Prediction of drug-resistance using genotypic and docking analysis among anti-retroviral therapy naïve and first-line treatment failures in Salem, Tamil Nadu, India.

The emergence of drug resistance among HIV-positive patients undergoing Anti- Retroviral Therapy (ART) with poor adherence to the HAART is a major concern in India. As the HIV accumulates the key mutations, the drug resistance occurs, that pose challenges to the ART regimens currently being used. Thus, the present study was carried out among the ART- naïve patients attending ART Centre at Salem district, Tamil Nadu, India. The mutations that concern the drug resistance were discriminated by determining the viral load before and after 6 months. The drug resistance was analyzed by HIV genotyping from the patients possessing a viral load of >1000 copies/mL after 6 months of ART. The mutations pertaining to drug resistance were analyzed by the online Stanford HIV Database. The 3D structures of the RT were modeled and the drugs used in the first-line regimens were docked to explore the effect of mutations on the binding pattern of the drugs. Among the 250 patients, the viral load data were obtained for 213 patients. The study found 23 patients with both virological and immunological failures and HIV drug mutations were also revealed by genotyping. The mutations of I135R/T/V/X, L178 I/M, M184V/I, D67N, K70R, and K103N were the most common among these 23 patients. The present study revealed that the NACO recommended first-line ART regimen is efficient in most of the patients attending ART center. The emergence of drug resistance of HIV variant is common even under the best circumstance of ART. This study reveals that there is a necessity for the implementation of improved and economically systematic attempt that allows the clinicians to make a rational choice of therapy regimen to overcome the first-line therapy failures among the ART- naive.

Development and characterization of a continuous macrophage cell line, LRTM, derived from thymus of Labeo rohita (Hamilton 1822).

A long-term thymic macrophage cell line from the thymus explants of Labeo rohita designated as LRTM (L. rohita thymic macrophages) was established, which has been maintained in culture for more than 1 yr. This cell line designated LRTM cells have been subcultured for 70 passages. The cells shape was initially long and elongated; with subsequent passages, the cells became short and epithelial like. The cells exhibited optimum growth in L-15 containing 10% fetal bovine serum and also in Dulbecco's modified Eagle's medium at 37°C with 5% CO2 and showed 85+-% viability after 12 mo storage in liquid nitrogen. In addition, cells showed nonspecific esterase and surface expression of Fc receptors for immunoglobulin G and classes I and II major histocompatibility complex antigens. These observations confirmed that this cell line had the morphologic and functional features as a macrophage. The cells exhibited phagocytic activity by engulfing yeast cells as well as fluorescent latex beads, which was demonstrated by scanning electron microscopy and Giemsa staining. The long-term cultured cells show rapid production of reactive oxygen and nitrogen intermediates following stimulation with lipopolysaccharides and phorbol miristate acetate (PMA). Mostly, all the cells were alpha napthyl esterase acetate positive. After stimulation with PMA and lipopolysaccharide, cultured fish macrophages produced reactive oxygen and nitrogen intermediates. The karyotype analysis showed that these cells have a tetraploid karyotype with 100 chromosomes in each cell, indicating that they are normal L. rohita cells. Amplification, sequencing, and alignment of fragments of two mitochondrial genes 12S rRNA from rohu confirmed that the cell line originated from L. rohita. This cell line should be useful for studying the role of thymic macrophages in differentiation and maturation of thymocytes and can be source of macrophage-specific enzymes and cytokines. The macrophage cell line will be invaluable in studies of pathogen/macrophage interactions, the mechanisms of macrophage antimicrobial effector functions and the contribution of macrophages to the specific immune responses of teleosts.

Studies on the cardioprotective role of gallic acid against AGE-induced cell proliferation and oxidative stress in H9C2 (2-1) cells.

Epidemiological studies have shown that high glucose levels and oxidative stress cause elevation of advanced glycation end products (AGEs) that are known to contribute to diabetic complications. Thus, agents that hamper reactive oxygen species (ROS) load can be used as a potential drug against AGEs-mediated complications. Hence, the present study investigated the protective role of gallic acid (GA) against the effects of AGEs in cardiac H9C2(2-1) cells. Exposure of cells to AGEs resulted in release of ROS (P < 0.05) with significant (P < 0.05) decline in antioxidant enzyme levels and increase in collagen (P < 0.01) content. In addition, the altered mitochondrial membrane potential (mmp) (P < 0.01) was also observed in cells exposed to AGEs, whereas AGEs-exposed cells pretreated with GA prevented the release of ROS, and there were no significant changes in the antioxidant status, collagen content and mmp. Thus, the results of the present study provide evidence that GA exhibits protective role against AGEs-induced cardiovascular complications probably through its free radical scavenging activity.

Postnatal growth, age estimation and development of foraging behaviour in the fulvous fruit bat Rousettus leschenaulti.

This study documents the postnatal growth, age estimation and development of the foraging behaviour of the fulvous fruit bat Rousettus leschenaulti under captive conditions. At birth, the young were naked and pink with closed eyes and folded pinnae. By day four of age, their eyes had opened and the pups began to move. The mean length of forearm in 5-day-old pups was 24.9 mm and body mass was 10.8 g, equivalent to 32.3% and 14.2% of the values from postpartum females. The length of forearm and body mass increased linearly until 45 and 50 days, respectively, and thereafter maintained an apparent stability. The epiphyseal gap of the fourth metacarpal-phalangeal joint increased until 15 days, then decreased linearly until 75 days and thereafter closed. Age was estimated quantitatively, based on linear changes observed in the length of the forearm and epiphyseal gap. Pups began to roost separately, but adjacent to their mothers when 30 days old and flew clumsily when they were about 40 days old. After attaining clumsy flight, the young bats made independent foraging attempts feebly by biting and licking small fruit pieces. Young bats were engaged in suckling as well as ingesting fruits when they were about 50 days old. Between 55 and 65 days, they flew well and fed on fruits. At the age of 75 days, the young bats were completely weaned and at two months, their foraging behaviour was similar to that of their mothers. There was no significant difference in the growth pattern of the young maintained in captivity compared with those under natural conditions.

Neurological recovery from closed head injury is impaired in diabetic rats.

Diabetes mellitus is a metabolic disorder associated with central nervous system impairments. Recent studies implicate oxidative stress mediated by reactive oxygen species (ROS) in the pathogenesis of diabetic complications. ROS have been shown to play role in the pathophysiology of brain injury. In the present study, closed head injury (CHI) was induced in diabetic rats to test the hypothesis that chronic oxidative stress exacerbates brain damage following CHI. Neurological recovery, edema, levels of low molecular weight antioxidants (LMWA), and markers of lipid peroxidation were determined at different intervals after injury. Diabetic rats (4 weeks after induction with streptozotocin) were subjected to CHI. Brain edema (percent water) and clinical status (neurological severity score) were assessed during 7 days. Brain LMWA were determined using cyclic voltammetry (CV) and HPLC-EC. In addition, conjugated dienes and thiobarbituric acid reactive substances (TBARS) were measured. Diabetic-CHI rats exhibited a lower rate of recovery and greater and more sustained edema (p < 0.01), as compared with the controls. At all times diabetic rats had higher levels of TBARS and conjugated dienes and lower concentrations of LMWA, and of vitamins C and E, suggesting chronic oxidative stress. At 5 min of CHI, the amounts of LMWA in control-CHI brains decreased (approximately 50%, p < 0.01) and returned to normal by 48 h and 7 days. In the diabetic-CHI brain only one class of LMWA slightly declined but remained low for 7 days. The present results support the hypothesis that diabetic rats are under chronic oxidative stress, and suffer greater neurological dysfunction, associated with further lipid peroxidation following CHI.

Increased hepatic lipid soluble antioxidant capacity as compared to other organs of streptozotocin-induced diabetic rats: a cyclic voltammetry study.

It has been suggested that oxidative stress plays an important role in the chronic complications of diabetes. The experimental findings regarding the changes in tissue antioxidant enzymes and lipid peroxidation of diabetic tissues have been inconsistent. Previous studies in our laboratory demonstrated that the reducing power of a specific tissue correlates with its low molecular weight antioxidant (LMWA) capacity. In the present study, the overall LMWA capacity (reducing equivalents) of plasma and tissues of streptozotocin (STZ)-induced diabetic rats (1-4 weeks) and insulin treated diabetic rats were measured by cyclic voltammetry. Levels of water and lipid soluble LMWA capacity progressively decreased in the diabetic plasma, kidney, heart and brain, while the diabetic liver, at 2, 3 and 4 weeks after STZ injection, showed a significant increase in the overall lipid soluble LMWA capacity (p < 0.001). Subsequently, analysis of specific components by high pressure liquid chromatography (electrochemical detection) showed decreased levels of ascorbic acid in plasma, kidney, heart and brain of diabetic animals. The alpha-tocopherol level dropped in all tissues, except for the liver in which there was a significant increase (p < 0.01 and p < 0.001 at 2-4 weeks). Lipid peroxidation was assessed by conjugated diene levels, which increased significantly in all diabetic tissues except the liver. Insulin treatment that was started after 3 weeks of diabetes and continued for 3 weeks showed no change in the conjugated dienes and in the overall LMWA capacity in all organs. Our results suggest a unique behavior of the liver in the STZ-induced diabetic rats to the stress and indicate its higher capacity to cope with oxidative stress as compared to other organs.

Fluorescence spectroscopic identification of 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis.

An attempt was made to study whether light-induced fluorescence spectroscopy could be exploited to discriminate premalignant and malignant tissues of hamster buccal pouch carcinogenesis from normal tissues during a 16 week regimen of tri-weekly topical application of 7,12-dimethylbenz[a]anthracene (DMBA) in liquid paraffin. Histologically, the DMBA-treated buccal mucosa showed hyperplastic changes at 4-6 weeks, papillomas at 8-10 weeks, early invasive carcinomas at 11-13 weeks and finally well-differentiated squamous cell carcinomas at 14-16 weeks of treatment. Acetone extracts of these different staged tissues with age matched control tissues were excited at 405 and 420 nm and the emissions were scanned from 430 and 440 to 700 nm respectively. The spectral profiles of control and transformed tissues were found to be different, each displaying their own characteristic prominent maxima and other spectral marks. The spectra of transformed tissues showed characteristic peaks around 620-630 nm which did not appear in control tissues and the fluorescent intensities at 630 nm [FI(630)nm] were significantly increased from early stages onwards when compared to controls. The spectra of DMBA carcinomas developed at the 18th week after withdrawal of DMBA application at the 10th week and carcinoma extract spiked with DMBA confirmed the peak around 620-630 could be attributed only to porphyrin compounds accumulated in transformed tissues. Furthermore, the ratios of FI(520)nm/FI(630)nm of transformed tissues were also significantly decreased when compared to control tissues. This diagnostic test had a very close resemblance with respect to histological studies. These results suggest that this technique using conventional light-induced fluorescence spectroscopy may be useful for early diagnosis of premalignant and malignant lesions of oral cavity.

Chemopreventive potential of dietary bioflavonoids against 20-methylcholanthrene-induced tumorigenesis.

The effect of dietary supplementation of flavonoidal compounds such as quercetin, rutin, luteolin and (+)-catechin on the incidence of fibrosarcoma induced by 20-methylcholanthrene (20-MC) in male Swiss albino mice was observed. Subcutaneous injection of 20-MC produced 100% tumor incidence and the onset of tumor appeared within 7 weeks, while flavonoid-treated mice (1% quercetin- and luteolin-mixed diets) produced tumors in the 9th week, and the tumor incidences in mice treated with quercetin- and luteolin-mixed diets were 52% and 60%, respectively. Subcutaneous administration of 20-MC along with the flavonoidal compounds (quercetin, luteolin) was found to have significant effect on tumor expression. The compounds rutin and (+)-catechin did not influence tumor expression in both experiments. Elevated levels of lipid peroxides, cytochrome P450 and decreased activity of glutathione-S-transferase (GST) were observed in the tumor bearing animals. Test-diet-treated animals showed reduction in the lipid peroxides and cytochrome P450, and increased activity of GST (P < 0.001). In vitro [3H]thymidine incorporation showed the inhibition of DNA synthesis in fibrosarcoma cells by the flavonoids. The possible mode of action of the flavonoidal compounds may be through their influence on the initiation and promotion phases of the carcinogenic process coupled with enhancement of the detoxification process.

Insulin-like effects of bis-glycinato oxovanadium (IV) complex on experimental diabetic rats.

The insulin-like activity of bis-glycinato oxovanadium (IV) complex on experimental diabetes has been studied. Rats made diabetic with streptozotocin, after one month, were fed ad libitum with bis-glycinato oxovanadium (IV) complex (30 mg/100 ml) for fifteen days. The altered blood glucose, urea, cholesterol, triglycerides, liver glycogen and the activities of liver enzymes such as hexokinase, lactate dehydrogenase and fructose-1, 6-bisphosphatase, were reverted to normal levels in bis-glycinato oxovanadium (IV) complex treated diabetic rats, thereby suggesting for the insulin-mimetic effect of bis-glycinato oxovanadium (IV) in experimental diabetes.