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Tumor-associated blood vessels differ from normal vessels and play key roles in tumor progression. We aimed to identify biomolecules that are expressed differentially in human bladder cancer-associated blood vessels to find novel biomarkers and mechanisms involved in tumor-associated angiogenesis. The transcriptome of tumor blood vasculature from human invasive bladder carcinoma (I-BLCA) and normal bladder tissue vasculature was compared using differential expression and unsupervised hierarchical clustering analyses. Pathway analysis identified up-regulation of genes involved in the proliferation, cell cycle, angiogenesis, inflammation, and transforming growth factor-β signaling in tumor blood vasculature. A common consensus gene expression signature was identified between bladder cancer tumor blood vasculature with tumor blood vasculature of other solid cancers, which correlated with the overall survival of patients with several of the solid cancers investigated in The Cancer Genome Atlas data set. In bladder tumor blood vasculature, the secreted factor angiopoietin-like protein 2 (ANGPTL2), was confirmed to be up-regulated by quantitative RT-PCR and immunohistochemical staining. The up-regulation of ANGPTL2 in plasma was also observed in non-invasive bladder carcinoma and I-BLCA. We semiquantitatively analyzed expression of ANGPTL2 in tissue microarrays from I-BLCA and surprisingly found an opposite correlation between staining intensity and progression-free survival. Our results indicate that ANGPTL2 might serve as a potential biomarker to predict progression-free survival in I-BLCA., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Knowledge about the clonal evolution of a tumor can help to interpret the function of its genetic alterations by identifying initiating events and events that contribute to the selective advantage of proliferative, metastatic, and drug-resistant subclones. Clonal evolution can be reconstructed from estimates of the relative abundance (frequency) of subclone-specific alterations in tumor biopsies, which, in turn, inform on its composition. However, estimating these frequencies is complicated by the high genetic instability that characterizes many cancers. Models for genetic instability suggest that copy number alterations (CNAs) can influence mutation-frequency estimates and thus impede efforts to reconstruct tumor phylogenies. Our analysis suggested that accurate mutation frequency estimates require accounting for CNAs-a challenging endeavour using the genetic profile of a single tumor biopsy. Instead, we propose an optimization algorithm, Chimæra, to account for the effects of CNAs using profiles of multiple biopsies per tumor. Analyses of simulated data and tumor profiles suggested that Chimæra estimates are consistently more accurate than those of previously proposed methods and resulted in improved phylogeny reconstructions and subclone characterizations. Our analyses inferred recurrent initiating mutations in hepatocellular carcinomas, resolved the clonal composition of Wilms' tumors, and characterized the acquisition of mutations in drug-resistant prostate cancers.

Background: Highly sensitive and specific urinary biomarkers for the early detection of bladder cancer (BC) to improve the performance of urinary cytology are needed., Objective: To investigate the usefulness of methylation markers in voided urine to identify BC presence and grade., Design, Settings, and Participants: Using genome-wide methylation strategies in Toronto, Canada and Liège, Belgium, we have identified differentially methylated genes (TWIST1, RUNX3, GATA4, NID2, and FOXE1) in low-grade vs. high-grade BC tissue and urine. We accrued urine samples from 313 patients using a 2:1 ratio in a case-control setting from Toronto, Canada, Halifax, Canada, and Zurich, Switzerland. We studied the usefulness of these 5 methylated genes to identify BC and discriminate cancer grade in voided urine specimens. Urinary cell sediment DNA was evaluated using qPCR-based MethyLight assay. Multivariable logistic regression prediction models were created., Results and Limitations: We included 211 BC patients (180 nonmuscle invasive) and 102 controls. In univariate analyses, all methylated genes significantly predicted BC vs. no BC, and high grade vs. low grade (all P < 0.05). In multivariable analysis, NID2, TWIST1, and age were independent predictors of BC (all P < 0.05). Sensitivity of NID2 and TWIST1 to predict BC and BC grade was 76.2% and 77.6%, respectively, whereas specificity was 83.3% and 61.1%, respectively. Multivariable models predicting BC overall and discriminating between high-grade and low-grade BC reached area under the receiver operating characteristics curves of 0.89 and 0.78, respectively., Conclusions: This multi-centric study in a real life scenario (different countries, techniques, and pathologists) supports the promise of epigenetic urinary markers in noninvasively detecting BC. With sensitivities and specificities in the range of 80%, the overall performance characteristics of this panel of methylated genes probably does not allow such signature to significantly alter clinical care at this stage but is worth further studying for instance in BC surveillance or screening in high-risk populations., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: We sought to externally validate recently published prostate cancer risk calculators incorporating multiparametric magnetic resonance imaging to predict clinically significant prostate cancer. We also compared the performance of these calculators to that of multiparametric magnetic resonance imaging naïve prostate cancer risk calculators., Materials and Methods: We identified men without a previous prostate cancer diagnosis who underwent transperineal template saturation prostate biopsy with fusion guided targeted biopsy between November 2014 and March 2018 at our academic tertiary referral center. Any Gleason pattern 4 or greater was defined as clinically significant prostate cancer. Predictors, which were patient age, prostate specific antigen, digital rectal examination, prostate volume, family history, previous prostate biopsy and the highest region of interest according to the PI-RADS™ (Prostate Imaging Reporting and Data System), were retrospectively collected. Four multiparametric magnetic resonance imaging prostate cancer risk calculators and 2 multiparametric magnetic resonance imaging naïve prostate cancer risk calculators were evaluated for discrimination, calibration and the clinical net benefit using ROC analysis, calibration plots and decision curve analysis., Results: Of the 468 men 193 (41%) were diagnosed with clinically significant prostate cancer. Three multiparametric magnetic resonance imaging prostate cancer risk calculators showed similar discrimination with a ROC AUC significantly higher than that of the other prostate cancer risk calculators (AUC 0.83-0.85 vs 0.69-0.74). Calibration in the large showed 2% deviation from the true amount of clinically significant prostate cancer for 2 multiparametric magnetic resonance imaging risk calculators while the other calculators showed worse calibration at 11% to 27%. A clinical net benefit was observed only for 3 multiparametric magnetic resonance imaging risk calculators at biopsy thresholds of 15% or greater. None of the 6 investigated prostate cancer risk calculators demonstrated clinical usefulness against a biopsy all strategy at thresholds less than 15%., Conclusions: The performance of multiparametric magnetic resonance imaging prostate cancer risk calculators varies but they generally outperform multiparametric magnetic resonance imaging naïve prostate cancer risk calculators in regard to discrimination, calibration and clinical usefulness. External validation in other biopsy settings is highly encouraged.

Tuberculosis (TB) is one of the major infectious diseases caused by Mycobacterium tuberculosis. The development of an effective and economical vaccine for controlling TB is essential especially for developing countries. Edible plants can serve as biofactories to produce vaccine antigens. In this study, 6 kDa early secretory antigenic target (ESAT-6) of M. tuberculosis was expressed in Brassica oleracea var. italica via Agrobacterium-mediated transformation to facilitate oral delivery of antigen. ESAT-6 gene was cloned using Gateway® cloning strategy. Transformation and presence of transgene was confirmed through PCR. Expression level of transgene was calculated via quantitative real-time PCR (qRT-PCR) and the maximum integrated transgene number was two. Maximum amount of total soluble fraction of ESAT-6 was evaluated by immunoblotting, estimated to accumulate up to 0.5% of total soluble protein. The recombinant ESAT-6 protein was further purified and detected using silver staining and Western blotting. ESAT-6 protein induced humoral immune response in mice immunized orally and subcutaneously. The expression of M. tuberculosis antigen in edible plants could aid in the development of cost-effective and oral delivery of an antigen-based subunit vaccine against TB. To the best our knowledge, it is the first report of expression of a vaccine antigen in broccoli., (© 2019 International Union of Biochemistry and Molecular Biology, Inc.)

Response classification after neoadjuvant chemotherapy in muscle-invasive bladder carcinoma is based on the TNM stage at radical cystectomy. We recently showed that histopathologic tumor regression grades (TRGs) add prognostic information to TNM. Our aim was to validate the prognostic significance of TRG in muscle-invasive bladder cancer in a multicenter setting. We enrolled 389 patients who underwent cisplatin-based chemotherapy before radical cystectomy in 8 centers between 2010 and 2016. Median follow-up was 2.2 years. TRG was determined in radical cystectomy specimens by local pathologists. Central pathology review was conducted in 20% of cases, which were randomly selected. The major response was defined as ≤pT1N0. The remaining patients were grouped into partial responders (≥ypT2N0-3 and TRG 2) and nonresponders (≥ypT2N0-3 and TRG 3). TRG was successfully determined in all cases, and interobserver agreement in central pathology review was high (κ=0.83). After combining TRG and TNM, 47%, 15%, and 38% of patients were major, partial, and nonresponders, respectively. Combination of TRG and TNM showed significant prognostic discrimination of overall survival (major responder: reference; partial responder: hazard ratio 3.5 [95% confidence interval: 1.8-6.8]; nonresponder: hazard ratio 6.1 [95% confidence interval: 3.6-10.3]). This discrimination was superior compared with TNM staging alone, supported by 2 goodness-of-fit criteria (P=0.041). TRG is a simple, reproducible histopathologic measurement of response to neoadjuvant chemotherapy in muscle-invasive bladder cancer. Integrating TRG with TNM staging resulted in significantly better prognostic stratification than TNM staging alone.

Mycobacterium tuberculosis causes tuberculosis in humans. The major disease burden of tuberculosis lies in developing countries. Lack of an effective vaccine for adults is one of the major hurdles for controlling this deadly disease. In the present study, 6 kDa early secretory antigenic target (ESAT-6) of M. tuberculosis was inducibly expressed in chloroplasts of Nicotiana tabacum. The expression of ESAT-6 in chloroplasts was controlled by T7 promoter that was activated by nuclear-generated signal peptide. Tobacco plants, containing nuclear component, were transformed via biolistic bombardment with pEXP-T7-ESAT-6 obtained by Gateway® cloning. Transformation and homoplasmic status of transplastomic plants was confirmed by polymerase chain reaction and Southern blotting. Plants were induced for protein expression by spraying with 5% ethanol for 1 day, 3 days, 7 days and 10 days. ESAT-6 protein was detected by immunoblot analysis and maximum protein was obtained for 10 days induced plants that was estimated to accumulate up to 1.2% of total soluble fraction of protein. Transplastomic plants showed completely normal morphology. Transplastomic and untransformed plants became slightly chlorotic upon prolonged exposure to ethanol until 10 days. Taken together, this data could help in the development of an antigen-based subunit vaccine against tuberculosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Formalin-fixed, paraffin-embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here, we developed a pressure cycling technology (PCT)-SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B-cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh-frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored between 1 and 15 years in a biobank and show a high degree of the proteome pattern similarity between two types of histological regions in small FFPE samples, that is, punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of a certain degree of biological variations. Applying the method to two independent DLBCL cohorts, we identified myeloperoxidase, a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Background: Online clinical risk prediction tools built on data from multiple cohorts are increasingly being utilized for contemporary doctor-patient decision-making and validation. This report outlines a comprehensive data science strategy for building such tools with application to the Prostate Biopsy Collaborative Group prostate cancer risk prediction tool., Methods: We created models for high-grade prostate cancer risk using six established risk factors. The data comprised 8492 prostate biopsies collected from ten institutions, 2 in Europe and 8 across North America. We calculated area under the receiver operating characteristic curve (AUC) for discrimination, the Hosmer-Lemeshow test statistic (HLS) for calibration and the clinical net benefit at risk threshold 15%. We implemented several internal cross-validation schemes to assess the influence of modeling method and individual cohort on validation performance., Results: High-grade disease prevalence ranged from 18% in Zurich (1863 biopsies) to 39% in UT Health San Antonio (899 biopsies). Visualization revealed outliers in terms of risk factors, including San Juan VA (51% abnormal digital rectal exam), Durham VA (63% African American), and Zurich (2.8% family history). Exclusion of any cohort did not significantly affect the AUC or HLS, nor did the choice of prediction model (pooled, random-effects, meta-analysis). Excluding the lowest-prevalence Zurich cohort from training sets did not statistically significantly change the validation metrics for any of the individual cohorts, except for Sunnybrook, where the effect on the AUC was minimal. Therefore the final multivariable logistic model was built by pooling the data from all cohorts using logistic regression. Higher prostate-specific antigen and age, abnormal digital rectal exam, African ancestry and a family history of prostate cancer increased risk of high-grade prostate cancer, while a history of a prior negative prostate biopsy decreased risk (all p-values < 0.004)., Conclusions: We have outlined a multi-cohort model-building internal validation strategy for developing globally accessible and scalable risk prediction tools.

Purpose: To evaluate the prognostic value of positive surgical margins (PSM) focality for the prediction of biochemical recurrence (BCR) in patients undergoing robotic-assisted radical prostatectomy (RARP) for prostate cancer., Methods: All men with clinically localized prostate cancer undergoing RARP in our tertiary referral centre between May 2005 and August 2016 were retrospectively identified. Patients with neoadjuvant therapy were excluded. Comparisons were made between cases with negative surgical margins (NSM), unifocal PSM (uPSM), and multifocal PSM (mPSM)., Results: From a total of 973 patients available for analysis, 315 (32%) had a PSM. In these patients, 190 had uPSM and 125 had mPSM. Focality of PSM was significantly associated with tumour stage and grade, preoperative PSA, and postoperative PSA persistence (all p < 0.001), but not with nerve sparing (NS) (p = 0.15). PSA persistence was found in 120 (12%) patients, resulting in 853 patients available for survival analyses with a median follow-up of 52 months. Both uPSM and mPSM were found to be independent predictors of BCR, conferring a hazard ratio of 1.9 (95% CI 1.3-3.0; p = 0.002) and 3.4 (95% CI 2.1-5.6; p < 0.001), respectively, when compared to NSM. In subgroup analyses, PSM was particularly predictive for BCR when patients underwent unilateral or bilateral NS (p ≤ 0.003)., Conclusions: Based on a large case series of RARP, we found PSM focality to be an independent predictor of BCR, with a 1.9- and 3.4-fold risk increase for BCR in case of uPSM and mPSM, respectively. PSM seems to be of particular prognostic relevance when NS has been performed.

Background: Most studies on adolescent screening, brief intervention, and referral to treatment (SBIRT) have examined substance use outcomes. However, it may also impact service use and comorbidity-an understudied topic. We address this gap by examining effects of SBIRT on health care use and comorbidities., Methods: In a randomized trial sample, we assessed 3 SBIRT care modalities: (1) pediatrician-delivered, (2) behavioral clinician-delivered, and (3) usual. Medical comorbidity and health care use were compared between a brief-intervention group with access to SBIRT for behavioral health (combined pediatrician and behavioral clinician arms) and a group without (usual care) over 1 and 3 years., Results: Among a sample of eligible adolescents ( n = 1871), the SBIRT group had fewer psychiatry visits at 1 year (incidence rate ratio [iRR] = 0.76; P = .05) and 3 years (iRR = 0.65; P < .05). Total outpatient visits did not differ in year 1. The SBIRT group was less likely to have mental health diagnoses (odds ratio [OR] = 0.69; 95% confidence interval [CI] = 0.48-1.01) or chronic conditions (OR = 0.66; 95% CI = 0.45-0.98) at 1 year compared with those in usual care. At 3 years, the SBIRT group had fewer total outpatient visits (iRR = 0.85; P < .05) and was less likely to have substance use diagnoses (OR = 0.64; 95% CI = 0.45-0.91) and more likely to have substance use treatment visits (iRR = 2.04; P < .01)., Conclusions: Providing SBIRT in pediatric primary care may improve health care use and health, mental health, and substance use outcomes. We recommend further exploring the effects of SBIRT on these outcomes., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2019 by the American Academy of Pediatrics.)

Systemic delivery of nucleic acids to the central nervous system (CNS) is a major challenge for the development of RNA interference-based therapeutics due to lack of stability, target specificity, non-permeability to the blood-brain barrier (BBB), and lack of suitable carriers. Using a designed bi-functional fusion protein TARBP-BTP in a complex with siRNA, we earlier demonstrated knockdown of target genes in the brain of both AβPP-PS1 (Alzheimer's disease, AD) and wild-type C57BL/6 mice. In this report, we further substantiate the approach through an extended use in AβPP-PS1 mice, which upon treatment with seven doses of β-secretase AβPP cleaving Enzyme 1 (BACE1) TARBP-BTP:siRNA, led to target-specific effect in the mouse brain. Concomitant gene silencing of BACE1, and consequent reduction in plaque load in the cerebral cortex and hippocampus (greater than 60%) in mice treated with TARBP-BTP:siRNA complex, led to improvement in spatial learning and memory. The study validates the efficiency of TARBP-BTP fusion protein as an efficient mediator of RNAi, giving considerable scope for future intervention in neurodegenerative disorders through the use of short nucleic acids as gene specific inhibitors.

Early and specific diagnosis of oxidative stress linked diseases as cardiac heart diseases remains a major dilemma for researchers and clinicians. MicroRNAs may serve as a better tool for specific early diagnostics and propose their utilization in future molecular medicines. We aimed to measure the microRNAs expressions in oxidative stress linked cardiac hypertrophic condition induced through stimulants as Endothelin and Isoproterenol. Cardiac hypertrophic animal models were confirmed by BNP, GATA4 expression, histological assays, and increased cell surface area. High oxidative stress (ROS level) and decreased antioxidant activities were assessed in hypertrophied groups. Enhanced expression of miR-152, miR-212/132 while decreased miR-142-3p expression was observed in hypertrophic condition. Similar pattern of these microRNAs was detected in HL-1 cells treated with H 2 O 2 . Upon administration of antioxidants, the miRNAs expression pattern altered from that of the cardiac hypertrophied model. Present investigation suggests that oxidative stress generated during the cardiac pathology may directly or indirectly regulate anti-hypertrophy pathway elements through microRNAs including antioxidant enzymes, which need further investigation. The down-regulation of free radical scavengers make it easier for the oxidative stress to play a key role in disease progression., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: To determine the pain of single versus multiple visit endodontic treatment after obturation in teeth with necrotic pulps and infected canals., Methods: The randomized controlled study was conducted from January to June 2016 at Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad, Pakistan. Dental patients aged 18-60 years of either gender were selected who were subjected to computer-generated randomisation. Both maxillary and mandibular single rooted teeth till second premolar were included. Single visit endodontic treatment to Group-I subjects was carried out by gaining access to canals, cleaning, shaping and obturation at same visit whereas in Group-II patients, who underwent multiple visits, cleaning and shaping was followed by placement of intra-canal medicament, while obturation was done 5 days later. Patients were recalled within 48 hours after obturation to record the pain score using visual analogue scale.., Results: Of the 60 patients, 30(50%) were in each of the two groups. There was no significant difference in post-operative pain in the two groups (p=0.8). The frequency of post-operative pain was not significant either (p>0.05). The post-operative mean pain score was 2.23±1.736 in Group-I and 2.38±1.94 in Group-II (p=0.8).., Conclusions: Treatment carried out in single visit or multiple visits showed no difference in pain frequency.

Objective: To compare the frequency of pain in root canal treatment using sodium hypochlorite and chlorhexidine as root canal irrigants., Methods: The cross-sectional study was carried out from January to June 2016 at the Pakistan Institute of Medical Sciences (PIMS), Islamabad, Pakistan, and comprised patients aged 18-40 years with irreversible pulpitis involving a posterior tooth. The patients were divided into two equal groups, with Group-I receiving 2% chlorhexidine, and Group-II receiving 5.25% sodium hypochlorite. Following the use of the irrigants, the selected teeth were evaluated for pain at 72 hours using Visual Analogue Scale., Results: There were 60 patients divided into groups of 30(50%) each. The overall mean age was 27.97±5.9 years. There were 32 (53.3%) females and 28 (46.6%) males. The mean post-operative pain score was 1.70±1.9 in Group-I and 1.90 ± 2.3 in Group-II (p=0.5). In Group-I, 25(83.3%) patients had no pain while in Group-II, 20(66.7%) had no pain (p=0.1)., Conclusions: There was no difference in post-operative pain at 72 hours between 2% chlorhexidine and 5.25% sodium hypochlorite when used as irrigants.

Background: As a gold standard, calcium hydroxide has always been used as a root canal medicament against root canal pathogens but its combination with chlorhexidine is not being employed in routine despite its documented efficacy and results. The objective of the study is to compare calcium hydroxide and combination of calcium hydroxide with 2% CHX on the basis of radiographic evidence of resolution of periapical radiolucency when they are used as an interappointment dressing /intracanal medicament for 14 days.., Methods: A total of 60 patients of either gender was included in the study. The age range was 15-60 years with the radiographic evidence of radiolucency of 2-4 mm in size. Both maxillary and mandibular teeth till first molar were selected. Teeth with previous endodontic treatment mentally handicapped and terminally ill patients were excluded. Selected patients were placed randomly into Group 1 (calcium hydroxide alone) and in Group 2 (calcium hydroxide in combination with 2% chlorhexidine gel). Obturation and permanent filling was done at 14th day. The patient was recalled after 90 days of obturation and a radiograph was taken. Final outcome was measured at the end of 90thday of obturation., Results: The mean of percentage reduction in size of apical radiolucency at 3 months was 91.03% for group 1 and 97.26% for group 2 with p-value 0.13 (not significant). The results suggested equivalent results after medication with 2%chlorhexidine with calcium hydroxide but 2% chlorhexidine still showed better results than the group without CHX in terms of healing., Conclusions: This study concluded that calcium hydroxide in combination with 2% chlorhexidene as intracanal medicament results in better resolution of periapical radiolucency in terms of percentage resolution of periapical radiolucency.

Alzheimer's disease (AD) is the most common neurodegenerative disease associated with progressive loss of cognitive function, personality, and behavior. The present study evaluates neuronal and astroglial metabolic activity, and neurotransmitter cycle fluxes in AβPP-PS1 mouse model of AD by using 1 H-[ 13 C]-nuclear magnetic resonance (NMR) spectroscopy together with an infusion of either [1,6- 13 C 2 ]glucose or [2- 13 C]acetate. The levels of N-acetyl-aspartate (NAA) and glutamate were found to be decreased in the cerebral cortex and hippocampus in AβPP-PS1 mice, when compared with wild type controls. The cerebral metabolic rate of acetate oxidation was increased in the hippocampus and cerebral cortex of AβPP-PS1 mice suggesting enhanced astroglial activity in AD. AβPP-PS1 mice exhibit severe reduction in glutamatergic and gamma-amino butyric acid (GABA)ergic neuronal metabolic activity and neurotransmitter cycling fluxes in the hippocampus, cerebral cortex, and striatum as compared with controls. These data suggest that metabolic activity of excitatory and inhibitory neurons is compromised across brain in AβPP-PS1 mouse model of AD.

It remains unclear to what extent tumor heterogeneity impacts on protein biomarker discovery. Here, we quantified proteome intra-tissue heterogeneity (ITH) based on a multi-region analysis of prostate tissues using pressure cycling technology and SWATH mass spectrometry. We quantified 6,873 proteins and analyzed the ITH of 3,700 proteins. The level of ITH varied depending on proteins and tissue types. Benign tissues exhibited more complex ITH patterns than malignant tissues. Spatial variability of ten prostate biomarkers was validated by immunohistochemistry in an independent cohort (n=83) using tissue microarrays. PSA was preferentially variable in benign prostatic hyperplasia, while GDF15 substantially varied in prostate adenocarcinomas. Further, we found that DNA repair pathways exhibited a high degree of variability in tumorous tissues, which may contribute to the genetic heterogeneity of tumors. This study conceptually adds a new perspective to protein biomarker discovery: it suggests that recent technological progress should be exploited to quantify and account for spatial proteome variation to complement biomarker identification and utilization., Competing Interests: Competing financial interests R.A. holds shares of Biognosys AG, which operates in the field covered by the article. The research group of R.A. is supported by SCIEX, which provides access to prototype instrumentation, and Pressure Biosciences, which provides access to advanced sample preparation instrumentation.

Background: Micro leakage around the margins of a restoration is believed to be one of the main causes of postoperative sensitivity. Dental amalgam is a non-insulating material and has the potential to transfer heat and cold causing irritation of the pulp. Different dentin tubule sealers are used under amalgam restoration to compensate for this post-operative sensitivity. This study was conducted to compare the frequency of sensitivity in amalgam restorations using copal varnish and dentin adhesive liner (dentin bonding agent)., Methods: A total of 60 patients of either gender, aged 18-40 years having class 1 carries in posterior teeth were included. Teeth with restorations, dentinal sensitivity and patients taking analgesic drugs for chronic pain conditions were excluded. The selected patients were placed randomly into Group A (copal varnish) & Group B (dentin adhesive liner), by using computer generated table of random numbers. Restored teeth were evaluated 1-month post operatively for sensitivity., Results: Mean age was 25.63±5.42 years. Out of 60 patients, 68.0% were females and 32.0% were males with a female to male ratio of 2:1. The mean post-operative pain score was 2.83±2.79 in Group A and in Group B, it was 1.43±2.14 with a p-value of 0.03. There was no pain on application of a cold stimulus in 14 (46.7%) patients in Group A (copal varnish) while in Group B (Dentin adhesive), no pain was seen in 23 (76.7%) patients with p-value of 0.02., Conclusions: This study concluded that dentin adhesive liner (dentin bonding agent) is better than copal varnish in reducing postoperative sensitivity in amalgam restorations.

Background: Prostate-specific antigen (PSA) test is of paramount importance as a diagnostic tool for the detection and monitoring of patients with prostate cancer. In the presence of interfering factors such as heterophilic antibodies or anti-PSA antibodies the PSA test can yield significantly falsified results. The prevalence of these factors is unknown., Methods: We determined the recovery of PSA concentrations diluting patient samples with a standard serum of known PSA concentration. Based on the frequency distribution of recoveries in a pre-study on 268 samples, samples with recoveries <80% or >120% were defined as suspect, re-tested and further characterized to identify the cause of interference., Results: A total of 1158 consecutive serum samples were analyzed. Four samples (0.3%) showed reproducibly disturbed recoveries of 10%, 68%, 166% and 4441%. In three samples heterophilic antibodies were identified as the probable cause, in the fourth anti-PSA-autoantibodies. The very low recovery caused by the latter interference was confirmed in serum, as well as heparin- and EDTA plasma of blood samples obtained 6 months later. Analysis by eight different immunoassays showed recoveries ranging between <10% and 80%. In a follow-up study of 212 random plasma samples we found seven samples with autoantibodies against PSA which however did not show any disturbed PSA recovery., Conclusions: About 0.3% of PSA determinations by the electrochemiluminescence assay (ECLIA) of Roche diagnostics are disturbed by heterophilic or anti-PSA autoantibodies. Although they are rare, these interferences can cause relevant misinterpretations of a PSA test result.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder, characterized by progressive loss of cognitive functions and memory. Excessive intake of aluminum chloride in drinking water is associated with amyloid plaques and neurofibrillary tangles in the brain, which are the hallmark of AD. We have evaluated brain energy metabolism in aluminum chloride (AlCl 3 ) mouse model of AD. In addition, effectiveness of Rasa Sindoor (RS), a formulation used in Indian Ayurvedic medicine, for alleviation of symptoms of AD was evaluated. Mice were administered AlCl 3 (40 mg/kg) intraperitoneally once a day for 60 days. The memory of mice was measured using Morris Water Maze test. The 13 C labeling of brain amino acids was measured ex vivo in tissue extracts using 1 H-[ 13 C]-NMR spectroscopy with timed infusion of [1,6- 13 C 2 ]glucose. The 13 C turnover of brain amino acids was analyzed using a three-compartment metabolic model to derive the neurotransmitter cycling and TCA cycle rates associated with glutamatergic and GABAergic pathways. Exposure of AlCl 3 led to reduction in memory of mice. The glutamatergic and GABAergic neurotransmitter cycling and glucose oxidation were found to be reduced in the cerebral cortex, hippocampus, and striatum following chronic AlCl 3 treatment. The perturbation in metabolic rates was highest in the cerebral cortex. However, reduction in metabolic fluxes was higher in hippocampus and striatum following one month post AlCl 3 treatment. Most interestingly, oral administration of RS (2 g/kg) restored memory as well as the energetics of neurotransmission in mice exposed to AlCl 3 . These data suggest therapeutic potential of RS to manage cognitive functions and memory in preclinical AD.

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by progressive loss of memory and cognitive function. The cerebral metabolic rate of glucose oxidation has been shown to be reduced in AD. The present study evaluated efficacy of dietary Amalaki Rasayana (AR), an Ayurvedic formulation used in Indian traditional system, in AbPP-PS1 mouse model of AD in ameliorating memory and neurometabolism, and compared with donepezil, a standard FDA approved drug for AD. The memory of mice was measured using Morris Water Maze analysis. The cerebral metabolism was followed by 13C labelling of brain amino acids in tissue extracts ex vivo using 1H-[13C]-NMR spectroscopy together with a short time infusion of [1,6-13C2]glucose to mice. The intervention with Amalaki Rasayana showed improved learning and memory in AbPP-PS1 mice. The 13C labelings of GluC4, GABAC2 and GlnC4 were reduced in AbPP-PS1 mice when compared with wild-type controls. Intervention of AR increased the 13C labelling of amino acids suggesting a significant enhancement in glutamatergic and GABAergic metabolic activity in AbPP-PS1 mice similar to that observed with donepezil treatment. These data suggest that AR has potential to improve memory and cognitive function in AD.

Background: The prognostic role of preoperative serum lipid levels in patients undergoing radical prostatectomy (RP) for clinically localized prostate cancer (PCa) is unclear. The aim of the present study was to investigate preoperative serum lipid levels in patients with clinically localized PCa undergoing RP and their association with clinicopathological features and oncological outcome., Methods: Preoperative lipid levels (total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides) and statin use from consecutive patients with clinically localized PCa undergoing RP in a tertiary referral center between 2008 and 2015 were recorded and patients were followed prospectively. Logistic regression analysis was used to test the association between lipid levels and clinicopathological parameters. Lipid values were analyzed both as continuous and dichotomized variables. Univariable and multivariable Cox regression analyses were performed to identify predictors for recurrence-free survival (RFS). Recurrence was defined as rising and verified PSA levels >0.1 ng/ml., Results: Our cohort consisted of 371 men with a median age of 63 years (range 41-78 years) and a median preoperative PSA value of 6.79 ng/ml (0.43-81.4 ng/ml). Median follow-up was 28 months (1-64). No association was found between lipid levels and adverse pathological characteristics such as ≥pT3, Gleason score ≥8, positive nodal status and positive surgical margins. Recurrence occurred in 49 patients (15.4%) at a median time of 18 months (2-51 month). Compared to low LDL cholesterol, high LDL cholesterol was associated with longer RFS in univariable analysis (continuous: Hazard Ratio (HR): 0.67, 95%-Confidence Interval (CI): 0.47-0.96, P = 0.03; 3 mM cut-point: HR: 0.44, 95%-CI: 0.24-0.79, P = 0.006). Neither levels of other lipids, nor statin use were associated with RFS. Preoperative LDL cholesterol remained an independent predictor for PCa recurrence in a multivariable model adjusted for age, preoperative PSA, statin use, tumor stage, Gleason score, nodal status and surgical margin status (continuous: HR: 0.66, 95%-CI: 0.44-0.99, P = 0.04; 3 mM cut-point: HR: 0.41, 95%-CI: 0.21-0.78, P = 0.007)., Conclusions: This is the first prospective study showing the potential adverse and independent prognostic role of low preoperative LDL cholesterol levels in patients with localized PCa undergoing RP. Prostate 77:549-556, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Microscopy image data of human cancers provide detailed phenotypes of spatially and morphologically intact tissues at single-cell resolution, thus complementing large-scale molecular analyses, e.g., next generation sequencing or proteomic profiling. Here we describe a high-resolution tissue microarray (TMA) image dataset from a cohort of 71 prostate tissue samples, which was hybridized with bright-field dual colour chromogenic and silver in situ hybridization probes for the tumour suppressor gene PTEN. These tissue samples were digitized and supplemented with expert annotations, clinical information, statistical models of PTEN genetic status, and computer source codes. For validation, we constructed an additional TMA dataset for 424 prostate tissues, hybridized with FISH probes for PTEN, and performed survival analysis on a subset of 339 radical prostatectomy specimens with overall, disease-specific and recurrence-free survival (maximum 167 months). For application, we further produced 6,036 image patches derived from two whole slides. Our curated collection of prostate cancer data sets provides reuse potential for both biomedical and computational studies.

Purpose: We externally validated a novel prostate cancer risk calculator based on data from the Swiss arm of the ERSPC and assessed whether the risk calculator (ProstateCheck) is superior to the PCPT-RC and SWOP-RC in an independent Swiss cohort., Materials and Methods: Data from all men who underwent prostate biopsy at an academic tertiary care center between 2004 and 2012 were retrospectively analyzed. The probability of having any prostate cancer or high grade prostate cancer (Gleason score 7 or greater) on prostate biopsy was calculated using the ProstateCheck. Risk calculator performance was assessed using calibration and discrimination, and additionally compared with the PCPT-RC and SWOP-RC by decision curve analyses., Results: Of 1,615 men 401 (25%) were diagnosed with any prostate cancer and 196 (12%) with high grade prostate cancer. Our analyses of the ProstateCheck-RC revealed good calibration in the low risk range (0 to 0.4) and moderate overestimation in the higher risk range (0.4 to 1) for any and high grade prostate cancer. The AUC for the discrimination of any prostate cancer and high grade prostate cancer was 0.69 and 0.72, respectively, which was slightly but significantly higher compared to the PCPT-RC (0.66 and 0.69, respectively) and SWOP-RC (0.64 and 0.70, respectively). Decision analysis, taking into account the harms of transrectal ultrasound measurement of prostate volume, showed little benefit for ProstateCheck-RC, with properties inferior to those of the PCPT-RC and SWOP-RC., Conclusions: Our independent external evaluation revealed moderate performance of the ProstateCheck-RC. Its clinical benefit is limited, and inferior to that of the PCPT-RC and SWOP-RC., (Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: To evaluate whether the rate of Gleason score (GS) upgrade on final pathology, the rate of positive surgical margins (PSM) and the rate of biochemical recurrence (BCR) after radical prostatectomy (RP) were different if prostate biopsy (PB) was graded by community pathologists (CP) as compared to specialized uro-pathologists (UP)., Methods: A consecutive series of patients undergoing RP in our institution between 2005 and 2013 were retrospectively reviewed. Any GS higher or lower in RP specimen as compared to PB GS was defined as GS upgrade or downgrade, respectively. Additionally, stratification for the new ISUP 2014 grading system was performed. Predictors of GS upgrade and PSMs and prognostic parameters for BCR were assessed by stepwise logistic regression models and by multivariable Cox regression analyses, respectively., Results: A total of 786 patients were available for analysis, and median follow-up was 36 months (1-101 months). A GS upgrade was found in 345 patients (43.9 %) and a GS downgrade in 91 patients (11.6 %). Discordance between PB GS and RP GS was significantly more frequent when grading had been performed by a CP (50.5 % upgrade, 9.0 % downgrade) than by a UP (33.1 % upgrade, 15.7 % downgrade, p < 0.001). CP evaluation was an independent predictor for GS upgrade (odds ratio [OR] 1.91, p < 0.001) and for PSMs (OR 1.69, p = 0.003), as well as an independent predictor of BCR (hazard ratio [HR] 1.65, p = 0.028)., Conclusions: Pathologic evaluation of PBs by a dedicated UP should be recommended to reduce the rate of biopsy undergrading, PSM and BCR after RP.

RNA interference represents a novel therapeutic approach to modulate several neurodegenerative disease-related genes. However, exogenous delivery of siRNA restricts their transport into different tissues and specifically into the brain mainly due to its large size and the presence of the blood-brain barrier (BBB). To overcome these challenges, we developed here a strategy wherein a peptide known to target specific gangliosides was fused to a double-stranded RNA binding protein to deliver siRNA to the brain parenchyma. The designed fusion protein designated as TARBP-BTP consists of a double-stranded RNA-binding domain (dsRBD) of human Trans Activation response element (TAR) RNA Binding Protein (TARBP2) fused to a brain targeting peptide that binds to monosialoganglioside GM1. Conformation-specific binding of TARBP2 domain to siRNA led to the formation of homogenous serum-stable complex with targeting potential. Further, uptake of the complex in Neuro-2a, IMR32 and HepG2 cells analyzed by confocal microscopy and fluorescence activated cell sorting, revealed selective requirement of GM1 for entry. Remarkably, systemic delivery of the fluorescently labeled complex (TARBP-BTP:siRNA) in ΑβPP-PS1 mouse model of Alzheimer's disease (AD) led to distinctive localization in the cerebral hemisphere. Further, the delivery of siRNA mediated by TARBP-BTP led to significant knockdown of BACE1 in the brain, in both ΑβPP-PS1 mice and wild type C57BL/6. The study establishes the growing importance of fusion proteins in delivering therapeutic siRNA to brain tissues., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: To see the efficiency, adequacy and accuracy of toluidine blue stained smears of FNAC of Breast thyroid and salivary glands swelling with comparison to conventional stained FNAC smears with Papanicolaou., Methods: A total of 114 aspirates from various sites were included in the study. The smears were stained with toluidine blue and conventional Papanicolaou stain and the cytomorphology of both the smears were compared. The values were tabulated and statistical tests of significance was applied., Results: Of the 114 aspirates included in our study the diagnostic accuracy by using papanicolaou was 78%, while it was upto 100% with supravital toluidine blue stained smears. The percentage of inadequacy was reduced to just 25%. The observations were statistically significant. Breast 37/48 (77%) and Salivary glands 11/48 (23%) respectively. The most commonly used categorization of a five-tier system was used for reporting of breast cytology, with categories ranging from insufficient materials (C1), benign (C2), atypical (C3), suspicious of malignancy (C4), or (C5) frankly malignant. Most of breast lesions were benign 25 (67.56%). There were only 9 (24.32%) malignant cases followed by 2 cases of C-4 and one case of C-3. Benign thyroid lesion were more frequent comprising of 51 (72.27%) cases. One case (1.5%) of papillary carcinoma was found while 13 case were follicular lesions. There were 4 (36.4%) cases of pleomorphic adenoma and 3 (27.3) cases of non-specific sialadenitis. There was one case (9%) of each lesion for mucoepidermoid carcinoma, adenoidcytic carcinoma and benign cyst., Conclusion: Toluidine blue stained study of FNAC improves the diagnostic accuracy by minimizing the smearing and drying artifact, loss of cell sample during fixation and staining which influences the diagnostic accuracy.

Objective: To evaluate the Bone Mineral Density (BMD) and diet of teachers of a Govt. College of Home Economics in Lahore., Methods: It was survey research. Purposive sampling technique was adopted for the selection of 50 teachers from Govt. College of Home Economics of age group 30 - 60 years., Results: About 46% of the subjects had BMD ratio in between -2.58 to -4.0 (Osteoporotic category). The root cause of low BMD ratio was not really age related but in majority of the sample it was due to sedentary life style and lack of awareness about the importance of exercise in relation to bone health., Conclusion: The total mineral and vitamin intake required for bone health (calcium, magnesium, phosphorus & vitamin D) was below the recommended, among majority of the sample.

Objective: To determine the frequency of placenta Previa in patients coming to a tertiary care unit with previously scarred and non-scarred uterus., Methods: A descriptive cross sectional study was carried on 114 cases who underwent caesarean sections (37 cases out of 645 cases with non scarred uterus and 77 cases from 721 cases with scarred uterus) in the department of obstetrics and gynecology Lady Willingdon Hospital from January 2008- December 2011., Results: Most patients (47.36%) were between 26-30 years age group, presented with gestational age between 36-40 weeks (70.17%), were mostly G2-4, while frequency of placenta Previa in non-scarred uterus was 32.45% (37 cases), and frequency in previously scarred uterus was 67.54% (77 cases). Major degree Previa was found in 88 cases (77.19%). There were 5.70% cases of placenta Previa from non-scarred uteruses and 10.67% cases of placenta Previa (10.67%) from already scarred uteruses. Stratification revealed a higher trend of the morbidity with the increase in number of previous caesarean sections., Conclusion: A significantly higher frequency of placenta Previa was found among patients coming to a tertiary care hospital with previously scarred uterus.

Aim: To determine the frequency of HER2/neu protein overexpression in gastric (group A), small intestine (group B), and colorectal (group C) adenocarcinoma., Methods: A descriptive, cross-sectional study was performed on 50 cases of gastrointestinal adenocarcinoma (stomach, small intestine, and colorectal); 11 from group A, 8 from group B, and 31 from group C. The samples were grossed and processed in the pathology department, and sections were stained with HE (hematoxylin and eosin stain) for histopathological confirmation of malignancy (well-differentiated, moderately-differentiated, and poorly-differentiated). The confirmed samples were processed for immunomarker study of HER2/neu., Results: HER2/neu protein overexpression was found in 33 (66%) patients overall (P = 0.000). Out of 33 HER2/neu positive subjects, 23 (69.6%) were from group C, while the remaining 10 (30%) were from group A. None of the patients from group B had positive HER2/neu protein overexpression. No protein overexpression or membrane staining in < 10% tumor cells was observed in 17 (34%) patients, which were labeled as score "0" and considered negative for HER2/neu protein overexpression. Faint/weak staining (in ≥ 10% of tumors cells) were observed in 8 (16%) patients and given the "1+" score. Similarly 13 (26%) patients reported moderate staining (in ≥ 10% tumor cells) and were thus labeled as "2+", and strong staining (in ≥ 10% tumors cells), labeled as "3+", was observed in 12 (24%) patients. Out of 50 patients, 26 (52%) were suffering from grade-II malignancy, 16 (32%) from grade-I, and 8 (16%) from grade-III. There was highly significant association between tumor grades and HER2/neu protein overexpression (P = 0.0000)., Conclusion: HER2/neu protein is credibly overexpressed in colon and gastric adenocarcinomas in immunohistochemistry. There is significant association between grade of tumor and HER2/neu protein overexpression.

Isolated hepatic tuberculosis without pulmonary or bowel involvement is a diagnostic challenge and can cause considerable morbidity. A young lady from Lahore presented with fever, pain in right hypochondria, nausea and weight loss. CT scan of abdomen showed multiple small hypodense non-enhancing lesions and a heterogeneous texture of liver. Biopsy confirmed the diagnosis of hepatic tuberculosis. It was concluded a case of isolated hepatic tuberculosis without evidence of other primary sites involvement. It is important to consider tuberculosis in the differential diagnosis when suspecting lymphoproliferative or metastatic diseases in a patient with vague symptoms and abnormal hepatic texture on CT.

Background: Premalignant and malignant lesions are not uncommon in Pakistani women, especially in the older age-groups, Aim: This study was conducted to determine the clinicopathological importance of conventional Papanicolaou (Pap) smears for the diagnosis of premalignant and malignant lesions of the cervix., Materials and Methods: Pap smears of 1000 women were examined from January 2007 to June 2009. Only cases with neoplastic cytology were included., Results: The overall frequency of normal, inadequate, neoplastic, and infective smears was 50%, 1.8%, 10.2%, and 38.3%, respectively. Most of the patients (67%) were in the postmenopausal age-group, with the mean age being 44.7±15.63 years. The commonest clinical signs/symptoms seen among the 102 patients with neoplastic gynecological lesions were vaginal discharge and abnormal bleeding (93/102;(91.2% and 62/102;60.7%). Of the 102 cases with neoplastic lesions 46 patients (45%) had low-grade squamous cell intraepithelial lesions (LSILs), 22 (21.5%) had high-grade squamous cell intraepithelial lesions (HSILs), 14 (13.7%) had squamous cell carcinoma, and 6 (5.8%) showed features of adenocarcinoma. Ten (9.8%) cases showed cytology of atypical squamous cells of undetermined significance (ASCUS) and four (3.9%) cases had atypical glandular cells of undetermined significance (AGUS)., Conclusion: We conclude that cervical smear examination is well suited for diagnosing neoplastic disease. It is clear that cervical neoplastic lesions are becoming a problem in Pakistan.

Dendritic cells (DCs) express cell surface lectins that are potentially involved in the recognition, uptake, and presentation of glycosylated foreign substances. A unique calcium-type (C-type) lectin, the macrophage galactose (Gal)-type C-type lectin (MGL/CD301) expressed on DCs, is thought to participate in the recognition of molecules from both altered self and pathogens due to its monosaccharide specificity for Gal and N-acetylgalactosamine (GalNAc). Although mice have two MGL genes, Mgl1 and Mgl2, their distinct roles have not been previously explored. The present report characterizes the properties of MGL2 by examining its distribution and its role in antigen presentation by DCs. We generated an MGL2-specific monoclonal antibody and examined MGL2 expression in tissues by immunohistochemistry and in isolated cells by flow cytometry. The cells reactive with this antibody were shown to be a portion of MGL1-expressing cells, mostly conventional DCs. Internalization of soluble polyacrylamide polymers (PAA) with alpha-GalNAc residues (GalNAc-PAA) by bone marrow-derived DCs (BM-DCs) was mediated by MGL2, as revealed by a comparison of Mgl1(-/-) and Mgl2(-/-) BM-DCs with wild-type BM-DCs. Biotinylated GalNAc-PAA conjugated to streptavidin (SAv) was more efficiently presented to SAv-primed T cells by BM-DCs than beta-N-acetylglucosamine-PAA conjugated to SAv or SAv alone as shown by thymidine uptake and cytokine production. This is the first report that demonstrates the involvement of GalNAc residues in antigen uptake and presentation by DCs that lead to CD4(+) T cell activation.

Inflammatory bowel disease is caused by abnormal inflammatory and immune responses to harmless substances, such as commensal bacteria, in the large bowel. Such responses appear to be suppressed under healthy conditions, although the mechanism of such suppression is currently unclear. The present study aimed to reveal whether the recognition of bacterial surface carbohydrates by the macrophage galactose-type C-type lectin-1, MGL1/CD301a, induces both the production and secretion of interleukin (IL)-10. Dextran sulfate sodium salt (DSS) was orally administrated to mice that lacked MGL1/CD301a (Mgl1(-/-) mice) and their wild-type littermates. Mgl1(-/-) mice showed significantly more severe inflammation than wild-type mice after administration of DSS. MGL1-positive cells in the colonic lamina propria corresponded to macrophage-like cells with F4/80-high, CD11b-positive, and CD11c-intermediate expression. These cells in Mgl1(-/-) mice produced a lower level of IL-10 mRNA compared with wild-type mice after the administration of DSS for 2 days. Recombinant MGL1 was found to bind both Streptococcus sp. and Lactobacillus sp. among commensal bacteria isolated from mesenteric lymph nodes of DSS-treated mice. Heat-killed Streptococcus sp. induced an increase in IL-10 secretion by MGL1-positive colonic lamina propria macrophages, but not the macrophage population from Mgl1(-/-) mice. These results strongly suggest that MGL1/CD301a plays a protective role against colitis by effectively inducing IL-10 production by colonic lamina propria macrophages in response to invading commensal bacteria.

The transforming growth factor beta 1 (TGF-beta1) is a multifunctional cytokine with several regulatory activities in tumor cells affecting growth, differentiation, and function. Alterations in gene expression, secretion, and regulation of TGF-beta1 may lead to a favorable environment for tumor development by angiogenesis stimulation and immune system suppression. We evaluated the influence of the TGFB1 polymorphisms by ARMS-PCR, Leu10Pro, and Arg25Pro, on prostate cancer (PCa) and benign prostatic hyperplasia (BPH). We assessed TGFB1 polymorphisms and their relation to mRNA levels (semi-quantitative RT-PCR) in blood samples as well as the implications in disease occurrence and progression. Peripheral blood samples from 175 patients were analyzed as to 92 BPH and 83 PCa. Samples obtained from 132 healthy males were used as negative controls. PCa patients with a Gleason score greater than 7 presented a higher frequency of the C allele (Leu10Pro). This allele was associated with a higher risk of developing PCa and BPH compared to the population (2.6 and 3.6 times higher, respectively). Patients with TGFB1 transcript levels equal to or more than 70% higher than control levels presented a 5.34 and 2.14-fold higher risk of having PCa and BPH, respectively, relative to the population. No association was detected between polymorphisms and mRNA levels. The C allele of the Leu10Pro polymorphism may predispose men to a more rapid cancer progression. Additionally, higher mRNA levels in the peripheral blood of PCa patients suggest that tumor cells may be disseminated in the circulation and could be used as a biomarker for extra-capsular invasion.

Objective: To establish the prevalence of dyslipidemia in the Iraqi adult population in Mosul, Iraq, according to the different recommended criteria., Methods: The study was carried out from October 2003 to April 2004, with 871 apparently healthy volunteers (413 males, 458 females) aged 20-70 year ([mean +/- SD] 41.2 +/- 13.8 year). Fasting blood specimens were collected from all subjects for measurement of serum lipid profile including triglycerides (TG), total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), and ratios of cholesterol such as TC:HDL-C, LDL-C:HDL-C, and TG:HDL-C. Classification was carried out according to the different cut-off levels as recommended by the 2001 American National Cholesterol Education Program (NCEP) III using thresholds of TG > or = 150 mg/dl (2.0 mmol/L), LDL-C > or = 100 mg/dl (2.6 mmol/L), HDL-C<40 mg/dl (1.04 mmol/L), and non-HDL-C> or 130 mg/dl (3.37 mmol/L). The criteria of the 1998 British Hyperlipidemia Association (BHA) were also followed using thresholds of TG > or =180 mg/dl (2.4 mmol/L), TC > or 194 mg/dl (5.0 mmol/L), LDL-C > or = 116 mg/dl (3.0 mmol/L), HDL-C< or =45 mg/dl (1.15 mmol/L), TC:HDL-C> or =5.0, HDL-C:DL-C> or =2.5, and TG:HDL-C> or =3.0., Results: Based on the American NCEP III criteria, the dyslipidemic states were noted with high TG (41.6%), high LDL-C (57.8%), low HDL-C (49.9%), and high non-HDL-C (56.8%) from the subjects. Based on the BHA criteria, high TG (24.5%), high TC (32.7%), high LDL-C (37.8%), high TC:HDL-C (30.9%), high LDL-C HDL-C (55.7%), and high TG:HDL-C (58.3%) were noted., Conclusion: Dyslipidemia is common in the Iraqi population, and this was identified at different recommendation criteria.

Manipulatable models of bladder development which interrogate specific pathways are badly needed. Such models will allow a systematic investigation of the multitude of pathologies which result from developmental defects of the urinary bladder. In the present communication, we describe a model in which mouse embryonic stem (ES) cells are directed to differentiate to form bladder tissue by specific interactions with fetal bladder mesenchyme. This model allows us to visualize the various stages in the differentiation of urothelium from ES cells, including the commitment to an endodermal cell lineage, with the temporal profile characterized by examining the induction of specific endodermal transcription factors (Foxa1 and Foxa2). In addition, final functional urothelial differentiation was characterized by examining uroplakin expression. It is well established that ES cells will spontaneously develop teratomas when grown within immunocompromised mouse hosts. We determined the specific mesenchymal to ES cell ratios necessary to dictate organ-specific differentiation while completely suppressing teratomatous growth. Embryonic mesenchyme is well established as an inductive tissue which dictates organ-specific programming of epithelial tissues. The present study demonstrates that embryonic bladder mesenchyme can also steer ES cells towards developing specific endodermal derived urothelium. These approaches allow us to capture specific stages of stem cell differentiation and to better define stem cell hierarchies.

Tissue recombination is a powerful method to evaluate the paracrine-signaling events that orchestrate the development of organs using the in vivo environment of a host rodent. Studies have reported the successful generation of primary cultures of rodent bladder urothelium, but none have reported their use to recapitulate bladder tissue with tissue recombination. We propose that primary cultured bladder urothelium, when recombined with inductive embryonic bladder mesenchyme, will form bladder tissue in a recombination model. Adult rat bladders were isolated and urothelium obtained. Sheets of bladder urothelium were re-suspended in collagen and maintained in tissue culture. After expansion (>20 passages), the urothelium was recombined with embryonic day-14 mouse bladder mesenchyme, then grafted beneath the renal capsule of immunocompromised mouse hosts. Grafts were harvested after 28 days. Control grafts were performed with bladder mesenchyme alone, cultured bladder urothelium alone, and collagen matrix alone. Final tissues were evaluated with staining and immunohistochemistry (H&E, Gomori's trichrome, broad-spectrum uroplakin, and smooth muscle actin alpha and gamma). Immunocytochemistry on cultured urothelium for broad-spectrum keratin, vimentin, and broad-spectrum uroplakin confirmed pure populations, void of mesenchymal contaminants. Staining of recombinant grafts demonstrated bladder tissue with mature urothelium and stromal differentiation. Control tissues were void of bladder tissue formation. We have successfully demonstrated that a chimeric bladder is formed from primary cultured bladder urothelium recombined with embryonic bladder mesenchyme. This is a powerful new tool for investigating the molecular mechanisms of bladder development and disease. Future applications may include the in vitro genetic manipulation of urothelium and examining those effects on growth and development in an in vivo environment., ((c) 2006 Wiley-Liss, Inc.)

Methansarcina mazei Gö1 DNA arrays were constructed and used to evaluate the genomic expression patterns of cells grown on either of two alternative methanogenic substrates, acetate or methanol, as sole carbon and energy source. Analysis of differential transcription across the genome revealed two functionally grouped sets of genes that parallel the central biochemical pathways in, and reflect many known features of, acetate and methanol metabolism. These include the acetate-induced genes encoding acetate activating enzymes, acetyl-CoA synthase/CO dehydrogenase, and carbonic anhydrase. Interestingly, additional genes expressed at significantly higher levels during growth on acetate included two energy-conserving complexes (the Ech hydrogenase, and the A1A0-type ATP synthase). Many previously unknown features included the induction by acetate of genes coding for ferredoxins and flavoproteins, an aldehyde:ferredoxin oxidoreductase, enzymes for the synthesis of aromatic amino acids, and components of iron, cobalt and oligopeptide uptake systems. In contrast, methanol-grown cells exhibited elevated expression of genes assigned to the methylotrophic pathway of methanogenesis. Expression of genes for components of the translation apparatus was also elevated in cells grown in the methanol medium relative to acetate, and was correlated with the faster growth rate observed on the former substrate. These experiments provide the first comprehensive insight into substrate-dependent gene expression in a methanogenic archaeon. This genome-wide approach, coupled with the complementary molecular and biochemical tools, should greatly accelerate the exploration of Methanosarcina cell physiology, given the present modest level of our knowledge of these large archaeal genomes.

The San Francisco Estuary Regional Monitoring Program (RMP) for Trace Substances is an innovative partnership among a regulatory agency, more than 70 regulated entities, and an independent scientific organization. The institutional arrangement behind the RMP has made the regulatory system increasingly responsive to emerging management needs, particularly with regard to the development of total maximum daily loads and ecosystem impairment assessment. Through multiagency partnerships within and outside the RMP institutional structure, major information gaps for several pollutants of concern have been narrowed, resulting in a successful consensus-based regulatory approach to managing copper and nickel mass inputs into the Estuary. Short-term research efforts, based upon monitoring results, helped identify the most cost-effective control and remediation options for various bioaccumulative substances. Additionally, adaptive changes to the monitoring program documented the existence of widespread aquatic toxicity in the Estuary that is apparently due to pesticide runoff from agricultural and urban areas. One of the most important contributions of this collaborative monitoring program is the deliberate and systematic adjustment of management and research questions that serve to influence and add relevance to the overall research agenda related to San Francisco Estuary ecosystem assessment.