Role of µ-Opioid Receptor Polymorphism in Patients of Rheumatoid Arthritis and their Correlation with Severity of Disease.

INTRODUCTION: With 1 billion tobacco users worldwide, nicotine dependence has a major impact on global health. Advances in medication development for nicotine dependence require an improved understanding of the neurobiology of this complex, relapsing brain disorder. AIMS: To study association of µ Opioid Receptor polymorphism in patients of rheumatoid arthritis and its correlation with severity of disease and prevalent alleles of the OPRM1 genes. MATERIAL AND METHODS: This is a case control study wherein all available patients and volunteers were recruited. 142 controls subjects with no known history of disease and 85 study group cases were included. RESULTS: Comparison of genotype frequencies showed a statistically significant difference between the studied groups (p<0.004). A statistically significant difference was found when the allelic frequencies between the two groups were compared (p<0.0001), with the 17T allele having a-1.7518 fold higher risk of having RA (risk ratio (RR)=1.7518, 95%CI of RR=1.2988-2.3627, OR =3.2914; 95%CI =1.9608-5.5251). Significant difference was also found when the allelic frequencies between the two groups were compared (p<0.0001), with the 118G allele having a 1.5-fold higher risk of developing RA (RR)=1.5801, 95%CI =1.3091-1.9071, OR=3.1357; 95%CI 2.1083-4.6638). CONCLUSION: The study definitely needs to be extended to larger cohort of patients and control samples and to a larger set of candidate µ opioid receptors. Extending the studies to a larger cohort will also allow genetic analyses of clinically defined endophenotypes observed in the patients of this chronic metabolic disease with attributes of autoimmune disorder and multiple symptoms in patients.

DOPA and DHN pathway orchestrate melanin synthesis in Aspergillus species.

Melanins are high molecular weight hydrophobic pigments that have been studied for their role in the virulence of fungal pathogens. We investigated the amount and type of melanin in 20 isolates of Aspergillus spp.; A. niger (n = 3), A. flavus (n = 5), A. tamarii (n = 3), A. terreus (n = 3), A. tubingensis (n = 3), A. sydowii (n = 3). Aspergillus spp. were identified by sequencing the internal transcribed spacer (ITS) region. Extraction of melanin from culture filtrate and fungal biomass was done and followed by qualitative and quantitative analysis of melanin pigment. Ultraviolet (UV), Fourier transformed infrared (FT-IR), and electron paramagnetic resonance (EPR) spectra analyses confirmed the presence of melanin. The melanin pathway was studied by analyzing the effects of inhibitors; kojic acid, tropolone, phthalide, and tricyclazole. The results indicate that in A. niger and A. tubingensis melanin was found in both culture filtrate and fungal biomass. For A. tamarii and A. flavus melanin was extracted from biomass only, whereas melanin was found only in culture filtrate for A. terreus. A negligible amount of melanin was found in A. sydowii. The maximum amount of melanin from culture filtrate and fungal biomass was found in A. niger and A. tamarrii, respectively. The DOPA (3,4-dihydroxyphenylalanine) pathway produces melanin in A. niger, A. tamarii and A. flavus, whereas the DHN (1,8-dihydroxynaphthalene) pathway produces melanin in A. tubingensis and A. terreus. It can be concluded that the amount and type of melanin in aspergilli largely differ from species to species.

Microscopic evaluation, molecular identification, antifungal susceptibility, and clinical outcomes in fusarium, Aspergillus and, dematiaceous keratitis.

PURPOSE: Fusarium, Aspergillus, and Dematiaceous are the most common fungal species causing keratitis in tropical countries. Herein we report a prospective study on fungal keratitis caused by these three fungal species. METHODOLOGY: A prospective investigation was undertaken to evaluate eyes with presumed fungal keratitis. All the fungal isolates (n = 73) obtained from keratitis infections were identified using morphological and microscopic characters. Molecular identification using sequencing of the ITS region and antifungal susceptibility tests using microdilution method were done. The final clinical outcome was evaluated in terms of the time taken for resolution of keratitis and the final visual outcome. The results were analyzed after segregating the cases into three groups, namely, Fusarium, Aspergillus, and Dematiaceous keratitis. RESULTS: Diagnosis of fungal keratitis was established in 73 (35.9%) cases out of 208 cases. The spectra of fungi isolated were Fusarium spp. (26.6%), Aspergillus spp. (21.6%), and Dematiaceous fungi (11.6%). The sequence of the ITS region could identify the Fusarium and Aspergillus species at the species complex level, and the Dematiaceous isolates were accurately identified. Using antifungal agents such as fluconazole, natamycin, amphotericin B, and itraconazole, the minimum inhibitory concentrations (MICs) for Fusarium spp. were >32 µ g/mL, 4-8 µ g/mL, 0.5-1 µ g/mL, and >32 µ g/mL, respectively. Antifungal susceptibility data showed that Curvularia spp. was highly resistant to all the antifungal agents. Overall, natamycin and amphotericin B were found to be the most effective antifungal agents. The comparative clinical outcomes in all cases showed that the healing response in terms of visual acuity of the Dematiaceous group was significantly good when compared with the Fusarium and Aspergillus groups (P < 0.05). The time required for healing in the Fusarium group was statistically significantly less when compared with the Aspergillus and Dematiaceous groups. CONCLUSION: This study demonstrates important differences in microscopic features of scraping material and antifungal susceptibility between the three groups. Early and accurate identification coupled with the MIC data, and thereby appropriate treatment is crucial for complete recovery.

Estrogen mediated protection of cytoskeleton against oxidative stress.

BACKGROUND & OBJECTIVES: Cytoskeletal proteins are deregulated during oxidative stress and cataract formation. However, estrogen which protects against cataract formation and harmful effects of oxidative stress has not been tested on the cytoskeleton of lens epithelial cells (LECs). The current study was undertaken to assess if the protection rendered to LECs by estrogen was mediated by preserving the cytoskeletal proteins. METHODS: Oxidative stress was induced by 50 µM of H 2 O 2 in cultured goat LECs (gLECs) and effect of 1 µM 17ß-estradiol (E 2 ) was tested. After treatment, morphological analysis of cells was carried out using haematoxylin-eosin staining and cell density was also quantified. Cell viability was determined using Hoechst (Ho), YO-Pro (YP) and propidium iodide (PI). F-actin and vimentin were localized using phalloidin and anti-vimentin antibody, respectively, and viewed under fluorescence microscopy. Vimentin was further analysed at protein level by Western blotting. RESULTS: H 2 O 2 led to increased condensation of nucleus, cell death and apoptosis but these were prevented with pre- and co-treatment of E 2 with increase in cell viability (P<0.001). E 2 also prevented H 2 O 2 mediated depolymerization of cytoskeleton but was not able to reverse the changes when given after induction of oxidative stress. INTERPRETATION & CONCLUSIONS: Our findings showed that E 2 helped in preventing deteriorating effect of H 2 O 2 , inhibited cell death, apoptosis and depolymerisation of cytoskeletal proteins in LECs. However, the exact mechanism by which estrogen renders this protection to cytoskeleton of lens epithelial cells remains to be determined.

Triphala and its constituents ameliorate visceral adiposity from a high-fat diet in mice with diet-induced obesity.

CONTEXT: In India, vaidyas (Ayurvedic physicians) traditionally administer triphala and its constituents as therapeutic agents for promoting digestion and satiety. OBJECTIVE: The research team performed the present study to investigate the effects of triphala and its constituents (T bellirica [bibhitaki], T chebula [haritaki], and E officinalis [amalaki]) on the dietary induction of obesity (diet-induced obesity [DIO]), and other symptoms of visceral obesity syndrome, in mice fed a high-fat diet (HFD). DESIGN: The research team obtained 42 fertile, male, Swiss albino mice, weighing 20 g each, and housed them individually in an approved small-animal facility, in a pathogen-free environment. The team generated DIO mice by feeding them a HFD. SETTING: The study took place at the Birla Institute of Technology and Science (BITS) in Pilani, India. INTERVENTION: The research team fed all mice, except those in a control group (ND), a HFD for 10 weeks beginning at 7 weeks of age, supplementing the HFDs with herbal treatments for 4 of the groups. The team divided the mice into six weight-matched groups of seven mice each: (1) normal diet (ND), (2) high-fat diet (HFD), (3) triphala (HFD+T), (4) amalaki (HFD+A), (5) haritaki (HFD+H), and (6) bibhitaki (HFD+B). OUTCOME MEASURES: The research team evaluated daily energy intake, fasting plasma glucose, serum lipid profile, and liver cytology. The team measured food and energy intake daily for 10 weeks and measured the body weight of each mouse every third day during the course of the experiment. The team drew blood samples at 2, 4, 8, and 10 weeks posttreatment and determined fasting plasma-glucose concentrations and fasting plasma concentrations of cholesterol, triglycerides (TG), LDL, HDL, and plasma alanine transaminase (ALT) using commercial kits. At the completion of the study, a pathologist examined the livers and diagnosed a fatty liver based on the presence of macrovesicular or microvesicular fat in the hepatocytes. RESULTS: The research team's results showed that mice fed a HFD for a 10-week period, supplemented with herbal preparation(s) of triphala or its constituents, resulted in significant reductions in body weight (P < .0001), energy intake, and percentage of body fat (P < .001), as compared with mice in the HFD group. Herbal treatment significantly improved the lipid profiles of the mice by lowering serum total cholesterol (Total-C), TG, and low-density lipoprotein cholesterol (LDL-C) and increasing levels of high-density lipoprotein cholesterol (HDL-C) as compared to the mice in the HFD group. The research team also found that herbal treatment attenuated glucose levels, oral glucose tolerance as measured by the oral glucose tolerance test (OGTT), and levels of ALT. In addition to treatment with its three individual components, treatment with a popular Ayurvedic formulation of triphala also reversed the pathological changes in liver tissue and decreased the relative weight of visceral adipose fat pads. CONCLUSIONS: The present findings suggest that triphala and its constituents can counter the effects of an environment (ie, high dietary intake of fats) and have the potential for use as antiobesity agents with desirable lipid-profile modulating properties.

Cx43, ZO-1, alpha-catenin and beta-catenin in cataractous lens epithelial cells.

Specimens of the anterior lens capsule with an attached monolayer of lens epithelial cells (LECs) were obtained from patients (n=52) undergoing cataract surgery. Specimens were divided into three groups based on the type of cataract: nuclear cataract, cortical cataract and posterior subcapsular cataract (PSC). Clear lenses (n=11) obtained from donor eyes were used as controls. Expression was studied by immunofluorescence, real-time PCR and Western blot. Statistical analysis was done using the student's t-test. Immunofluorescence results showed punctate localization of Cx43 at the cell boundaries in controls, nuclear cataract and PSC groups. In the cortical cataract group, cytoplasmic pools of Cx43 without any localization at the cell boundaries were observed. Real-time PCR results showed significant up-regulation of Cx43 in nuclear and cortical cataract groups. Western blot results revealed significant increase in protein levels of Cx43 and significant decrease of ZO-1 in all three cataract groups. Protein levels of alpha-catenin were decreased significantly in nuclear and cortical cataract group. There was no significant change in expression of beta-catenin in the cataractous groups. Our findings suggest that ZO-1 and alpha-catenin are important for gap junctions containing Cx43 in the LECs. Alterations in cell junction proteins may play a role during formation of different types of cataract.

Alterations in intestinal permeability and endotoxemia in severe acute pancreatitis.

BACKGROUND: Intestinal permeability increases early in the course of acute pancreatitis and is associated with sepsis and organ failure. AIM: To evaluate the intestinal permeability (IP) and anti-endotoxin antibodies immunoglobulin G and A (AEA IgG and A) in severe acute pancreatitis (SAP) as compared to healthy controls and determine their significance in relation to various complications of SAP. METHODS: IP was measured by urinary lactulose/mannitol (LM) excretion ratio and anti-endotoxin antibodies by Endocab ELISA kit at days one and seven of admission (D1 and D7). RESULTS: Thirty one patients of SAP [mean age (42.0 +/- 15.8) years, APACHE II scores (8.8 +/- 5.4) and CT severity index (6.4 +/- 2.0)] were included in this study. Infected pancreatic necrosis was detected in 13 (42%) patients of whom three died. Six died of persistent organ failure. Median values of LM ratio at D1 and D7 were similar to those in healthy controls. Patients experiencing complications [organ failure (4/9, 44%), infected pancreatic necrosis (5/10, 50%) and death (1/2, 50%)] manifested a substantial increase in their intestinal permeability at D7. Anti-endotoxin antibodies IgG were lower (p = 0.003) in patients than the controls at admission. AEA IgG were lower (p = 0.03) in non-survivors as compared to survivors at D7. CONCLUSION: Patients experiencing complications of severe acute pancreatitis showed an increase in intestinal permeability. Higher endotoxemia predicted poor outcome in severe acute pancreatitis.

Fungal scleral keratitis caused by Phomopsis phoenicicola.

We report a case of scleral keratitis caused by Phomopsis phoenicicola. Pterygium surgery was a predisposing factor, and the patient was treated with natamycin and fluconazole eye drops and oral fluconazole. The fungus was identified by sequencing of the internal transcribed spacer (ITS) region of the fungal ribosomal DNA (rDNA) locus and confirmed on the basis of its typical pycnidia and conidia.