Interaction of tacrolimus through hedgehog signaling pathway: An in vitro evaluation using rat hepatocytes.

Tacrolimus (TAC) is the drug of choice in immunosuppressive therapy for organ transplantation; however, adverse effects are still a major concern. The current study aims to decipher the short-term exposure of TAC on rat hepatocytes in relation to activation of hedgehog (HH) signaling pathway. Time dependent study was conducted using primary rat hepatocytes treated with TAC (36 µM) for 6, 12, 24 and 48 h. Western blot analysis was performed using cell lysate in order to analyze the regulation of HH pathway proteins including HHIP, SMO, PTCH, IHH, SHH, and GLI transcription factors. The study revealed change in protein expression of HH signaling molecules with activation of HH pathway, due to downregulation of HHIP, and enrichment of HH ligands with activation of SMO and GLI transcription factors. It is therefore, concluded that short term TAC exposure leads to upregulation of HH pathway in liver, which may initially act to repair the liver damage but can worsen the condition in case of prolonged immunosuppressive therapy. This insight could lead to understand association of off target effects of immunosuppressive drugs and occurrence of other liver diseases in transplant patients when it comes to long term immunosuppressive therapy. These findings also illuminate a novel direction that use of HH inhibitor might provide a therapeutic strategy for immune suppression related liver disorders.

Effect of Short-Term Tacrolimus Exposure on Rat Liver: An Insight into Serum Antioxidant Status, Liver Lipid Peroxidation, and Inflammation.

Tacrolimus (TAC) is an immunosuppressive drug, optimally used for liver, kidney, and heart transplant to avoid immune rejection. In retrospect, a multitude of studies have reported effects of TAC, such as nephrotoxicity, diabetes, and other complications. However, limited information is available regarding short-term exposure of TAC on the liver. Therefore, the present study was designed to unravel the effects of short-term exposure of TAC on a rat model. The animal model was established by TAC administration for 6, 12, 24, and 48 h time points. Liver histopathological changes were observed with PAS-D, reticulin stain, and immunostaining of PCNA and CK-7 coupled with glycogen quantification in a liver homogenate. TUNEL assay was performed to evaluate the DNA damage in the liver. Concentration of GSH and activities of SOD and CAT in the serum were measured to assess the antioxidant status, whereas liver tissue MDA level was measured as a biomarker of oxidative stress. Hepatic gene expression analysis of IL-10, IL-13, SOCS-2, and SOCS-3 was performed by RT-PCR. Results revealed marked changes in liver architecture of all TAC-treated groups, as evidenced by sinusoid dilation, hepatocyte derangement, glycogen deposition, and collapsed reticulin fibers. Significant increase in PCNA and CK-7 immunostaining along with the presence of TUNEL-positive cells was revealed in treatment groups as compared to the control group. Serum antioxidant enzyme status was markedly decreased, whereas the liver MDA level was increased in TAC treatment groups indicating oxidative stress induction. The gene expression profile of cytokines was significantly upregulated in treatment groups highlighting an inflammatory response. In conclusion, results of the current study propose that even a short-term TAC exposure can induce change in antioxidant status and lipid peroxidation. Therefore, these factors should be considered to avoid and minimize immunosuppression-related issues in a prolonged course of treatment.

Assessment of Genetic Variability in the Coding Sequence of Melatonin Receptor Gene (MTNR1A) in Tropical Arid Sheep Breeds of India.

Seasonal behaviour in sheep, which varies in tropical and temperate environmental conditions, is a matter of study, because it can provide a clue to address the problem of seasonality in sheep. Melatonin receptor is the membrane-bound G-coupled receptor, sensing the message of photoperiodic cues thorough melatonin. Restriction fragment length polymorphism (RFLP) studies were carried out to assess the variability of gene at G612A and C606T SNPs in MTNR1A gene, which have been studied to be markers for out-of-season breeding. Allelic frequency distribution corresponded to higher frequency of GG and CC genotype, in tropical arid sheep breed in comparison with temperate region sheep breed. PCR amplification of MTNR1A gene of 30 animals was performed and single nucleotide polymorphisms (SNPs) identification was carried out using Lasergene software. Seven SNPs/mutations were identified, but most of them were synonymous, except the one G706A, leading to substitution of valine by isoleucine. Polyphen-2 analysis of G706A mutation revealed that it is a benign mutation. Two important SNPs C426T and G555A, which were identified in temperate sheep breeds, could not be traced in Magra and Marwari breeds of sheep. Thus, the Magra and Marwari breeds of tropical, arid region demonstrated the presence of both polymorphic SNPs markers G612A and C606T, associated with out-of-season breeding. GG and CC genotypes were having a higher prevalence in the studied population.

Menopausal Health Status of Women of Kapilvastu District of Nepal.

BACKGROUND: Women face numerous health problems in their post-reproductive and menopausal years, including issues such as pelvic pain, incontinence and obstetric fistula. In Nepal, the importance of these health issues is increasing with the aging of the population, yet women are often unable to access adequate health care due to entrenched gender structures and misconceptions regarding menopause. This study aimed to describe the prevalence of menopause, the associated health problems and their severity amongst women aged 40-60 years in Kapilvastu. METHODS: A descriptive study was conducted in Kapilvastu district. Multi stage cluster sampling technique was adopted: Illakas of Kapilvastu were identified, then one Village Development Committee (VDC) from each Illaka of the district, and two wards from each of these VDCs, were selected randomly. RESULTS: The median age of onset of menopause was 47 years (25 th and 75 percentiles = 43 and 50 years), and >90% of women had entered menopause by 54. All symptoms in the Menopause Rating Scale were experienced to some extent by at least 30% of women, even amongst the premenopausal group. The most common problems experienced by menopausal women were: sexual problems (81.7%, 95%CI: 78.0-85.4), physical and mental exhaustion (81.5%, 95%CI: 77.8-85.2) and joint and muscular discomfort (78.5%, 95%CI: 74.6-82.4). The symptoms most frequently deemed "severe" or "very severe" were: sexual problems (40.5%), joint and muscular discomfort (27.4%), and sleep problems (21.0%). th CONCLUSIONS: Middle-aged women in Kapilvastu experienced menopausal symptoms to a substantial degree. The prevalence and severity of these symptoms increased with the menopausal transition, and with increasing age. Menopause itself did not appear to be the primary risk factor for typical "menopausal" symptoms.

Genetic polymorphisms of CYP2E1 and GSTM1 loci and susceptibility to anti-tuberculosis drug-induced hepatotoxicity.

BACKGROUND: Host genetic factors that influence predisposition to anti-tuberculosis drug-induced hepatotoxicity (DIH) are not clear in the Indian population. OBJECTIVE: To investigate the possible association of DIH with polymorphism at the RsaI site of the 5-prime untranslated region of CYP2E1 and GSTM1 'null' mutations. METHODS: In this prospective study, 113 tuberculosis (TB) patients with DIH and 201 TB patients receiving anti-tuberculosis treatment without developing hepatotoxicity (non-DIH) constituted cases and controls, respectively. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to analyse genetic polymorphisms of CYP450 2E1 at the RsaI site and 'null' GSTM1 mutations. PCR-RFLP results were compared between 185 non-DIH and 105 DIH patients RESULTS: A high frequency of c1c1 genotypes of CYP2E1 was commonly encountered, and the difference between DIH and non-DIH patients was not significant (75.14% vs. 77.14%). The genotypic distribution of c2c2 was significantly higher in the DIH than in the non-DIH group (4.8% vs. 0.5%, OR 8.58, P = 0.03). However, adjustment for age, sex and serum albumin differences yielded an OR of 2.75, making it non-significant (P = 0.26). Homozygous 'null' mutation frequencies at the GSTM1 gene in DIH and non-DIH patients were observed that were not significantly different (40% and 37%, respectively, P = 0.61). CONCLUSION: RsaI variants of the CYP2E1 gene and GSTM1 'null' mutation were not associated with risk of DIH in a north Indian population.

Assessment of district health system within inter-sectoral context in Nepal.

BACKGROUND: Inter-sectoral coordination has been one of the different factors in the district health system that produces efficient output which has been identified by the Alma Ata declaration as an essential component to achieve notion of 'Health for All'. This study was therefore aimed to describe the major four key functions of the health systems and to find out the situation of inter-sectoral coordination in Nepal. METHODS: A mixed method with Focus Group Discussion (FGD) and In-Depth Interview with relevant personnel to collect the majority of the data was carried out from June 2012 to November 2012 from six districts selected purposively based on the health performance indicators. RESULTS: The major findings in relation to the key functions of district health systems showed that the overall management of the district health system happens under the leadership of chief of District Health Office of Public health office with the cooperation of all the personnel in different sections in a predetermined pattern and inter-sectoral coordination and collaboration exist only to a very limited extent. CONCLUSIONS: The major constraints for inter-sectoral coordination to be effective is lack of its planning and enforcement where inter-sectoral coordination could be important for both preventive and promotive health care, waste management, water supply and sanitation, health service utilization, pesticides and human health, agriculture and nutrition, air pollution. The main components in the district health system needs an immediate attention and inter-sectoral effort should be initiated from the central level and implemented in all the levels.

Effectiveness and efficiency of Aama Surakshya Karyakram in terms of barriers in accessing maternal health services in Nepal.

BACKGROUND: The Family Health Division along with the MoHP developed a policy that recommended provision of incentives to all delivering mother by removing the parity condition and termed Safe Delivery Incentive Programme (SDIP) to make it more appropriate. The SDIP was branded as AamaSurakshyaKaryakram (ASK). The main objective of the study was to find out the effectiveness and efficiency of AamaSurakshyaKaryakram to address barrier in accessing maternal health services in Nepal. METHODS: An exploratory and cross sectional descriptive study was conducted by quantitative and qualitative tools and techniques. To provide comprehensive coverage, five districts have been selected representing four development (eastern, central, western and far-western) and three (mountain, hill and flat) ecological region were selected. RESULTS: Out of 47 exit client interviews conducted in this study, 51 percent were done in Sunsari, followed by Sarlahi (17%), Dadeldhura (17%), and Arghakhanchi (15%). Most of these mothers (94%) delivered their children in the hospitals, and rest (6%) in PHCCs. Sixty percent mothers were in the age group of 20-25 years, while 45 percent were from Tarai/Madhesi group followed by Brahmins/Chhetries group (34%). Total 70% mothers were found to be literate. 55% mothers were found to be visiting health facilities during labour pain. 2% mothers were visiting heath facilities before labour pain started. Rest mothers were visiting health facilities after one or two days of labour pain. Total 70% mothers were able to reach the health facility within 60 minutes, while 13 percent mothers were able to reach the facility more than 3 hours, and 17% were in between. All mothers who visited PHCCs were able to reach the facility within 60 minutes while analyzing health facility-wise. CONCLUSIONS: Mothers delivered at home as they were not well prepared to go to health facility. Lack of transportation facility hindered for institutional delivery. None of them figured out that there was a provision of transport incentive; they only knew that there was a cash payment, but they didn't know exactly for what specific purpose mothers were receiving such payments. Ask found to be effective and efficient in order to address barriers occurring inside the health facility and financial barrier except geo-graphical barrier in accessing maternal health services in Nepal.

Assessment of medical and health institutions registered as research centres in Nepal.

BACKGROUND: There were several reports in the various mass media regarding misuse of the word "research" by various organization and it has been argued that putting the word "Research" in any agency's name made easier to get the various medical from abroad without paying or paying minimum or discounted government tax than others. The objectives of the study was to find out the status Medical and Health institutions designated as Research Centres in Nepal. METHODS: A cross sectional prospective study was carried in medical and health institutions of Nepal. Data regarding the registration of institutions/organizations having the word "research" in its name were collected from concerned registered organizations. Formative evaluation method was adopted in which information were added continuously, organized systematically and analysed periodically during the evaluation period. RESULTS: There were altogether 370 health related research centers registered in MoI and CDO at the district level till 31 December 2010. These research centers were located in 33 districts of Nepal. Among these there were 65 (82%) health facilities and 305 (18%) NGOs designated as research centers. The region wise the range of number of research centers among five regions was 4 to 283. The highest number of research centers level. The highest number of research centers was found in Kathmandu district. Out of 370 research centers, 85 research centers (72 from among health facility related research centers and 13 from among NGO related research centers) were selected randomly for evaluation purpose, which represented 23 percent of sample selection. The sample selection was not less than 20 percent in each category of research centers. One fifth research centers were found to conduct health related research progam. Among which majority (more than 50%) of NGO related research centers was found to conduct health research. There were few (14%) health facility related research centers that actually conducted health related research program. The study also shows that majority 73 (86%) of the research centers didn't start the research yet. CONCLUSIONS: Forty percent of the research centers in Nepal didn't know the actual reason for putting the word "research" into their signboard. A regulation has to be made to safeguard and maintain the integrity of research in Nepal.

The Flvr-encoded murine oligoadenylate synthetase 1b (Oas1b) suppresses 2-5A synthesis in intact cells.

Resistance to flavivirus-induced disease in mice is conferred by the autosomal gene Flv, identified as 2'-5' oligoadenylate synthetase 1b (Oas1b). Resistant mice express a full-length Oas1b protein while susceptible mice express the truncated Oas1btr. In this study, Oas1b was shown to be an inactive synthetase. Although the Oas/RNase L pathway was previously shown to have an antiviral role during flavivirus infections, Oas1b protein inhibited Oas1a in vitro synthetase activity in a dose-dependent manner and reduced 2-5A production in vivo in response to poly(I:C). These findings suggest that negative regulation of 2-5A by inactive Oas1 proteins may fine tune the RNase L response that if not tightly controlled could cause significant damage in cells. The results also indicate that flavivirus resistance conferred by Oas1b is not mediated by 2-5A. Instead, Oas1b inhibits flavivirus replication by an alternative mechanism that overrides the proviral effect of reducing 2-5A accumulation and RNase L activation.

Prevalence of methicillin resistant Staphylococcus aureus (MRSA) among skin infection cases at a hospital in Chitwan, Nepal.

This study was carried out to see the methicillin resistance of Staphylococcus aureus isolated from patients with skin and soft tissue infection. In this study, a total of 600 S. aureus isolates isolated from 1,334 specimens from skin and soft tissue infection were included. For the screening of methicillin resistant of S. aureus (MRSA), specimens were inoculated on lipovitellin mannitol salt agar (LMSA) and MRSA were screened by testing resistance to oxacillin in AST plate following the standard bacteriological technique. The overall prevalence of MRSA was 68.0% (408/600). MRSA infection was higher among patients above 30 years of age. Tibeto-Burmans had high MRSA infection rate (72.5%) than Indo-Aryans (56.8%) and the rate was significantly higher among males (75.0%) than females (63.4%) (p<0.05). MRSA isolation rate was higher from wound (76.9%), followed by purulent exudates (67.7%) and abscesses (64.1%). The high prevalence of MRSA found in this study might be due to hospital based specimen.

Prevalence of asymptomatic bacteriuria among elderly diabetic patients residing in Chitwan.

BACKGROUND: A retrospective study was done on Asymptomatic Bacteriuria (ASB), among elderly diabetic patients residing in Chitwan, Nepal from 1st January 2006 to 12th December 2006. Diabetic patients above 40 years of age were included in this study where as below 40 years of age group were excluded. OBJECTIVE: To study the prevalence of ASB among elderly diabetic patients for the Bacteriological profile and their antibiotic susceptibility pattern. MATERIALS AND METHODS: Urine sample was collected from 636 diabetic patients above 40 years age attending College of Medical Sciences Teaching Hospital for regular blood sugar estimation. Culture and Antibiotic susceptibility test (AST) were performed using standard microbiological technique. ASB was determined as presence of > 105(5) bacteria/ml in a culture of urine sample. RESULTS: The prevalence of ASB among diabetic patients in Bharatpur was found to be 9.43% (12.07% in females and 5.08% in males). The most common bacterial isolate was Escherichia coli (41.66%). Nitrofurantoin, Cefotaxime and Chloramphenicol were the most effective drugs for the majority of isolates. CONCLUSION: The study shows necessity of regular urine culture to detect ASB, along with blood sugar estimation for diabetic patients to prevent them from further complications due to it.

Role of 2-5A-dependent RNase-L in senescence and longevity.

Senescence is a permanent growth arrest that restricts the lifespan of primary cells in culture, and represents an in vitro model for aging. Senescence functions as a tumor suppressor mechanism that can be induced independent of replicative crisis by diverse stress stimuli. RNase-L mediates antiproliferative activities and functions as a tumor suppressor in prostate cancer, therefore, we examined a role for RNase-L in cellular senescence and aging. Ectopic expression of RNase-L induced a senescent morphology, a decrease in DNA synthesis, an increase in senescence-associated beta-galactosidase activity, and accelerated replicative senescence. In contrast, senescence was retarded in RNase-L-null fibroblasts compared with wild-type fibroblasts. Activation of endogenous RNase-L by 2-5A transfection induced distinct senescent and apoptotic responses in parental and Simian virus 40-transformed WI38 fibroblasts, respectively, demonstrating cell type specific differences in the antiproliferative response to RNase-L activation. Replicative senescence is a model for in vivo aging; therefore, genetic disruption of senescence effectors may impact lifespan. RNase-L-/- mice survived 31.7% (P<0.0001) longer than strain-matched RNase-L+/+ mice providing evidence for a physiological role for RNase-L in aging. These findings identify a novel role for RNase-L in senescence that may contribute to its tumor suppressive function and to the enhanced longevity of RNase-L-/- mice.

Prevalence of asymptomatic bacteriuria in school going children in Pokhara valley.

OBJECTIVES: There were mainly two objectives of the study. One was to detect bacteriuria in school going children in Pokhara valley and the other was to identify the causative organisms in various age groups in children. MATERIALS AND METHODS: This study was carried out in the Microbiology laboratory of The School of Pharmaceutical and Biomedical Sciences, Simalchaur, Pokhara University, Nepal. A total of 502 urine samples of 5 to 13 years children from different schools of Pokhara valley were screened to see asymptomatic bacteriuria during January 2005 to June 2005. A sterile wide mouth container was given to each student to collect mid-stream urine samples. All the urine samples were transported to the Microbiology laboratory within half an hour to one hour. The samples were processed for microscopical examination to observe for turbidity and the presence of protein and sugar by dipstick method, microscopical examination to see pus cells, RBCs, epithelial cells, casts and crystals, culture of urine samples on Blood agar and MacConkey agar to identify the potential pathogens. The antibiotic sensitivity test was performed for those bacteria which were grown on culture. The colony count was evaluated and organisms were identified by biochemical tests. RESULT: Out of 502 samples, 7(1.39%) samples grew the bacterial pathogens that are responsible to cause urinary tract infection. Among them Escherichia coli 4(57.14%) was the predominant bacterial pathogen. CONCLUSION: Study of asymptomatic bacteriuria is important as found in the present study in which Escherichia coli was the most frequently incriminated as the causative agents.

Prevalence of Moraxella catarrhalis infections of the lower respiratory tract in elderly patients.

OBJECTIVES: To determine: *rate of isolation of Moraxella catarrhalis in elderly patients having lower respiratory tract infection *The antibiotic susceptibility pattern of Moraxella catarrhalis isolates. *The predisposing factors responsible for Moraxella catarrhalis infections of lower respiratory tract. MATERIAL AND METHOD: This is a prospective study carried out at Manipal Teaching Hospital, Pokhara during May 2002 to April 2003. A total of 716 sputum samples collected from patients with suspected lower respiratory tract infection were investigated. The samples were subjected to Gram stain, culture and sensitivity. Sputum samples were inoculated on blood and chocolate agar and incubated at 37 degrees C for 18 to 24 hours in candle jar containing 3-5% Co2. For the identification of bacterial isolates, the cultural and colonial characteristics were recorded and identified with the use of biochemical test. The susceptibility of bacterial isolates to antimicrobial agent was performed following Kirby-Bauer method. RESULT: Out of the 716 samples, 355 (49.58%) grew normal commensals of the upper respiratory tract. Respiratory pathogens were recovered from 361 (50.41%) samples. The most common respiratory pathogen was H. influenzae (26. 86%), followed by S. pneumoniae (21.16%). Moraxella catarrhalis accounted for 6.90%. Rate of isolation of Moraxella catarrhalis was higher in males than in females. Out of 25 isolates 16 were from males and 9 were from females. Frequency of isolation of Moraxella catarrhalis was more frequently seen in age group 61-70 years. It was most commonly recovered in winter. The most effective antibiotics were amoxicillin-clavulanate and ceftriaxone and least was ampicillin. CONCLUSION: Moraxella catarrhalis isolation from sputum especially in persons above 60 years of age and in the absence of other well established pathogens should not be disregarded as they can cause lower respiratory tract infection in these individuals.

Interactions between a lipase and charged surfactants--a comparison between bulk and interfaces.

The interaction between a charged surfactant and a lipase has been investigated by several methods. Interactions in aqueous bulk phase was studied by NMR and by microcalorimetry. Surface tension and neutron reflectivity were used for studies at the air-water interface. Interactions at the interface between a hydrophobic solid surface and water was investigated by ellipsometry. The results obtained are as follows. The cationic surfactant, tetradecyltrimethylammonium bromide (iodide in the NMR experiments), showed strong interaction at the air-water and the hydrophobic solid-water interfaces but no clear indication of an interaction in bulk phase was seen. The anionic surfactant showed no interaction with the lipase neither at the interfaces, nor in bulk. The difference in behavior of the system cationic surfactant-lipase in bulk and at the interfaces may be due to the change in enzyme conformation that is known to occur at interfaces between water and an apolar phase.

An unusual foreign body in the mastoid process.

The interest in reporting this case is the unusual manner in which the foreign body entered the mastoid process without any clinical symptoms and remained covered by the skin from outside. Plastic refill of the dot pen (metallic end) pierced the mastoid bone in a child is rather atypical due to a fall.