its downstream targets SOD1 and TRX by CS supplementation.CS supplementation with or without exercise improves exercise endurance capacity by activating the skeletal muscle metabolic regulators and a coordinated antioxidant response. Consequently, CS can be used as a potent natural exercise mimetic.

Despite chickpea being the third important grain legume, there is a limited availability of genomic resources, especially of the expressed sequence tag (EST)-based markers. In this study, we generated 822 chickpea ESTs from immature seeds as well as exploited 1,309 ESTs from the chickpea database, thus utilizing a total of 2,131 EST sequences for development of functional EST-SSR markers. Two hundred and forty-six simple sequence repeat (SSR) motifs were identified from which 183 primer pairs were designed and 60 validated as functional markers. Genetic diversity analysis across 30 chickpea accessions revealed ten markers to be polymorphic producing a total of 29 alleles and an observed heterozygosity average of 0.16 thereby exhibiting low levels of intra-specific polymorphism. However, the markers exhibited high cross-species transferability ranging from 68.3 to 96.6% across the six annual Cicer species and from 29.4 to 61.7% across the seven legume genera. Sequence analysis of size variant amplicons from various species revealed that size polymorphism was due to multiple events such as copy number variation, point mutations and insertions/deletions in the microsatellite repeat as well as in the flanking regions. Interestingly, a wide prevalence of crossability-group-specific sequence variations were observed among Cicer species that were phylogenetically informative. The neighbor joining dendrogram clearly separated the chickpea cultivars from the wild Cicer and validated the proximity of C. judaicum with C. pinnatifidum. Hence, this study for the first time provides an insight into the distribution of SSRs in the chickpea transcribed regions and also demonstrates the development and utilization of genic-SSRs. In addition to proving their suitability for genetic diversity analysis, their high rates of transferability also proved their potential for comparative genomic studies and for following gene introgressions and evolution in wild species, which constitute the valuable secondary genepool in chickpea.

Catharanthus roseus (L.) G. Don, commonly known as Madagascar periwinkle, is a plant of great medicinal value. In this study microsatellite markers were developed and utilized for analysis of genetic diversity in C. roseus. In order to isolate microsatellite sequences, two small insert genomic libraries of C. roseus cv. Nirmal were constructed and screened with CA, CT, GC and GCG oligonucleotide repeats. Sixty-five microsatellite motifs were identified, from which 38 functional STMS primer pairs were designed and validated. Out of these, 24 STMS markers were used to evaluate the genetic polymorphism in 37 genotypes, which comprised of 32 accessions of C. roseus, a single accession each of two related species (C. trichophyllus and C. pusillus) and one accession each of three related genera (Vinca minor, Thevetia peruviana and Nerium indicum). The 24 STMS markers detected 26 loci with two markers amplifying more than one locus. A total of 124 alleles were amplified in the 37 genotypes ranging from 2 to 10 alleles with an average of 4.76 alleles per locus. The high average expected heterozygosity (HE) value of 0.56 and observedheterozygosity(HO)valueof0.52establishedtheefficiencyoftheSTMSmarkersfordiscriminatingthe C. roseusgenotypes.NeiandLi's similarity coefficients were calculated and a UPGMA-based dendrogram was constructed which clearly distinguished all genotypes except two pairs. Sequence analysis of the length variant alleles at three STMS loci revealed that the variation in the copy number of repeat motifs was the major source of length polymorphism within C. roseus. However, isolated point mutations and indels in the microsatellite flanking regions (MFRs) of homologus loci from other species also contributed to size homoplasy and allelic size variation. This study is the first report of microsatellite development and utilization in C. roseus, providing significant insights into its genome structure and organization. # 2006 Elsevier Ireland Ltd. All rights reserved.

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

Hypoxia is a well-known threat to neuronal cells and triggers the pathophysiological syndromes in extreme environments such as high altitudes and traumatic conditions such as stroke. Among several prophylactic molecules proven suitable for ameliorating free radical damage, NAP (an octapeptide with initial amino acids: asparagine/N, alanine/A, and proline/P) can be considered superlative, primarily due to its high permeability into brain through blood-brain barrier and observed activity at femtomolar concentrations. Several mechanisms of action of NAP have been hypothesized for its protective role during hypoxia, yet any distinct mechanism is unknown. Oxidative stress is advocated as the leading event in hypoxia; we, therefore, investigated the regulation of key antioxidant genes to understand the regulatory role of NAP in providing neuroprotection. Primary neuronal culture of rat was subjected to cellular hypoxia by limiting the oxygen concentration to 0.5% for 72 h and observing the prophylactic efficacies of 15fM NAP by conventional cell death assays using flow cytometry. We performed real-time quantitative polymerase chain reaction to comprehend the regulatory mechanism. Further, we validated the significantly regulated candidates by enzyme assays and immunoblotting. In the present study, we report that NAP regulates a major clad of cellular antioxidants and there is an involvement of more than one route of action in neuroprotection during hypoxia.

This chapter presents the definition and brief history of enzymatic biosensors and biomarkers, their types, and measurement for diagnosis of diseases. The glucose biosensors including noninvasive and implantable sensors for diabetes; cholesterol biosensors are also described. The enzymatic biosensors for oxidative stress biomarkers, viz., superoxide, thiol, nitric oxide, and its metabolites, viz., NO2- and NO3-, are discussed. In addition, the development of biosensors for cytochrome c using cytochrome oxidase and reductase for the measurement of cytochrome c release from mitochondria, and thereby apoptosis in cancer cells, is demonstrated. Simultaneous measurement of biomarkers and bienzymatic and enzyme-inhibition-based biosensors are also presented. Because of high cost and less stability of the enzymes, the enzyme mimetics, especially metalloporphyrin-based biosensors, are highlighted. Advantages of screen-printed functionalized electrodes and nanocomposite-based electrochemical biosensors are also described. The characterization, optimization, interferences and their elimination, and the recent applications of electrochemical biosensors are discussed.

'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.

Oxidative stress is a condition when the concentration of free radicals and reactive molecular species rise above certain level in living systems. This condition not only perturbs the normal physiology of the system but also has been implicated in many diseases in humans and other animals. Hydrogen peroxide (H2O2) is known to be involved in induction of oxidative stress and has also been linked to a variety of ailments such as inflammation, rheumatoid arthritis, diabetes, and cancer in humans. It is one of the more stable reactive molecular species present in living systems. Because of its stability and links with various diseases, sensing the level of H2O2 can be of great help in diagnosing these diseases, thereby easing disease management and amelioration. Nanoceria is a potent candidate in free radical scavenging as well as sensing because of its unique redox properties. These properties have been exploited, in the reported work, to sense and quantify peroxide levels. Nanoceria has been synthesized using different capping agents: Hexamethylene-tetra-amine (HMTA) and fructose. CeO2-HMTA show rhombohedral and cubic 6.4 nm particles whereas CeO2-fructose are found to be spherical with average particle diameter size 5.8 nm. CeO2-HMTA, due to the better exposure of the active (200) and (220) planes relative to (111) plane, exhibits superior electrocatalytic activity toward H2O2 reduction. Amperometric responses were measured by increasing H2O2 concentration. The authors observed a sensitivity of 21.13 and 9.6 μA cm(-2) mM(-1) for CeO2-HMTA and CeO2-fructose, respectively. The response time of 4.8 and 6.5 s was observed for CeO2-HMTA and CeO2-fructose, respectively. The limit of detection is as low as 0.6 and 2.0 μM at S/N ratio 3 for CeO2-HMTA and CeO2-fructose, respectively. Ceria-HMTA was further tested for its antioxidant activity in an animal cell line in vitro and the results confirmed its activity.

This work presents a novel electrochemical assay for the collective measurement of nitric oxide (NO) and its metabolites nitrite (NO 2 − ) and nitrate (NO 3 − ) 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, NO 2 − measurement showed linearity of 100 nM to 1 mM with a detection limit of 100 nM for NO 2 − and sensitivity of 96.4 nA μM −1 . Then, the concentration of NO 3 − was measured after its enzymatic conversion into NO 2 − . Using this assay, the concentrations of NO, NO 2 − , and NO 3 − 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.

Silk cocoon membrane (SCM) is an insect engineered structure. We studied the electrical properties of mulberry (Bombyx mori) and non-mulberry (Tussar, Antheraea mylitta) SCM. When dry, SCM behaves like an insulator. On absorbing moisture, it generates electrical current, which is modulated by temperature. The current flowing across the SCM is possibly ionic and protonic in nature. We exploited the electrical properties of SCM to develop simple energy harvesting devices, which could operate low power electronic systems. Based on our findings, we propose that the temperature and humidity dependent electrical properties of the SCM could find applications in battery technology, bio-sensor, humidity sensor, steam engines and waste heat management.