Identification and differential expression of serotransferrin and apolipoprotein A-I in the plasma of HIV-1 patients treated with first-line antiretroviral therapy.

BACKGROUND: Plasma proteins are known to interfere the drug metabolism during therapy. As limited information is available regarding the role of plasma proteins in HIV drug resistance during ART in HIV/AIDS patients, the present study aimed to identify and characterize the differentially expressed plasma proteins in the drug resistant and drug respondent groups of HIV-1 infected patients with > 6 years of first line ART. METHODS: Four-drug resistant (treatment failure) and four-drug respondent (treatment responder) patients were selected for plasma proteomic analysis based on viral load and drug resistance associated mutations from a cohort study designed on the first line ART patients who were enrolled in the antiretroviral therapy center, Sarojini Naidu Medical College, Agra, India from December 2009 to November 2016. After depleting high abundant proteins, plasma proteins were resolved using two-dimensional gel electrophoresis on IPG strips, pH range of 3-10. Spots were selected in the gel based on the density of staining which was common in the drug resistant and drug respondent groups separately. The fold change of each spot was calculated using image-J. Each protein spot was identified using the matrix assisted laser desorption/ionization-time of flight/time of flight (MALDI-TOF/TOF) after tryptic digestion. Peptide peaks were identified through flex analysis version 3.3, and a search against a protein data base using the internal Mascot. Gene ontology study was completed through STRING v.11 and Panther15.0. RESULTS: Out of eight spots from 2D gel samples analyzed by MALDITOF/TOF, two proteins were found to have significant score (> 56) after Flex analysis. These two proteins were identified to be apolipoprotein A1 and serotransferrin. The fold change expression of these two proteins were analyzed in drug resistant and drug respondent group. Apolipoprotein-A1 and serotransferrin were observed to be expressed 1.76 and 1.13-fold more respectively in drug respondent group compared to drug resistant group. The gene ontology analysis revealed the involvement of these two proteins in various important physiological processes. CONCLUSION: Apolipoprotein A-I and serotransferrin were found to be expressed more in drug respondent group compared to drug resistant group.

Effect of persistent and transient hypothyroidism on histoarchitecture and antioxidant defence system in rat brain.

The present study reports the effect of 6-n-propylthiouracil (PTU)-induced hypothyroidism on oxidative stress parameter, lipid peroxidation (LPx) and major antioxidant enzyme expressions such as superoxide dismutase (SOD: SOD1 and SOD2) and catalase (CAT) in cerebral cortex rat brain during postnatal persistent (90 days PTU treatment from birth) and transient (30 days PTU treatment from birth followed by PTU withdrawal for 60 days) hypothyroidism. Enhanced level of LPx was observed in transient hypothyroid rats with respect to control and persistent hypothyroid rats. Significantly increased activity of SOD and expression of SOD1 were observed in cerebral cortex of both persistent and transient hypothyroid rats as compared to control. However, unaltered translated level of SOD2 was observed among the groups. Activity of CAT was increased in transient hypothyroid rats, whereas translate level of CAT was increased in both the regions of persistent as well as transient hypothyroidism. The histoarchitecture of cerebral cortex clearly showed a decline in neuronal migration with neurons packed together in both persistent and transient hypothyroid rats as compared to control. These results suggest that deprivation of thyroid hormone modulates redox status and causes oxidative stress in rat brain cerebral cortex during postnatal development and maturation.

Hypothyroidism alters antioxidant defence system in rat brainstem during postnatal development and adulthood.

The present investigation was carried out to evaluate alterations in oxidative stress parameter [lipid peroxidation (LPx)] and antioxidant enzyme activities [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] in rat brainstem in response to neonatal hypothyroidism during development (from birth to 7, 15 and 30 days old) and adulthood (90 days old). Hypothyroidism in rats was induced by feeding the lactating mothers (from the day of parturition till weaning, 25 days old) or directly to the pups with 0.05 % [6-n-propyl 2-thiouracil (PTU)] in drinking water. Increased level of LPx was observed in brainstem of 7 days old hypothyroid rats, accompanied by augmented activities of SOD and GPx. In 15 and 30 days old hypothyroid rat brainstem, a significant decline in LPx was observed. Significantly increased activities of CAT and GPx were observed in 15 and 30 days PTU-treated rats. Decreased level of LPx was observed in brainstem of rats treated with PTU from birth to 30 days followed by withdrawal up to 90 days of age (transient hypothyroidism) as compared to control and persistent treatment of PTU up to 90 days of age. Activities of CAT and GPx were decreased in persistent hypothyroid rats of 90 days old with respect to control and transient hypothyroid rats. On the other hand, SOD activity was decreased in both persistent and transient hypothyroid rats with respect to control rats. These results suggest that the PTU-induced neonatal hypothyroidism modulates the antioxidant defence system during postnatal development and adulthood in brainstem of rats.

Modulation of antioxidant enzyme expression by PTU-induced hypothyroidism in cerebral cortex of postnatal rat brain.

This study aimed to elucidate the effect of 6-n-propylthiouracil (PTU)-induced hypothyroidism on oxidative stress parameters and expression of antioxidant enzymes in cerebral cortex of rat brain during postnatal development. A significant decrease in levels of lipid peroxidation and H(2)O(2) were seen in 7 and 30 days old PTU-treated rats with respect to their controls. Significantly decreased activities of superoxide dismutase (SOD) and catalase (CAT) along with the translated products of SOD1 and SOD2 were observed in 7, 15 and 30 days old PTU-treated rats as compared to their respective controls. However, increase in translated product of CAT was seen in all age groups of PTU-treated rats. Glutathione peroxidase activity was decreased in 7 days and increased in 15 days old PTU-treated rats with respect to their control groups. Histological sections clearly show a decline in neuronal migration with neurons packed together in the hypothyroid group as compared to the control.

Curcumin differentially regulates the expression of superoxide dismutase in cerebral cortex and cerebellum of L-thyroxine (T4)-induced hyperthyroid rat brain.

The present investigation was aimed to elucidate the effect of curcumin on lipid peroxidation (LPx) and superoxide dismutase (SOD) in L-thyroxine (T4)-induced oxidative stress in cerebral cortex and cerebellum of rat brain. Elevated level of LPx in cerebral cortex declined to control level on supplementation of curcumin to T4-treated rats. On the other hand, unaltered LPx level in T4-treated rats showed a significantly decreased level of LPx on supplementation of curcumin. The increased activity of SOD and translated products of SOD1 and SOD2 in cerebral cortex of T4-treated rats was ameliorated on supplementation of curcumin. The decreased activity of SOD and protein expression of SOD1 in cerebellum of T4-treated rats were ameliorated on administration of curcumin. On the other hand, SOD2 expression was not influenced either by T4-treated or by curcumin supplementation to T4-treated rats. Results of the present investigation reveal that the regulation of expression of SOD by curcumin in different regions (cerebral cortex and cerebellum) of rat brain is different under hyperthyroidism.

SWATH-MS analysis of plasma proteins among Indian HIV-1 infected patients.

The identification and characterization of plasma proteins in drug resistant and drug sensitive in HIV-1 infected/AIDS patients were carried out using the SWATH-MS protocol. In total, 204 proteins were identified and quantified, 57 proteins were differentially expressed, out of which 25 proteins were down regulated and 32 proteins were up regulated in drug resistant patients. Six proteins such as complement C4-A, immunoglobulin heavy variable 1-2, carboxylic ester hydrolase, fibulin-1, immunoglobulin lambda constant7, secreted phosphoprotein 24 were differentially expressed in individuals with drug resistant HIV as compared to individuals with drug sensitive HIV. Gene ontology of 57 differentially expressed proteins was analysed and documented.

Expression of antioxidant genes in renal cortex of PTU-induced hypothyroid rats: effect of vitamin E and curcumin.

The present study was undertaken to investigate the effect of vitamin E and curcumin on the expression of antioxidant genes in 6-propyl-2-thiouracil (PTU)-induced hypothyroid rat renal cortex. The levels of lipid peroxidation and protein carbonylation were increased in hypothyroid rat kidney. Co-administration of vitamin E and curcumin to hypothyroid rats resulted in amelioration of lipid peroxidation level, whereas curcumin alone alleviated the protein carbonylation level. The mRNA levels of SOD1 and SOD2 were decreased in hypothyroid rats. Decreased level of SOD1 transcripts was observed in hypothyroid rats supplemented with curcumin alone or co-administrated with vitamin E. Translated products of SOD1 and SOD2 in hypothyroid rats was elevated in response to supplementation of both the antioxidants. Decreased SOD1 and SOD2 activities in hypothyroid rats compared to control were either unaltered or further decreased in response to the antioxidants. Expressions of CAT at transcript and translate level along with its activity were down regulated in hypothyroid rats. Administration of vitamin E to hypothyroid rats resulted in elevated CAT mRNA level. In contrast, expression of CAT protein was elevated in response to both the antioxidants. However, CAT activity was unaltered in response to vitamin E and curcumin. GPx1 and GR mRNA level and the activity of glutathione peroxidase (GPx) were not affected in response to induced hypothyroidism. The activity of GPx was increased in response to vitamin E treatment, whereas decreased GR activity in hypothyroid rats was further declined by the administration of antioxidants. The over all results suggest that vitamin E and curcumin differentially modulate the altered antioxidant defence mechanism of rat kidney cortex under experimental hypothyroidism.

Hypothyroidism modulates renal antioxidant gene expression during postnatal development and maturation in rat.

In the present study effects of 6-n-propyl thiouracil (PTU)-induced hypothyroidism on renal antioxidant defence system during postnatal development (from birth to 7, 15 and 30days old) and on adult rats were reported. Hypothyroidism in rats was induced by feeding the lactating mothers (from the day of parturition till weaning, 25days old) or directly to the pups with 0.05% PTU in drinking water. The activities of Cu/Zn-superoxide dismutase (SOD1) and glutathione peroxidase (GPx) were increased in 30days old hypothyroid rats with respect to their respective controls, on the other hand, levels of translated products and activities of Mn-superoxide dismutase (SOD2) and catalase (CAT) were decreased in hypothyroid rats of all age groups as compared to their respective control rats. SOD1 activity remained unchanged in persistent (PTU-treatment from birth to 90days old) hypothyroid rats as compared to euthyroid. However, a decreased activity of SOD1 was recorded in transient (PTU-treatment from birth to 30days then withdrawal till 90days old) hypothyroid rats with respect to control rats. The mRNA level, protein expression and activity of SOD2 and CAT were significantly decreased in persistent hypothyroid rats as compared to euthyroid rats. The activity of GPx was significantly increased in both persistent and transient hypothyroid rats with respect to euthyroid rats. The present study indicates modulation of antioxidant defence status of rat kidney during postnatal development and maturation by hypothyroidism.

Induction of oxidative stress and inhibition of superoxide dismutase expression in rat cerebral cortex and cerebellum by PTU-induced hypothyroidism and its reversal by curcumin.

The present study was carried out to elucidate the effectiveness of curcumin in ameliorating the expression of superoxide dismutase (SOD) in cerebral cortex and cerebellum of rat brain under 6-propyl-2-thiouracil (PTU)-induced hypothyroidism. Induction of hypothyroidism in adult rats by PTU resulted in augmentation of lipid peroxidation (LPx), an index of oxidative stress in cerebellum but not in cerebral cortex. Curcumin-supplementation to PTU-treated (hypothyroid) rats showed significant reduction in the level of LPx in both the regions of brain. The decreased translated products (SOD1 and SOD2) and the unchanged activity of SOD in cerebral cortex of PTU-treated rats were increased on supplementation of curcumin to the hypothyroid rats. Declined translated products of SOD1 and SOD2 in cerebellum of PTU-treated rats were alleviated on administration of curcumin to hypothyroid rats. On the other hand, the decreased activity of SOD in cerebellum of PTU-treated rats was further declined on administration of curcumin to the hypothyroid rats. Results of the present investigation indicate that curcumin differentially modulates the expression of superoxide dismutase in rat brain cortex and cerebellum under PTU-induced hypothyroidism.

Genomic signatures of protease and reverse transcriptase genes from HIV-1 subtype C isolated from first-line ART patients in India.

Genomic signatures of the protease and reverse transcriptase gene of HIV-1 from HIV infected North Indian patients who were under ART from 1 to ≤ 7 years were analyzed. The DNA from plasma samples of 9 patients and RNA from 57 patients were isolated and subjected to amplification for the protease and reverse transcriptase gene of HIV-1 subtype C. Then sequencing was carried out following the WHO dried blood spot protocol. The drug resistance mutation patterns were analyzed using the HIV Drug Resistance Database, Stanford University, USA. Lamivudine-associated drug-resistance mutations such as M184V/M184I, nevirapine-associated drug resistance mutations Y181C and H221Y, and efavirenz-associated drug resistance mutations M230I were observed in reverse transcriptase gene of archived DNA of two HIV-1 infected patients. No mutation was observed in the remaining 7 patients. Various computational tools and websites like viral epidemiological signature pattern analysis (VESPA), hyper mutation, SNAP version 2.1.1, and entropy were utilized for the analysis of the signature pattern of amino acids, hyper mutation, selection pressure, and Shannon entropy in the protease and reverse transcriptase gene sequences of the 9 archived DNA, 56 protease gene and 51 reverse transcriptase gene from the HIV-1 DNA amplified sequences of RNA. The HIV-1 Subtype-C (Gene bank accession number: AB023804) and first isolate HXB2 (Gene bank accession number: K03455.1) was taken as reference sequence. The signature amino acid sequences were identified in the protease and reverse transcriptase gene, no hyper mutation, highest entropy was marked in the amino acid positions and synonymous to non-synonymous nucleotide ratio was calculated in the protease and reverse transcriptase gene of 9 archived DNA sequences, 56 protease and 51 reverse transcriptase gene sequences of HIV-1 Subtype C isolates.

Regulation of expression of antioxidant enzymes by vitamin E and curcumin in L-thyroxine-induced oxidative stress in rat renal cortex.

The present study investigates the antioxidative effects of vitamin E and curcumin against L-thyroxine (T(4))-induced oxidative stress in renal cortex of adult male rats. Rats were made hyperthyroid by administration of L-thyroxine (0.0012%) in their drinking water for 30 days. Vitamin E (200 mg/kg body weight/day) and curcumin (30 mg/kg body weight/day) were supplemented singly or in combination orally for 30 days along with L-thyroxine treatment. The elevated level of oxidative stress parameters (lipid peroxidation and protein carbonylation) and decline level of small antioxidant molecules (reduced glutathione and ascorbic acid) in renal cortex of T(4)-treated rats were restored back by supplementation of vitamin E or/and curcumin. Increased superoxide dismutase and catalase activities in kidney cortex of T(4)-treated rats were ameliorated in response to vitamin E or/and curcumin treatment. The elevated translated product of Cu/Zn-SOD, Mn-SOD and catalase in T(4)-treated rats were differentially reduced by the administration of vitamin E and curcumin independently or in combination. Cu/Zn-SOD expression was ameliorated by both vitamin E and curcumin independently or in combination, whereas Mn-SOD expression was ameliorated by the supplementation of vitamin E or curcumin independently. However, the expression of catalase was alleviated by only supplementation of vitamin E to T(4)-treated rats. The results suggest that both vitamin E and curcumin may play an important role in protecting T(4)-induced oxidative stress in rat renal cortex by differentially modulating the activities of antioxidant enzymes and oxidative stress parameters.

Nano-Neurotheranostics: Impact of Nanoparticles on Neural Dysfunctions and Strategies to Reduce Toxicity for Improved Efficacy.

Nanotheranostics is one of the emerging research areas in the field of nanobiotechnology offering exciting promises for diagnosis, bio-separation, imaging mechanisms, hyperthermia, phototherapy, chemotherapy, drug delivery, gene delivery, among other uses. The major criteria for any nanotheranostic-materials is 1) to interact with proteins and cells without meddling with their basic activities, 2) to maintain their physical properties after surface modifications and 3) must be nontoxic. One of the challenging targets for nanotheranostics is the nervous system with major hindrances from the neurovascular units, the functional units of blood-brain barrier. As blood-brain barrier is crucial for protecting the CNS from toxins and metabolic fluctuations, most of the synthetic nanomaterials cannot pass through this barrier making it difficult for diagnosing or targeting the cells. Biodegradable nanoparticles show a promising role in this aspect. Certain neural pathologies have compromised barrier creating a path for most of the nanoparticles to enter into the cells. However, such carriers may pose a risk of side effects to non-neural tissues and their toxicity needs to be elucidated at preclinical levels. This article reviews about the different types of nanotheranostic strategies applied in nervous dysfunctions. Further, the side effects of these carriers are reviewed and appropriate methods to test the toxicity of such nano-carriers are suggested to improve the effectiveness of nano-carrier based diagnosis and treatments.

Detection of Drug Resistance Mutations in the Reverse Transcriptase Gene of HIV-1-Infected North Indian Population Failing First-Line Antiretroviral Therapy "A Follow-Up Cohort Study".

We aim to characterize the drug resistance mutations in reverse transcriptase gene of HIV-1 subtype C-infected North Indian population in those who are failing first-line antiretroviral therapy (ART) and if these mutations are associated with mortality. We also attempted the assessment of switch over to second-line antiretroviral therapy in these patients. Based on the immunological marker CD4 count (<350 cubic/mm), 192 HIV/AIDS patients were selected and viral load was estimated in those who were enrolled from December 2009 to November 2016. Based on viral load, genotyping was carried out in 57 HIV-1 isolates (VL ≥1,000 copies/mL) by sequencing and drug resistance mutations were examined through the Stanford HIV Drug Resistance Database, USA. Among them, 21 (36.84%) first-line ART failure patients were shifted to second-line ART. These patients were followed for a period wide ranging from 10 months to 11 years. Drug resistance mutation M184V (ATG to GTA) (63.15%) associated with lamivudine and abacavir and K103N (AAG or AAA to AAU) (36.84%) associated with efavirenz and nevirapine were predominantly identified in first-line ART failure patients. During follow-up, it was observed that 3 out of 21 who were in second-line ART died, whereas 9 out of 36 died who were in the first-line ART. No mutation could be associated with mortality although TAM-2 mutations were absent in patients who died. This study indorses the need for a facility for viral load estimation and resistance monitoring in each treatment failure patient and availability of appropriate antiretroviral therapies.

Effect of Xylocarpus granatum Bark Extract on Amelioration of Hyperglycaemia and Oxidative Stress Associated Complications in STZ-Induced Diabetic Mice.

Xylocarpus granatum is a medicinal mangrove plant, traditionally used for the treatment of diarrhoea, cholera, fever, dyslipidaemia, inflammation, etc. The present study was aimed to evaluate the in vitro antidiabetic (α-glucosidase inhibition assay) and antioxidant (ABTS scavenging and metal chelating assay) activities of ethanol, methanol, and aqueous extracts of leaves and barks of X. granatum followed by in vivo antidiabetic and antioxidant evaluation of ethanol bark extracts in streptozotocin- (STZ-) induced diabetic mice. The in vitro evaluation revealed higher α-amylase inhibition and ABTS scavenging activities in ethanol bark extracts of X. granatum (XGEB). Administration of XGEB at 100 and 200 mg/kg BW doses to STZ-induced diabetic mice resulted in significant decrease (P < 0.05) in blood glucose, triglyceride (TG), total cholesterol (TC), serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transminase (SGPT), and urea levels in the serum of the extract administered groups as compared to diabetic control group. The levels of SOD, CAT, GPx, GR, and GST in liver along with LPx, SOD, GST, and GR activities in brain tissues were found to be ameliorated in XGEB treated diabetic mice. Histopathological alternations of liver tissues were also found to be restored in XGEB treated diabetic groups. The HPLC fingerprint analysis of XGEB revealed the presence of simple polyphenols, isoflavone, and flavonol-like compounds. The DSC and UV-VIS analysis also confirmed the presence of phenolic compounds in XGEB. The GC-MS analysis of XGEB showed the presence of a number of bioactive compounds. These results demonstrated the beneficial effect of XGEB in controlling hyperglycaemia and ameliorating oxidative stress associated complications associated with diabetes.

Possible activation of NRF2 by Vitamin E/Curcumin against altered thyroid hormone induced oxidative stress via NFĸB/AKT/mTOR/KEAP1 signalling in rat heart.

Oxidative stress is implicated in both hypo- and hyper-thyroid conditions. In the present study an attempt has been made to elucidate possible interaction between vitamin E or/and curcumin (two established antioxidants) with active portion (redox signaling intervening region) of nuclear factor erythroid 2-related factor 2 (NRF2) as a mechanism to alleviate oxidative stress in rat heart under altered thyroid states. Fifty Wistar strain rats were divided into two clusters (Cluster A: hypothyroidism; Cluster B: hyperthyroidism). The hypo- (0.05% (w/v) propylthiouracil in drinking water) and hyper- (0.0012% (w/v) T4 in drinking water) thyroid rats in both clusters were supplemented orally with antioxidants (vitamin E or/and curcumin) for 30 days. Interactive least count difference and principal component analyses indicated increase in lipid peroxidation, reduced glutathione level, alteration in the activities and protein expression of antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase under altered thyroid states. However, the expression of stress survival molecules; nuclear factor κB (NFκB) and the serine-threonine kinase B (Akt), in hyper-thyroidism only points towards different mechanisms responsible for either condition. Co-administration of vitamin E and curcumin showed better result in attenuating expression of mammalian target for rapamycin (mTOR), restoration of total protein content and biological activity of Ca2+ ATPase in hyperthyroid rats, whereas, their individual treatment showed partial restoration. Since NRF2 is responsible for activation of antioxidant response element and subsequent expression of antioxidant enzymes, possible interactions of both vitamin E or/and curcumin with the antioxidant enzymes, NRF2 and its regulator Kelch ECH associating protein (KEAP1) were studied in silico. For the first time, a modeled active portion of the zipped protein NRF2 indicated its interaction with both vitamin E and curcumin. Further, curcumin and vitamin E complex showed in silico interaction with KEAP1. Reduction of oxidative stress by curcumin and/or vitamin E may be due to modulation of NRF2 and KEAP1 function in rat heart under altered thyroid states.

Effect of methylene blue on oxidative stress and antioxidant defence parameters of rat hepatic and renal tissues.

The objective of the present study was to investigate the effect of administration of 1 mM methylene blue (MB) in drinking water for 30 days on hepatic and renal antioxidant status in female adult Wistar strain rats (n = 5). MB failed to induce significant change in any of the measured antioxidant defence parameters namely, superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH). However, a marginally significant (P < 0.05) increase in the level of lipid peroxidation (LPx) was recorded in liver, while a reduction (P < 0.05) in its level in the kidney was noticed. Serum alanine amino transferase (AlaAT) and creatinine levels significantly (P < 0.001) decreased in MB treated rats without any change in blood urea nitrogen (BUN) level. Our findings suggest that the effect of MB as administered in the present study was tissue specific with regard to the level of LPx, however, in general, it does not impair liver and kidney functions as evidenced by serum parameters.