Moderate Stress Level among Patients Undergoing Hemodialysis in a Tertiary Care Centre.

Introduction: Stress is the response of body to any change. The end stage of renal disease and the process of haemodialysis treatment are long-term stressors that alter patients' well-being and everyday lifestyle. The aim of this study was to find out the prevalence of moderate stress levels among patients undergoing hemodialysis in a tertiary care centre. Methods: A descriptive cross-sectional study was conducted among patients undergoing hemodialysis from 1 October 2021 to 30 September 2022. Ethical clearance from the Institutional Review Committee. Patients undergoing maintenance haemodialysis for at least 3 months were included in the study. Convenience sampling method was used. The point estimate was calculated at a 95% Confidence Interval. Results: Among 118 patients, 82 (69.49%) (61.18-77.80, 95% Confidence Interval) had moderate stress levels, out of which 51 (62.20%) were male and 31 (37.80%) were female. Conclusions: The prevalence of moderate stess level was found to be higher than other studies done in similar settings. Keywords: hemodialysis; prevalence; psychiatric disorders.

WFS1 Gene-associated Diabetes Phenotypes and Identification of a Founder Mutation in Southern India.

CONTEXT: Wolfram syndrome (WFS) is a rare autosomal recessive disorder characterized by juvenile-onset diabetes, diabetes insipidus, optic atrophy, deafness, and progressive neurodegeneration. However, due to the progressive nature of the disease and a lack of complete clinical manifestations, a confirmed diagnosis of WFS at the time of onset of diabetes is a challenge. OBJECTIVE: With WFS1 rare heterozygous variants reported in diabetes, there is a need for comprehensive genetic screening strategies for the early diagnosis of WFS and delineating the phenotypic spectrum associated with the WFS1 gene variants in young-onset diabetes. METHODS: This case series of 11 patients who were positive for WFS1 variants were identified with next-generation sequencing (NGS)-based screening of 17 genemonogenic diabetes panel. These results were further confirmed with Sanger sequencing. RESULTS: 9 out of 11 patients were homozygous for pathogenic/likely pathogenic variants in the WFS1 gene. Interestingly, 3 of these probands were positive for the novel WFS1 (NM_006005.3): c.1107_1108insA (p.Ala370Serfs*173) variant, and haplotype analysis suggested a founder effect in 3 families from Southern India. Additionally, we identified 2 patients with young-onset diabetes who were heterozygous for a likely pathogenic variant or a variant of uncertain significance in the WFS1 gene. CONCLUSION: These results project the need for NGS-based parallel multigene testing as a tool for early diagnosis of WFS and identify heterozygous WFS1 variants implicated in young-onset diabetes.

Prevalence of latent structural heart disease in Nepali schoolchildren.

BACKGROUND: The present study aimed to quantify the burden of structural heart disease in Nepali children. METHODS: We performed a school-based cross-sectional echocardiographic screening study with cluster random sampling among children 5-16 years of age. RESULTS: Between December 2012 and January 2019, 6573 children (mean age 10.6 ± 2.9 years) from 41 randomly selected schools underwent echocardiographic screening. Structural heart disease was detected in 14.0 per 1000 children (95% CI 11.3-17.1) and was congenital in 3.3 per 1000 (95% CI 2.1-5.1) and rheumatic in 10.6 per 1000 (95% CI 8.3-13.4). Rates of rheumatic heart disease were higher among children attending public as compared to private schools (OR 2.8, 95% CI 1.6-5.2, p = 0.0001). CONCLUSION: Rheumatic heart disease accounted for three out of four cases of structural heart disease and was more common among children attending public as compared to private schools.

Prevalence of Elevated D-dimer Levels in Confirmed COVID-19 Cases in Intensive Care Unit of a Tertiary Care Centre of Western Nepal.

INTRODUCTION: D-dimer is currently the best available marker for COVID-19 associated hemostatic abnormalities. This study aims to find out the prevelance of elevated D-dimer levels in confirmed COVID-19 cases in intensive care unit of a tertiary care hospital of western Nepal. METHODS: This descriptive cross-sectional study was conducted among 95 patients admitted to COVID Intensive Care Unit of a teriary care centre from August 2020 to January 2021 after taking ethical clearence from Institutional Review Committee in order to determine the D-dimer levels in confirmed COVID-19 cases. D-dimer value was measured at the admission and the highest D-dimer value was recorded during the course of hospital stay with the risk of mortality in confirmed COVID-19 cases. The normal range of D-dimer was taken as <0.35 mg/dl as per our hospital laboratory standards. Convenience sampling method was used. Data entry and descriptive analysis were done in Statistical Package for the Social Sciences version 25.0, point estimate at 95% Confidence Interval was calculated along with frequency and proportion for binary data. RESULTS: Out of total 95 cases of COVID-19 included in this study, 25 (89.3%) patients with age ≥ 65 years and 42 (62.69%) patients aged <65 years had elevated D-dimer on admission. Data showed that 29 (67.4%) patients having elevated D-dimer at admission had mortality. CONCLUSIONS: Elevated D-dimer levels was frequently seen in patients admitted in Intensive Care Unit with COVID-19. Our study suggested that measurement of D-dimer may guide in clinical decision making.

Mid-term outcome of children with latent rheumatic heart disease in eastern Nepal.

INTRODUCTION: Systematic echocardiographic screening of children in regions with an endemic pattern of rheumatic heart disease allows for the early detection of valvular lesions suggestive of subclinical rheumatic heart disease. The natural course of latent rheumatic heart disease is, however, incompletely understood at this time. METHODS: We performed a prospective cohort study of children detected to have echocardiographic evidence of definite or borderline rheumatic heart disease according to the World Heart Federation Criteria. RESULTS: Among 53 children found to have definite (36) or borderline (17) rheumatic heart disease, 44 (83%) children underwent follow-up at a median of 1.9 years (IQR 1.1-4.5). The median age of the children was 11 years (IQR 9-14) and 34 (64.2%) were girls. Among children with definite rheumatic heart disease, 21 (58.3%) were adherent to secondary antibiotic prophylaxis, 7 (19.4%) were not, information on adherence was missing in 2 (5.6%) children and 6 (16.7%) were lost to follow-up. Regression of disease was observed in 10 children (27.8%), whereas 20 children (55.6%) had stable disease. Among children adherent to secondary prophylaxis, seven (33.3%) showed regression of disease. Among children with borderline disease, seven (41.2%) showed regression of disease, three (17.6%) progression of disease, four (23.5%) remained stable and three (17.6%) were lost to follow-up. On univariate analysis, we identified no predictors of disease regression, and no predictors for lost to follow-up or non-adherence with secondary antibiotic prophylaxis. CONCLUSION: Definite rheumatic heart disease showed regression in one in four children. Borderline disease was spontaneously reversible in less than half of the children and progressed to definite rheumatic heart disease in one in five children. TRIAL REGISTRATION NUMBER: NCT01550068.

Effectiveness of Systematic Echocardiographic Screening for Rheumatic Heart Disease in Nepalese Schoolchildren: A Cluster Randomized Clinical Trial.

Importance: Echocardiographic screening allows for early detection of subclinical stages of rheumatic heart disease among children in endemic regions. Objective: To investigate the effectiveness of systematic echocardiographic screening in combination with secondary antibiotic prophylaxis on the prevalence of rheumatic heart disease. Design, Setting, and Participants: This cluster randomized clinical trial included students 9 to 16 years of age attending public and private schools in urban and rural areas of the Sunsari district in Nepal that had been randomly selected on November 17, 2012. Echocardiographic follow-up was performed between January 7, 2016, and January 3, 2019. Interventions: In the experimental group, children underwent systematic echocardiographic screening followed by secondary antibiotic prophylaxis in case they had echocardiographic evidence of latent rheumatic heart disease. In the control group, children underwent no echocardiographic screening. Main Outcomes and Measures: Prevalence of the composite of definite or borderline rheumatic heart disease according to the World Heart Federation criteria in experimental and control schools as assessed 4 years after intervention. Results: A total of 35 schools were randomized to the experimental group (n = 19) or the control group (n = 16). After a median of 4.3 years (interquartile range [IQR], 4.0-4.5 years), 17 of 19 schools in the experimental group (2648 children; median age at follow-up, 12.1 years; IQR, 10.3-12.5 years; 1308 [49.4%] male) and 15 of 16 schools in the control group (1325 children; median age at follow-up, 10.6 years; IQR, 10.0-12.5 years; 682 [51.5%] male) underwent echocardiographic follow-up. The prevalence of definite or borderline rheumatic heart disease was 10.8 per 1000 children (95% CI, 4.7-24.7) in the control group and 3.8 per 1000 children (95% CI, 1.5-9.8) in the experimental group (odds ratio, 0.34; 95% CI, 0.11-1.07; P = .06). The prevalence in the experimental group at baseline had been 12.9 per 1000 children (95% CI, 9.2-18.1). In the experimental group, the odds ratio of definite or borderline rheumatic heart disease at follow-up vs baseline was 0.29 (95% CI, 0.13-0.65; P = .008). Conclusions and Relevance: School-based echocardiographic screening in combination with secondary antibiotic prophylaxis in children with evidence of latent rheumatic heart disease may be an effective strategy to reduce the prevalence of definite or borderline rheumatic heart disease in endemic regions. Trial Registration: ClinicalTrials.gov Identifier: NCT01550068.

Anaphylactic reactions due to pantoprazole: case report of two cases.

BACKGROUND: Drug-induced hypersensitivity reaction is of great clinical significance in therapeutics. The objective of this reporting of two cases is to show that anaphylaxis reaction can occur with pantoprazole. CASE SUMMARIES: A 38-year-old female reported to the emergency ward in a critical condition, with a history of periorbital edema, edema of the skin, pruritus, nausea, vomiting, and difficulty breathing 20 minutes after ingestion of a pantoprazole 40 mg tablet. A 32-year-old female reported to the emergency ward in a critical condition, with complaints of rashes all over the body, itching on the whole body, and swollen lips and eyes after ingestion of a pantoprazole 40 mg tablet. CONCLUSION: It is necessary for all health care providers to know that pantoprazole can cause anaphylaxis, which is a life-threatening reaction, and to be cautious while prescribing it.

Iodine Status among Subclinical and Overt Hypothyroid Patients by Urinary Iodine Assay: A Case-Control Study.

OBJECTIVES: The objective of the study was to assess the differences of iodine status as measured by urinary iodine excretion (UIE) between cases of hypothyroidism and healthy controls. MATERIALS AND METHODS: The study was conducted in cases with subclinical hypothyroidism (n = 58) and overt hypothyroidism (n = 41) and compared with age- and sex-matched healthy euthyroid controls (n = 52) attending Universal College of Medical Sciences Teaching Hospital, Bhairahawa, Nepal. Serum free triiodothyronine (fT3), free thyroxine (fT4), and thyroid-stimulating hormone (TSH) were estimated by competitive ELISA and sandwich ELISA, respectively (Diametra, Italy). The urinary iodine concentration (UIC) in urine samples was estimated by ammonium persulfate digestion method recommended by the WHO. RESULTS: A significantly higher median UIC was observed among cases of subclinical hypothyroidism (224.90 µg/l) and overt hypothyroidism (281.0 µg/l) as compared to the controls (189.90 µg/l) (P = 0.0001, P = 0.001). Serum TSH in the cases of subclinical hypothyroid was higher, whereas fT3 was lower as compared to controls (P = 0.028, P = 0.0001), respectively. Similarly, serum TSH in the cases of overt hypothyroid was higher and fT3 and fT4 were lower as compared to controls (P = 0.0001, P = 0.0001, P = 0.015), respectively. There was positive correlation of UIC with TSH (r = 0.269, P = 0.0001), whereas negative correlation was seen with fT3 (r = -0.328, P = 0.0001) and fT4 (r = -0.145, P = 0.076). The test of multiple regression has shown that fT3 (ß = -0.262, P = 0.012) as an independent predictor in association with UIE in cases. CONCLUSION: Excessive iodine intake was found in hypothyroid patients as assessed by UIE concluding that it may trigger the thyroid hypofunction. Cohort studies to generate further evidence should be done to explore potential mechanism of hypothyroidism in excess iodine intake.

Prevalence of Subclinical Rheumatic Heart Disease in Eastern Nepal: A School-Based Cross-sectional Study.

IMPORTANCE: Although rheumatic heart disease has been nearly eradicated in high-income countries, 3 in 4 children grow up in parts of the world where it is still endemic. OBJECTIVES: To determine the prevalence of clinically silent and manifest rheumatic heart disease as a function of age, sex, and socioeconomic status and to estimate age-specific incidence. DESIGN, SETTING, AND PARTICIPANTS: In this school-based cross-sectional study with cluster sampling, 26 schools in the Sunsari district in Eastern Nepal with 5467 eligible children 5 to 15 years of age were randomly selected from 595 registered schools. After exclusion of 289 children, 5178 children were enrolled in the present study from December 12, 2012, through September 12, 2014. Data analysis was performed from October 1, 2014, to April 15, 2015. EXPOSURES: Demographic and socioeconomic characteristics were acquired in a standardized interview by means of a questionnaire customized to the age of the children. A focused medical history was followed by a brief physical examination. Cardiac auscultation and transthoracic echocardiography were performed by 2 independent physicians. MAIN OUTCOMES AND MEASURES: Rheumatic heart disease according to the World Heart Federation criteria. RESULTS: The median age of the 5178 children enrolled in the study was 10 years (interquartile range, 8-13 years), and 2503 (48.3%) were female. The prevalence of borderline or definite rheumatic heart disease was 10.2 (95% CI, 7.5-13.0) per 1000 children and increased with advancing age from 5.5 (95% CI, 3.5-7.5) per 1000 children 5 years of age to 16.0 (95% CI, 14.9-17.0) in children 15 years of age, whereas the mean incidence remained stable at 1.1 per 1000 children per year. Children with rheumatic heart disease were older than children without rheumatic heart disease (median age [interquartile range], 11 [9-14] years vs 10 [8-13] years; P = .03), more commonly female (34 [64.2%] vs 2469 [48.2%]; P = .02), and more frequently went to governmental schools (40 [75.5%] vs 2792 [54.5%]; P = .002). Silent disease (n = 44) was 5 times more common than manifest disease (n = 9). CONCLUSIONS AND RELEVANCE: Rheumatic heart disease affects 1 in 100 schoolchildren in Eastern Nepal, is primarily clinically silent, and may be more common among girls. The overall prevalence and the ratio of manifest to subclinical disease increase with advancing age, whereas the incidence remains stable at 1.1 per 1000 children per year. Early detection of silent disease may help prevent progression to severe valvular damage.

Association between serum uric acid, urinary albumin excretion, and glycated hemoglobin in Type 2 diabetic patient.

BACKGROUND: Diabetes mellitus (DM) is a chronic disease characterized by insulin deficiency or peripheral resistance resulting in hyperglycemia. Poor glycemic control leads to diabetic complications. Hyperuricemia has been reported with increased risk of renal insufficiency. The aim of this study was to evaluate the relationship between serum uric acid concentration, degree of urinary albumin excretion (UAE) and glycated hemoglobin (HbA1c) in Type 2 DM (T2DM) patients. MATERIALS AND METHODS: Serum uric acid concentrations, urine microalbumin, and HbA1c were measured in fifty T2DM patients. We then evaluated relationship between uric acid concentrations, degree of UAE and glycemic control as well as other confounding variables. RESULTS: Serum uric acid concentration correlated positively with UAE (r = 0.323, P < 0.05), age (r = 0.337, P < 0.05), age at onset (r = 0.341, P < 0.05), and duration of DM (r = 0.312, P < 0.05). Multiple regression analysis demonstrated that serum uric acid concentration (ß = 0.293, P < 0.0001), duration of DM (ß = 0.261, P < 0.0001), HbA1c (ß = 0.173, P < 0.005), and systolic blood pressure (ß = 0.268, P < 0.005) were independent determinants of UAE. CONCLUSIONS: Serum uric acid concentration is associated with microalbuminuria and HbA1c in T2DM patients.

Selective targeting of FAK-Pyk2 axis by alpha-naphthoflavone abrogates doxorubicin resistance in breast cancer cells.

Despite an initial positive response, breast cancer cells inevitably acquire resistance to doxorubicin (Dox). Alpha-naphthoflavone (ANF) is a well-known chemopreventive agent; however, its anti-cancer properties have not been established. We examined the therapeutic efficacy of ANF in doxorubicin-resistant MCF-7 (MCF-7/Dox) breast cancer cells and investigated its underlying molecular mechanisms of action. MCF-7/Dox cells expressed constitutively active forms of the tyrosine kinases: focal adhesion kinase (FAK-Y397) and protein tyrosine kinase 2 beta (Pyk2- Y579/580) compared with parental MCF-7 cells. ANF significantly enhanced the sensitivity of MCF-7/Dox cells to Dox cytotoxicity in vitro and when co-administered in vivo. This ANF-mediated chemosensitization has dual mechanisms of action: (a) intracellular Dox retention via suppression of P-glycoprotein pump activity, and (b) inhibition of clonogenic cell survival via de-phosphorylation of FAK, Pyk2, and EGF-induced Akt in MCF-7/Dox cells and tumor xenografts. Because of its strong chemosensitization action, broad safety profile, and health benefits, ANF is an attractive anti-cancer drug with therapeutic implications to circumvent drug resistance in breast cancer patients.

Gatifloxacin versus ofloxacin for the treatment of uncomplicated enteric fever in Nepal: an open-label, randomized, controlled trial.

BACKGROUND: Fluoroquinolones are the most commonly used group of antimicrobials for the treatment of enteric fever, but no direct comparison between two fluoroquinolones has been performed in a large randomised trial. An open-label randomized trial was conducted to investigate whether gatifloxacin is more effective than ofloxacin in the treatment of uncomplicated enteric fever caused by nalidixic acid-resistant Salmonella enterica serovars Typhi and Paratyphi A. METHODOLOGY AND PRINCIPAL FINDINGS: Adults and children clinically diagnosed with uncomplicated enteric fever were enrolled in the study to receive gatifloxacin (10 mg/kg/day) in a single dose or ofloxacin (20 mg/kg/day) in two divided doses for 7 days. Patients were followed for six months. The primary outcome was treatment failure in patients infected with nalidixic acid resistant isolates. 627 patients with a median age of 17 (IQR 9-23) years were randomised. Of the 218 patients with culture confirmed enteric fever, 170 patients were infected with nalidixic acid-resistant isolates. In the ofloxacin group, 6 out of 83 patients had treatment failure compared to 5 out of 87 in the gatifloxacin group (hazard ratio [HR] of time to failure 0.81, 95% CI 0.25 to 2.65, p = 0.73). The median time to fever clearance was 4.70 days (IQR 2.98-5.90) in the ofloxacin group versus 3.31 days (IQR 2.29-4.75) in the gatifloxacin group (HR = 1.59, 95% CI 1.16 to 2.18, p = 0.004). The results in all blood culture-confirmed patients and all randomized patients were comparable. CONCLUSION: Gatifloxacin was not superior to ofloxacin in preventing failure, but use of gatifloxacin did result in more prompt fever clearance time compared to ofloxacin. TRIAL REGISTRATION: ISRCTN 63006567 (www.controlled-trials.com).

Nelfinavir suppresses insulin signaling and nitric oxide production by human aortic endothelial cells: protective effects of thiazolidinediones.

BACKGROUND: In human immunodeficiency virus 1 (HIV-1)-infected individuals, exposure to a protease inhibitor (PI)-based highly active antiretroviral therapy (HAART) regimen increases cardiovascular disease and endothelial dysfunction. However, the mechanisms of PI-induced effects on endothelial cells (ECs) are not known. Furthermore, strategies to suppress these deleterious outcomes of PIs need to be developed. Insulin-induced PI3K/Akt signaling and endothelial nitric oxide (NO)-synthase (eNOS) phosphorylation regulates NO production by ECs that maintain vascular homeostasis. We evaluated whether nelfinavir (NEL), a potent HIV-1 PI that suppresses Akt phosphorylation, can alter insulin-induced NO production in human aortic endothelial cells (HAECs) and whether insulin sensitization of HAECs via the peroxisome proliferator-activated receptor-gamma agonists, thiazolidinediones, can ameliorate these side effects. METHODS: Real-time NO production in HAECs was monitored by fluorimetric dyes DAF-FM DA and DAF-2 DA. Immunodetection studies were used to determine the phosphorylation of Akt, eNOS, insulin receptor-ß (IR-ß), insulin receptor substrate-1 (IRS-1), and PI3K/p85α. Expression of eNOS messenger RNA was measured by reverse transcription polymerase chain reaction. RESULTS: In vitro exposure (72 hours) of HAECs to NEL (0.25-2 µg/mL) decreased both basal (2.5-fold) and insulin-induced NO production (4- to 5-fold). NEL suppressed insulin-induced phosphorylation of both Akt and eNOS at serine residues 473 and 1177, respectively. NEL decreased tyrosine phosphorylation of IR-ß, IRS-1, and PI3K. Coexposure to troglitazone (TRO; 250 nM) ameliorated the suppressive effects of NEL on insulin signaling and NO production. Coexposure to TRO also increased eNOS expression in NEL-treated HAECs. CONCLUSION: Our findings indicate that treatment with potent insulin sensitizers may protect against PI-mediated endothelial dysfunction during long-term HAART.

Clinical profile and outcome of patients presenting with mushroom poisoning in a tertiary care center of eastern Nepal.

INTRODUCTION: Accidental mushroom poisoning is constantly seen and regularly reported from all over world. Exact magnitude of problem and its clinical profile in Nepal is not well known. This study was done to evaluate clinical profile and treatment outcome of patients presenting with mushroom poisoning in the department of internal medicine, BPKIHS, Dharan. METHODS: It is a prospective observational study conducted in department of internal medicine, BPKIHS, Dharan from 1st January 2008 to 31st December 2009. Informed consent was taken. All the patients were subjected to necessary laboratory investigation. They were followed up at 1 week and 1 month after discharge. RESULTS: All together 60 patients were analyzed. Majority of subjects 56 (93.3%) were from rural areas. Vomiting and diarrhea were the two most common presentations seen in 56 (93.3%) subjects. The latent period for the symptoms were >6 hours in 4 (6.7%) and <6 hours in 56 (93.3%) subjects. Fulminant hepatic failure was seen in 6 (10%) subjects and among them 4 (66.7%) expired. After admission 3 (5%) subjects developed GI bleeding. Average duration of hospital stay was 4.6 days. In follow up recovery was complete in all subjects who survived the acute phase of poisoning. CONCLUSIONS: Especially in patients coming during rainy season mushroom poisoning should be considered in the differential diagnosis of acute gastroenteritis. Mortality is high in subjects with FHF whereas recovery is complete in subjects who survived the acute phase.

Low-dose methylmercury-induced oxidative stress, cytotoxicity, and tau-hyperphosphorylation in human neuroblastoma (SH-SY5Y) cells.

Acute neurotoxic effects of high-dose methylmercury (MeHg) in humans have been well documented in the scientific literature. However, low-dose effects are less well described. This study was designed to evaluate the effects of low-dose MeHg (<100 nM) on human brain cells in a tissue culture model. Neuroblastoma (NB) cells (SH-SY5Y) were used in the cell culture model to study low-dose effects of MeHg on cell growth, cell survival, reactive oxygen species (ROS), and the phosphorylation of tau protein, as a measure of potential markers of cellular events associated with tauopathies. When cells were incubated in culture with MeHg (50 and 100 nM), there were significant decreases in cell viability as well as significant increase in ROS generation as determined by fluorescent dye analysis (H(2)DCFDA). Furthermore, a concomitant decrease in glutathione levels to 25% of control was observed at both 50 and 100 nM MeHg. In addition, the level of phosphorylated tau was significantly increased after treatment at both 50 and 100 nM MeHg, compared with controls. Pretreatment of NB cells with the antioxidant, N-acetylcysteine (1.25 mM) and the calpain inhibitor, MDL-28170 (10 µM), significantly attenuated the effects of MeHg (50 and 100 nM) on cell viability as well as on tau phosphorylation. These results indicate that low-dose MeHg toxicity may be related to an induction of tau phosphorylation through an oxidative stress-dependent mechanism and that blockade of this pathway may attenuate the toxic effects of MeHg.

Prognostic significance of cytoplasmic SOX9 in invasive ductal carcinoma and metastatic breast cancer.

SOX9, a high mobility group (HMG) box transcription factor, is required for development, differentiation and lineage commitment. It is known to exert its effects through nuclear translocation, such as cell cycle changes in response to retinoic acid treatment in breast cancer cells. However, it is not known whether SOX9 has prognostic significance in human breast cancer. Over-expression and cytoplasmic sequestration of nuclear proteins are implicated in tumor progression. To determine whether SOX9 has any prognostic significance in human breast cancer, its expression and subcellular localization were analyzed in more than 200 human breast carcinomas (BCs). SOX9 mRNA expression data for human BCs were computed from microarray studies available in public databases and correlated with known poor prognostic parameters of BCs. SOX9 protein expression and its correlation with Ki-67 staining in human BCs were assessed using immunohistochemistry. Higher SOX9 mRNA levels were significantly associated with estrogen receptor negative (P ≤ 0.001) and higher grade (P ≤ 0.01) human breast tumors. Patients with higher SOX9 mRNA level had significantly shorter overall survival (P ≤ 0.0001). SOX9 protein, which is normally nuclear, was instead localized in the cytoplasm of 25-30% invasive ductal carcinomas (IDCs) and lymph node metastases. Its cytoplasmic accumulation significantly correlated with enhanced proliferation in breast tumors (Kendall's tau = 0.337 with a P value < 0.0001). Cytoplasmic SOX9 can serve as a valuable prognostic marker for IDCs and metastatic breast cancer. Its significant correlation with breast tumor cell proliferation implies that SOX9 directly contributes to the poor clinical outcomes associated with invasive breast cancer.

Recyclable single-stranded DNA template for synthesis of siRNAs.

RNA interference is a post-transcriptional silencing mechanism triggered by the bioavailability and/or exogenous introduction of double-stranded RNA (dsRNA) into cells. Here we describe a novel method for the synthesis of siRNA in a single vessel. The method employs in vitro transcription and a single-stranded DNA (ssDNA) template and design, which incorporates upon self-annealing, two promoters, two templates, and three loop regions. Using this method of synthesis we generated efficacious siRNAs designed to silence both exogenous and endogenous genes in mammalian cells. Due to its unique design the single-stranded template is easily amenable to adaptation for attachment to surface platforms for synthesis of siRNAs. A siRNA synthesis platform was generated using a 3' end-biotinylated ssDNA template tethered to a streptavidin coated surface that generates stable siRNAs under multiple cycles of production. Together these data demonstrate a unique and robust method for scalable siRNA synthesis with potential application in RNAi-based array systems.

Studies on molecular mechanisms of growth inhibitory effects of thymoquinone against prostate cancer cells: role of reactive oxygen species.

Thymoquinone (TQ), an active ingredient of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against a variety of experimental tumors. However, the precise mechanism of action of TQ is not known. We investigated the mechanism of action of TQ in androgen receptor (AR)-independent (C4-2B) and AR naïve (PC-3) prostate cancer cells, as models of aggressive prostate cancers. Exposure (24-48 h) to TQ (25-150 micromol/L) inhibited the growth of both C4-2B and PC-3 cells, with IC(50) values of approximately 50 and 80 micromol/L, respectively. Within one hour, TQ increased reactive oxygen species (ROS) levels (3-fold) and decreased glutathione (GSH) levels (60%) in both cell types. Pretreatment with N-acetylcysteine (NAC) inhibited both TQ-induced ROS generation and growth inhibition. TQ did not increase the activity of caspases and the caspase inhibitor, z-VAD-FMK did not decrease TQ-induced apoptosis. Furthermore, although TQ treatment resulted in the activation of Jun kinase (JNK), pretreatment with the JNK inhibitor, SP600125, did not protect cells from TQ. However, TQ significantly up-regulated the expressions of growth arrest and DNA damage inducible gene (GADD45alpha) and apoptosis-inducing factor-1 and down-regulated the expressions of several Bc12-related proteins, such as BAG-1, Bcl2, Bcl2A1, Bcl2L1 and BID. In C4-2B cells, TQ dose dependently inhibited both total and nuclear AR levels (4-5 fold) and AR-directed transcriptional activity (10-12 fold). Interestingly, this suppressive effect on AR was not prevented by NAC, which clearly suggested that TQ-induced cytotoxicity is not due to changes in AR regulation. These data suggest that TQ-induced cell death is primarily due to increased ROS generation and decreased GSH levels, and is independent of AR activity.