Search

Competing Interests: Conflict of interest: None declared.

Background: GBM is an aggressive grade 4 primary brain tumor (BT), with a 5%-13% 5-year survival. Most human GBMs manifest as immunologically "cold" tumors or "immune deserts," yet the promoting or suppressive roles of specific lymphocytes within the GBM tumor microenvironment (TME) is of considerable debate., Methods: We used meticulous multiparametric flow cytometry (FC) to determine the lymphocytic frequencies in 102 GBMs, lower-grade gliomas, brain metastases, and nontumorous brain specimen. FC-attained frequencies were compared with frequencies estimated by "digital cytometry." The FC-derived data were combined with the patients' demographic, clinical, molecular, histopathological, radiological, and survival data., Results: Comparison of FC-derived data to CIBERSORT-estimated data revealed the poor capacity of digital cytometry to estimate cell frequencies below 0.2%, the frequency range of most immune cells in BTs. Isocitrate dehydrogenase (IDH) mutation status was found to affect TME composition more than the gliomas' pathological grade. Combining FC and survival data disclosed that unlike other cancer types, the frequency of helper T cells (Th) and cytotoxic T lymphocytes (CTL) correlated negatively with glioma survival. In contrast, the frequencies of γδ-T cells and CD56bright natural killer cells correlated positively with survival. A composite parameter combining the frequencies of these 4 tumoral lymphocytes separated the survival curves of GBM patients with a median difference of 10 months (FC-derived data; P < .0001, discovery cohort), or 4.1 months (CIBERSORT-estimated data; P = .01, validation cohort)., Conclusions: The frequencies of 4 TME lymphocytes strongly correlate with the survival of patients with GBM, a tumor considered an immune desert., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Motivation: Single-cell RNA-sequencing technologies have greatly enhanced our understanding of heterogeneous cell populations and underlying regulatory processes. However, structural (spatial or temporal) relations between cells are lost during cell dissociation. These relations are crucial for identifying associated biological processes. Many existing tissue-reconstruction algorithms use prior information about subsets of genes that are informative with respect to the structure or process to be reconstructed. When such information is not available, and in the general case when the input genes code for multiple processes, including being susceptible to noise, biological reconstruction is often computationally challenging., Results: We propose an algorithm that iteratively identifies manifold-informative genes using existing reconstruction algorithms for single-cell RNA-seq data as subroutine. We show that our algorithm improves the quality of tissue reconstruction for diverse synthetic and real scRNA-seq data, including data from the mammalian intestinal epithelium and liver lobules., Availability and Implementation: The code and data for benchmarking are available at github.com/syq2012/iterative&#95;weight&#95;update&#95;for&#95;reconstruction., (© The Author(s) 2023. Published by Oxford University Press.)

The rampant variability in codon bias existing between bacterial genomes is expected to interfere with horizontal gene transfer (HGT), a phenomenon that drives bacterial adaptation. However, delineating the constraints imposed by codon bias on functional integration of the transferred genes is complicated by multiple genomic and functional barriers controlling HGT, and by the dependence of the evolutionary outcomes of HGT on the host's environment. Here, we designed an experimental system in which codon composition of the transferred genes is the only variable triggering fitness change of the host. We replaced Escherichia coli's chromosomal folA gene encoding dihydrofolate reductase, an essential enzyme that constitutes a target for trimethoprim, with combinatorial libraries of synonymous codons of folA genes from trimethoprim-sensitive Listeria grayi and trimethoprim-resistant Neisseria sicca. The resulting populations underwent selection at a range of trimethoprim concentrations, and the ensuing changes in variant frequencies were used to infer the fitness effects of the individual combinations of codons. We found that when HGT causes overstabilization of the 5'-end mRNA, the fitness contribution of mRNA folding stability dominates over that of codon optimality. The 5'-end overstabilization can also lead to mRNA accumulation outside of the polysome, thus preventing the decay of the foreign transcripts despite the codon composition-driven reduction in translation efficiency. Importantly, the fitness effects of mRNA stability or codon optimality become apparent only at sub-lethal levels of trimethoprim individually tailored for each library, emphasizing the central role of the host's environment in shaping the codon bias compatibility of horizontally transferred genes., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Competing Interests: Conflict of interest: None declared.

Background: Although BNT162b2 vaccine-efficacy analyses have been published, the effectiveness of the vaccine in preventing coronavirus disease 2019 given confirmed exposure has not been previously demonstrated, even though it has policy implications, such as the need for self-quarantine when exposure has occurred., Methods: In a retrospective cohort study, we used data collected between 20 December 2020 and 17 March 2021 from the second largest healthcare provider in Israel to analyze the probability of an additional household infection occurring within 10 days after an index infection. In model 1, vaccine effectiveness was described for Fully Vaccinated individuals (7 or more days from second dose) vs either Unvaccinated individuals or those Recently Vaccinated Once (0-7 days from the first dose, presumably still unprotected). Secondary analyses included correction for differing testing rates. In model 2, we conducted a separate analysis of households comprised of only adults with the same vaccination status., Results: A total of 173 569 households were included, of which 6351 had an index infection (mean [standard deviation] age, 58.9 [13.5] years); 50% were women. Adjusted vaccine effectiveness of Fully Vaccinated compared with Unvaccinated participants was 80.3% (95% confidence interval [CI], 73.5-85.4) and 82.0% (95% CI, 75.6-86.8) compared with those Recently Vaccinated Once., Conclusions: The BNT162b2 vaccine is effective in high-risk real-life exposure scenarios, but the protection afforded in these settings is lower than that previously described. Individuals with a confirmed significant exposure to severe acute respiratory syndrome are still at risk of being infected even if fully vaccinated., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Objectives: The choice of a bioprosthetic valve (BV) over a mechanical valve (MV) in middle-aged adults in the mitral position is still under debate. Each valve type has benefits and drawbacks. We examined the mid-term survival of patients aged 50-70 years after BV versus MV mitral valve replacement (MVR)., Methods: We conducted a multicentre, retrospective analysis of patients aged 50-70 years undergoing MVR from 2005 to December 2018 in 4 medical centres in Israel. To control for between-group differences, we used propensity-adjusted analysis. The primary end point was all-cause mortality. Secondary end points included reoperation, cerebrovascular accident and bleeding., Results: During the study period, 2125 MVR procedures were performed. Of these, 796 were eligible for inclusion [539 (67.8%) MV replacement and 257 (32.2%) BV]. The mean age was 61.0 ± 5.4. There were 287 deaths during 4890 person-years of follow-up. The adjusted hazard ratio was (1.13 [0.85-1.49], P = 0.672). There was also no difference in the secondary end points. Subgroup analysis of patients aged 50-64 years showed a higher risk of mortality with BV (hazard ratio = 1.50 [1.07-2.1], P = 0.018). Reoperation was a strong predictor of mortality during the study period (72.2%)., Conclusions: In patients aged 50-70 years, we found an interaction between age and MV or BV outcomes-those younger than 65 years gained a mortality advantage with MV, while outcomes were similar in the 65-70 age group. this supports the current guidelines recommending using MV in patients <65 years of age., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Benthic microbes are key organisms in the oligotrophic Southeastern Mediterranean Sea (SEMS), yet their abundance, activity, and diversity in this rapidly changing basin are not fully understood. We investigated the prokaryotic and microfungal communities throughout years 2018-2020 at 27 stations (6-1900 m water depths, down to 20 cm below the sediment surface), in two transects with distinct downslope transport regimes, and along the eutrophic coastline. We estimated microbial abundance with flow cytometry, secondary production as leucine assimilation, and sequenced marker genes (the 16S rRNA and internal transcribed spacer) to assess diversity indices. The highest abundance (0.21 × 108 cells gr-1 sediment) was estimated at slope stations where we assumed substantial transport rates and found an accumulation of organic carbon. Secondary production was the highest nearshore (12 ± 4 ng C gr-1 h-1), and markedly declined offshore (0.5 ± 0.9 ng C gr-1 h-1). Populations of archaea (dominant Nitrososphaeria and Nanoarchaeia) and diverse bacteria were stable over three years, and taxonomic composition was dictated mainly by depth gradients. Saprotrophic and pathotrophic microfungi Ascomycota (70% ± 23%) and Basidiomycota (16% ± 18%) were prevalent, whereas parasitic chytrids were abundant nearshore. Our results highlight the role of downslope transport, which enriched the typical deep-sea communities with anaerobic lineages, in shaping microbial populations near the continental slope., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Deep targeted sequencing technologies are still not widely used in clinical practice due to the complexity of the methods and their cost. The Molecular Inversion Probes (MIP) technology is cost effective and scalable in the number of targets, however, suffers from low overall performance especially in GC rich regions. In order to improve the MIP performance, we sequenced a large cohort of healthy individuals ( n  = 4417), with a panel of 616 MIPs, at high depth in duplicates. To improve the previous state-of-the-art statistical model for low variant allele frequency, we selected 4635 potentially positive variants and validated them using amplicon sequencing. Using machine learning prediction tools, we significantly improved precision of 10-56.25% ( P  < 0.0004) to detect variants with VAF > 0.005. We further developed biochemically modified MIP protocol and improved its turn-around-time to ∼4 h. Our new biochemistry significantly improved uniformity, GC-Rich regions coverage, and enabled 95% on target reads in a large MIP panel of 8349 genomic targets. Overall, we demonstrate an enhancement of the MIP targeted sequencing approach in both detection of low frequency variants and in other key parameters, paving its way to become an ultrafast cost-effective research and clinical diagnostic tool., (© The Author(s) 2022. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

DNA-protein interactions play essential roles in all living cells. Understanding of how features embedded in the DNA sequence affect specific interactions with proteins is both challenging and important, since it may contribute to finding the means to regulate metabolic pathways involving DNA-protein interactions. Using a massive experimental benchmark dataset of binding scores for DNA sequences and a machine learning workflow, we describe the binding to DNA of T7 primase, as a model system for specific DNA-protein interactions. Effective binding of T7 primase to its specific DNA recognition sequences triggers the formation of RNA primers that serve as Okazaki fragment start sites during DNA replication., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aims: The evidence are not conclusive that a small incremental increase in door-to-balloon (D2B) time leads to a significant increase in death of ST-elevation myocardial infarction (STEMI) patients. In a previous study, we described a quality improvement intervention that reduced D2B time in 333 patients with STEMI. The aim of the current study was to compare mortality rates of the patients, before and after the intervention., Methods and Results: We examined the survival of 133 consecutive patients with STEMI treated prior to an intervention to decrease D2B time and 200 treated after the intervention. The mortality rate was the same before and after the quality intervention. The median D2B time for the entire cohort was 55 min. The number of patients with D2B time >55 min prior to the intervention was 82/133 (61%) and after the intervention 74/200 (37%) P < 0.00001. Thirty-day mortality among the patients with D2B time ≤55 min was 5/178 (2.8%) and among those with D2B time >55 min was 15/155 (9.7%), P < 0.008. The hazard ratio for 30-day mortality when the D2B time was >55 min was 3.7 (1.3-10.4)., Conclusion: Mortality and non-fatal complications did not differ significantly between STEMI patients before and after a quality improvement intervention. However, the number of patients treated within 55 min from arrival was significantly higher after the intervention; and coronary intervention within this time was associated with a lower death rate., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

The marine macroalgae Ulva sp. is considered an ecosystem engineer in rocky shores of temperate waters worldwide. Ulva sp. harbors a rich diversity of associated microbial epibionts, which are known to affect the algae's typical morphological development and 'health'. We examined the interaction between airborne microbes derived from atmospheric aerosols and Ulva ohnoi growth and physiological state. Specifically, we measured U. ohnoi growth rates and photosynthetic efficiency (Fv/Fm), alongside its microbial epibionts abundance, activity and diversity following dust (containing nutrients and airborne microorganisms) or UV-treated dust (only nutrients) amendments to filtered seawater. Parallel incubations with epibionts-free U. ohnoi (treated with antibiotics that removed the algae epibionts) were also tested to specifically examine if dust-borne microbes can replenish the epibiont community of U. ohnoi. We show that viable airborne microbes can restore U. ohnoi natural microbial epibionts communities, thereby keeping the seaweed alive and 'healthy'. These results suggest that microbes delivered through atmospheric aerosols can affect epiphyte biodiversity in marine flora, especially in areas subjected to high annual atmospheric dust deposition such as the Mediterranean Sea., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Objectives: We evaluated associations of endobronchial stenting with airway bacterial colonization, the antimicrobial resistance profile, hospitalizations for pneumonia and survival in lung transplant recipients., Methods: This is a retrospective single-centre study of 582 recipients of lung transplant during 2002-2018. We compared outcomes of 57 patients (9.7%) who received endobronchial stents (intervention group) to a control group of 57 patients without stents who were matched one to one for age, sex, year of transplantation, unilateral/bilateral transplantation and underlying disease., Results: For the intervention compared to the control group, airway colonization was more common for Pseudomonas (86% vs 35%, P < 0.001), Acinetobacter (21% vs 7%, P = 0.05), Klebsiella (21% vs 5%, P = 0.02) and Staphylococcus species (11% vs 0%, P = 0.02). The respective proportions of patients with positive bronchoalveolar lavage cultures on the third post-transplantation day, the day of stent insertion and 6-month post-stent insertion were 47.4%, 50.9% and 65.4% for Pseudomonas sp.; 15.8%, 12.3% and 3.8% for Klebsiella sp.; and 8.8%, 5.3% and 5.8% for Acinetobacter sp. The mean number of hospitalizations for pneumonia per patient was higher, without statistical significance, in the intervention than the control group (1.5 ± 1.7 vs 0.9 ± 1.5, P = 0.1). Kaplan-Meier survival curves did not show a statistically significant difference between the intervention group and the entire group without endobronchial stents (n = 525) (P = 0.4)., Conclusions: Lung transplant recipients with endobronchial stents were more likely to be colonized with pathologic bacteria and having pneumonia; however, stent placement was not associated with increased long-term mortality with appropriate stent maintenance., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Objective: Nephrectomy, the standard of care for localized renal cell carcinoma (RCC), may lead to kidney function loss. Our goal was to identify prognostic biomarkers of postoperative renal function using metabolomics., Methods: Metabolomics data from benign kidney parenchyma were collected prospectively from 138 patients with RCC who underwent nephrectomy at a single institution. The primary endpoint was the difference between the postoperative and preoperative estimated glomerular filtration (eGFR) rate divided by the elapsed time (eGFR slope). eGFR slope was calculated ∼2   years post-nephrectomy (GFR1), and at last follow-up (GFR2). A multivariate regularized regression model identified clinical characteristics and abundance of metabolites in baseline benign kidney parenchyma that were significantly associated with eGFR slope. Findings were validated by associating gene expression data with eGFR slope in an independent cohort ( n  =   58)., Results: Data were compiled on 78 patients (median age 62.6 years, 65.4% males). The mean follow - up was 25 ± 3.4 months for GFR1 and 69.5 ± 23.5 months for GFR2 and 17 (22%) and 32 (41%) patients showed eGFR recovery, respectively. Nephrectomy type, blood lipids, gender and 23 metabolites from benign parenchyma were significantly associated with eGFR slope. Some metabolites associated with eGFR slope overlapped with previously reported chronic kidney disease - related processes. Subgroup analysis identified unique 'metabolite signatures' by older age, nephrectomy type and preoperative eGFR., Conclusions: Nephrectomy type, gender, blood lipids and benign parenchyma metabolites at nephrectomy were associated with long-term kidney function. On further study, these metabolites may be useful as potential biomarkers and to identify novel therapeutic targets for malignancy-associated renal disease., (© The Author(s) 2020. Published by Oxford University Press on behalf of ERA-EDTA.)

Objective: The purpose of this study was to determine if medication cost transparency alerts provided at time of prescribing led ambulatory prescribers to reduce their use of low-value medications., Materials and Methods: Provider-level alerts were deployed to ambulatory practices of a single health system from February 2018 through April 2018. Practice sites included 58 primary care and 152 specialty care clinics totaling 1896 attending physicians, residents, and advanced practice nurses throughout western Washington. Prescribers in the randomly assigned intervention arm received a computerized alert whenever they ordered a medication among 4 high-cost medication classes. For each class, a lower cost, equally effective, and safe alternative was available. The primary outcome was the change in prescribing volume for each of the 4 selected medication classes during the 12-week intervention period relative to a prior 24-week baseline., Results: A total of 15 456 prescriptions for high-cost medications were written during the baseline period including 7223 in the intervention arm and 8233 in the control arm. During the intervention period, a decrease in daily prescribing volume was noted for all high-cost medications including 33% for clobetasol propionate (p < .0001), 59% for doxycycline hyclate (p < .0001), 43% for fluoxetine tablets (p < .0001), and a non-significant 3% decrease for high-cost triptans (p = .65). Prescribing volume for the high-cost medications overall decreased by 32% (p < .0001)., Conclusion: Medication cost transparency alerts in an ambulatory setting lead to more cost-conscious prescribing. Future work is needed to predict which alerts will be most effective., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Gram-negative bacteremia is a major cause of morbidity and mortality in hospitalized patients. Data to guide the duration of antibiotic therapy are limited., Methods: This was a randomized, multicenter, open-label, noninferiority trial. Inpatients with gram-negative bacteremia, who were afebrile and hemodynamically stable for at least 48 hours, were randomized to receive 7 days (intervention) or 14 days (control) of covering antibiotic therapy. Patients with uncontrolled focus of infection were excluded. The primary outcome at 90 days was a composite of all-cause mortality; relapse, suppurative, or distant complications; and readmission or extended hospitalization (>14 days). The noninferiority margin was set at 10%., Results: We included 604 patients (306 intervention, 298 control) between January 2013 and August 2017 in 3 centers in Israel and Italy. The source of the infection was urinary in 411 of 604 patients (68%); causative pathogens were mainly Enterobacteriaceae (543/604 [90%]). A 7-day difference in the median duration of covering antibiotics was achieved. The primary outcome occurred in 140 of 306 patients (45.8%) in the 7-day group vs 144 of 298 (48.3%) in the 14-day group (risk difference, -2.6% [95% confidence interval, -10.5% to 5.3%]). No significant differences were observed in all other outcomes and adverse events, except for a shorter time to return to baseline functional status in the short-course therapy arm., Conclusions: In patients hospitalized with gram-negative bacteremia achieving clinical stability before day 7, an antibiotic course of 7 days was noninferior to 14 days. Reducing antibiotic treatment for uncomplicated gram-negative bacteremia to 7 days is an important antibiotic stewardship intervention., Clinical Trials Registration: NCT01737320., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Improving lung cancer risk assessment is required because current early-detection screening criteria miss most cases. We therefore examined the utility for lung cancer risk assessment of a DNA Repair score obtained from OGG1, MPG, and APE1 blood tests. In addition, we examined the relationship between the level of DNA repair and global gene expression., Methods: We conducted a blinded case-control study with 150 non-small cell lung cancer case patients and 143 control individuals. DNA Repair activity was measured in peripheral blood mononuclear cells, and the transcriptome of nasal and bronchial cells was determined by RNA sequencing. A combined DNA Repair score was formed using logistic regression, and its correlation with disease was assessed using cross-validation; correlation of expression to DNA Repair was analyzed using Gene Ontology enrichment., Results: DNA Repair score was lower in case patients than in control individuals, regardless of the case's disease stage. Individuals at the lowest tertile of DNA Repair score had an increased risk of lung cancer compared to individuals at the highest tertile, with an odds ratio (OR) of 7.2 (95% confidence interval [CI] = 3.0 to 17.5; P  < .001), and independent of smoking. Receiver operating characteristic analysis yielded an area under the curve  of 0.89 (95% CI = 0.82 to 0.93). Remarkably, low DNA Repair score correlated with a broad upregulation of gene expression of immune pathways in patients but not in control individuals., Conclusions: The DNA Repair score, previously shown to be a lung cancer risk factor in the Israeli population, was validated in this independent study as a mechanism-based cancer risk biomarker and can substantially improve current lung cancer risk prediction, assisting prevention and early detection by computed tomography scanning., (© The Author(s) 2020. Published by Oxford University Press.)

Capsulotomy is necessary to facilitate instrument manoeuvrability within the joint capsule in many arthroscopic hip surgical procedures. In cases where a clear indication for capsular closure does not exist, surgeon's preference and experience often determines capsular management. The purpose of this study was to assess the influence of capsular closure on clinical outcome scores and satisfaction in patients who underwent hip arthroscopy surgery for femoroacetabular impingement (FAI) and labral tear. Data were prospectively collected and retrospectively analysed for hip arthroscopy surgeries with a minimum 2 years follow-up. Patients with developmental dysplasia of the hip, previous back or hip surgeries, and degenerative changes to this hip and secondary gains were excluded. Demographic data, intraoperative findings and patient-reported outcome scores were recorded, including the Modified Harris Hip Score (MHHS) and Hip Outcome Score (HOS). A total of 29 and 35 patients were included in the non-closure and closure groups, respectively. The mean follow-up time was over 3 years for both groups. The mean pre-operative and post-operative HOS scores and MHHS scores did not significantly differ between groups (pre-operative HOS: 65.6 and 66.3, P = 0.898; post-operative HOS: 85.4 and 87.2, P = 0.718; pre-operative MHHS: 63.2 and 58.4, P = 0.223; post-operative MHHS: 85.7 and 88.7, P = 0.510). Overall patient satisfaction did not differ significantly between groups (non-closure 86.3%, closure group 88.6%; P = 0.672). Capsular closure did not significantly influence satisfaction or clinical outcome scores in patients who underwent arthroscopic hip surgery for FAI or labral tear., (© The Author(s) 2019. Published by Oxford University Press.)

Introduction: There is paucity of data regarding lipid goal attainment after coronary-artery bypass graft surgery (CABG) and its impact on adverse outcomes. We aimed to investigate the attainment of lipid goals and the association between plasma lipid levels achieved after CABG and mortality., Methods: Retrospective analysis of 1230 patients undergoing CABG. Mortality was examined in relation to most-recent lipid levels attained, categorized by clinically-relevant thresholds, and according to the improvement from pre-operative levels., Results: Low-density lipoprotein cholesterol (LDL-C) < 70 mg/dL was attained by 44% of the patients. After multivariable adjustment, the hazard ratio for long-term mortality was 1.33 (95% confidence interval, 1.05-1.67) and 1.97 (1.55-2.50) for patients attaining LDL-C 70-100 mg/dL and >100 mg/dL, respectively, compared with LDL-C < 70 mg/dL. The hazard ratio was 1.42 (1.07-1.88) and 1.73 (1.33-2.23) for patients attaining high-density lipoprotein cholesterol (HDL-C) 40-50 mg/dL and <40 mg/dL, respectively, compared with HDL-C > 50 mg/dL; and 1.11 (0.85-1.45) and 4.28 (1.89-9.68) for patients with triglycerides 200-500 mg/dL and >500 mg/dL compared with triglycerides <200 mg/dL. A progressive stepwise association was seen between the cumulative status of the lipid measures achieved and long-term mortality, with the lowest risk observed in those with optimal level of all lipid measures ( p < 0.0001). Improvement in any of the lipid measures from pre-operative to latest documented levels was associated with reduced mortality., Conclusions: Lack of attainment of optimal levels of routine lipid measures after CABG was common and associated both independently and additively with long-term mortality, emphasizing the importance of addressing plasma lipid profile as both a risk marker and a treatment target after CABG.

Aim: To investigate the characteristics long-term prognostic implications (up to ∼2.2 years) of atrial fibrillation (AF) compared to sinus rhythm (SR), between acute and chronic heart failure (HF) with reduced (HFrEF < 40%), mid-range (HFmrEF 40-49%), and preserved (HFpEF ≥ 50%) ejection fraction (EF)., Methods and Results: Data from the observational, prospective, HF long-term registry of the European Society of Cardiology were analysed. A total of 14 964 HF patients (age 66 ± 13 years, 67% male; 53% HFrEF, 21% HFmrEF, 26% HFpEF) were enrolled. The prevalence of AF was 27% in HFrEF, 29% in HFmrEF, and 39% in HFpEF. Atrial fibrillation was associated with older age, lower functional capacity, and heightened physical signs of HF. Crude rates of mortality and HF hospitalization were higher in patients with AF compared to SR, in each EF subtype. After multivariable adjustment, the hazard ratio of AF for HF hospitalizations was: 1.036 (95% CI 0.888-1.208, P = 0.652) in HFrEF, 1.430 (95% CI 1.087-1.882, P = 0.011) in HFmrEF, and 1.487 (95% CI 1.195-1.851, P < 0.001) in HFpEF; and for combined all-cause death or HF hospitalizations: 0.957 (95% CI 0.843-1.087, P = 0.502), 1.302 (95% CI 1.055-1.608, P = 0.014), and 1.365 (95% CI 1.152-1.619, P < 0.001), respectively. In patients with HFrEF, AF was not associated with worse outcomes in those presenting with either an acute or a chronic presentation of HF., Conclusions: The prevalence of AF increases with increasing EF but its association with worse cardiovascular outcomes, remained significant in patients with HFpEF and HFmrEF, but not in those with HFrEF.

Bartonella is a genetically diverse group of vector-borne bacteria. Over 40 species have been characterized to date, mainly from mammalian reservoirs and arthropod vectors. Rodent reservoirs harbor one of the largest Bartonella diversity described to date, and novel species and genetic variants are continuously identified from these hosts. Yet, it is still unknown if this significant genetic diversity stems from adaptation to different niches or from intrinsic high mutation rates. Here, we explored the vertical occurrence of spontaneous genomic alterations in 18 lines derived from two rodent-associated Bartonella elizabethae-like strains, evolved in nonselective agar plates under conditions mimicking their vector- and mammalian-associated temperatures, and the transmission cycles between them (i.e., 26 °C, 37 °C, and alterations between the two), using mutation accumulation experiments. After ∼1,000 generations, evolved genomes revealed few point mutations (average of one-point mutation per line), evidencing conserved single-nucleotide mutation rates. Interestingly, three large structural genomic changes (two large deletions and an inversion) were identified over all lines, associated with prophages and surface adhesin genes. Particularly, a prophage, deleted during constant propagation at 37 °C, was associated with an increased autonomous replication at 26 °C (the flea-associated temperature). Complementary molecular analyses of wild strains, isolated from desert rodents and their fleas, further supported the occurrence of structural genomic variations and prophage-associated deletions in nature. Our findings suggest that structural genomic changes represent an effective intrinsic mechanism to generate diversity in slow-growing bacteria and emphasize the role of prophages as promoters of diversity in nature.

Background Familial hypercholesterolemia is characterized by markedly increased low-density lipoprotein cholesterol and risk for premature atherosclerotic cardiovascular disease. Models of care vary and reflect differing health policies and resources. The availability of electronic databases may enable better identification and assessment of familial hypercholesterolemia in the community. Methods A regional healthcare database was utilized to identify patients with a high probability of familial hypercholesterolemia, clinically defined by age-dependent-peak low-density lipoprotein cholesterol cutoffs and exclusion of secondary causes of severe hypercholesterolemia. Clinical characteristics, low-density lipoprotein cholesterol goal attainment, and treatment gaps were investigated. Results Probable familial hypercholesterolemia was diagnosed in 1932 of 685,314 individuals (1:355; median age 47 years). Atherosclerotic cardiovascular disease was present in 16.3% of adults (38% in males aged 50-74 years). Median peak low-density lipoprotein cholesterol was 264 mg/dl (interquartile range 252-288). Statins and/or ezetimibe were prescribed to 83% of patients and high-intensity statins to 53%, whereas prescriptions were filled in 57% and 40% cases respectively over the last six months, p < 0.001. Treatment gaps were wider among ethnic minorities, younger individuals, and those without atherosclerotic cardiovascular disease. Low-density lipoprotein cholesterol < 100 mg/dl was attained in 10.1% overall and 28.7% of those with atherosclerotic cardiovascular disease. Predictors of low-density lipoprotein cholesterol goal attainment included recent issue of high-intensity statins, presence of atherosclerotic cardiovascular disease, diabetes, older age and lack of smoking. Conclusions The population with high probability for familial hypercholesterolemia was characterized by low attainment of low-density lipoprotein cholesterol treatment goals despite high prescription rates of lipid-lowering medications. Low utilization of intensified therapies, non-adherence, and ethnic disparities were contributing factors. These findings emphasize the need to improve awareness and quality of care of familial hypercholesterolemia in the community.

Background: Autonomic control of the cardiovascular system may be impaired in type 2 diabetes and is associated with increased morbidity and mortality. Parameters obtained during stress testing may reflect early stages of cardiac autonomic dysfunction and provide prognostic information in asymptomatic type 2 diabetes., Methods: We performed maximal exercise treadmill testing in 594 patients with type 2 diabetes without known coronary heart disease. The prognostic significance of physiological parameters associated with autonomic dysfunction was assessed, including chronotropic incompetence (<80% heart rate reserve), abnormal heart rate recovery at 1 minute <18 beats/minute, and resting tachycardia >100 beats/minute. Cox proportional hazards analysis was used to determine the association of exercise parameters with a composite outcome of all-cause mortality, myocardial infarction or stroke., Results: Resting heart rate >100 beats/minute was observed in 18% of patients, chronotropic incompetence in 30% and heart rate recovery at 1 minute <18 beats/minute in 35%. Over 79 ± 16 months, there were 72 (12%) events. Each parameter was significantly associated with event risk in an adjusted multivariate analysis: chronotropic incompetence (hazard ratio 1.89, 95% confidence interval 1.18-3.01; P = 0.008), resting heart rate ≥100 beats/minute (hazard ratio 1.97, 95% confidence interval 1.19-3.26; P = 0.008) and heart rate recovery at 1 minute <18 beats (hazard ratio 1.77, 95% confidence interval 1.12-2.81; P = 0.015). A progressive relationship between the number of abnormal parameters and event risk was observed (log rank P < 0.001)., Conclusions: Chronotropic incompetence, resting tachycardia and reduced heart rate recovery are independently and additively associated with long-term mortality, myocardial infarction or stroke in type 2 diabetes without known coronary heart disease., (© The European Society of Cardiology 2015.)

The purpose of this study is to demonstrate the assessment of intra-capsular femoral head and neck tumors, and to describe the arthroscopic surgical technique used to resect and fill the bone defects. Three cases of benign femoral head and neck lesions are presented. Two benign enchondromas and one benign osteochondroma were resected arthroscopically. Traction was used in one case. Modified Harris Hip Score improved in all three cases to scores of 95 or greater with an average improvement of 16 points with a minimum follow up of 15 months. Arthroscopic surgical resection of intra-capsular femoral hip lesions offers an effective alternative to open resection. This technique offered good outcomes in the limited cohort. We suggest that arthroscopic resection of intra-capsular femoral hip lesions be considered in relevant cases as an alternative to open resection.

Gene expression is a multi-step process involving many layers of regulation. The main regulators of the pathway are DNA and RNA binding proteins. While over the years, a large number of DNA and RNA binding proteins have been identified and extensively studied, it is still expected that many other proteins, some with yet another known function, are awaiting to be discovered. Here we present a new web server, BindUP, freely accessible through the website http://bindup.technion.ac.il/, for predicting DNA and RNA binding proteins using a non-homology-based approach. Our method is based on the electrostatic features of the protein surface and other general properties of the protein. BindUP predicts nucleic acid binding function given the proteins three-dimensional structure or a structural model. Additionally, BindUP provides information on the largest electrostatic surface patches, visualized on the server. The server was tested on several datasets of DNA and RNA binding proteins, including proteins which do not possess DNA or RNA binding domains and have no similarity to known nucleic acid binding proteins, achieving very high accuracy. BindUP is applicable in either single or batch modes and can be applied for testing hundreds of proteins simultaneously in a highly efficient manner., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: To investigate the natural history of hearing loss (HL) in Israeli military aviators and its risk factors., Methods: Audiometric results of aviators with available audiometry at ages 30 and 40 years, and up to their last available audiometry were retrieved. HL DEFINITION: pure-tone threshold (PTT) of 30 dB or higher in at least one frequency in at least one ear, moderate-to-severe (M-S) HL as PTT of 45 dB or higher, and suspected noise-induced HL (NIHL) as HL at 3 to 6 kHz. Potential risk factors for HL were assessed by the χ(2) test and logistic regression models., Results: 298 aviators were included. Rates of HL increased with age, up to 57.5% at age 50. All M-S HL at age 50 years had clues on previous examinations, but new HL was observed in all ages. Age was found as a statistically significant risk factor, but aircraft type was not. Total flying hours were found to be an independent risk factor for suspected NIHL only. Self-reported earplug use was not found to protect from HL, with methodological limitations., Conclusions: HL is prevalent in aviators. Age and total flying hours are risk factors, the latter probably only for NIHL, whereas aircraft type is not., (Reprint & Copyright © 2016 Association of Military Surgeons of the U.S.)

Background: Impaired cardiorespiratory fitness (CRF) is a potent risk factor for mortality in diabetes, and may modify the relation between adiposity and mortality. We evaluated the interaction between CRF and abdominal adiposity distribution with all-cause mortality, myocardial infarction or stroke in patients with diabetes., Methods: We studied 294 type 2 diabetics without known coronary artery disease. CRF was quantified in metabolic equivalents by maximal treadmill testing, and categorized as low CRF (first tertile) or high CRF (second and third tertiles). Abdominal fat was quantified as subcutaneous or visceral adipose tissue from non-enhanced computed tomography scans. Association of CRF, adiposity distribution and their interaction with all-cause mortality, myocardial infarction or stroke was assessed by Cox proportional-hazard models., Results: There were 31 (11%) events during 62 ± 12 months. Low CRF was significantly associated with event risk before and after adjustment for each measure of adiposity (hazard ratio 3.79, 95% confidence interval 1.79-8.01, p < 0.001). CRF level was inversely correlated with subcutaneous (r = -0.44, p < 0.001) but not visceral adipose tissue (r = -0.06, p = 0.31). Absolute event rates increased progressively across visceral adipose tissue tertiles, but decreased across subcutaneous tertiles. However, within each tertile of both adiposity measures, increased events were observed in the low compared with the high CRF group; this trend was also observed in an adjusted multivariate proportional hazards model., Conclusions: Although subcutaneous and visceral adipose tissues differed in their association with CRF levels and absolute event rates, lower baseline CRF in type 2 diabetics was significantly associated with higher risk of all-cause mortality, myocardial infarction or stroke, regardless of abdominal adiposity pattern., (© The European Society of Cardiology 2014.)

The spatial variability in the composition of bacterioplankton communities from the Levantine basin off the Israeli coast (south-eastern Mediterranean Sea) was studied using water samples from nine stations down to 1880 m depth. An nMDS (non-metric multidimensional scaling) ordination plot of ARISA (automated rRNA intergenic spacer analyses) fingerprints performed on 39 water samples revealed three groups of communities based on depth (epi, meso and bathypelagial waters), while the stations' geographical location did not seem to have an effect on the distribution of bacterioplankton assemblages. Interestingly, communities from the two deep pelagic zones were further grouped depending on the three prevailing water masses in the Levantine basin, and depth, salinity, temperature and silicic acid were significantly related to the variations of the bacterial community compositions. Cloning of internal transcribed spacer (ITS) sequences of dominant ARISA peaks showed that abundant bacteria in all depths were ubiquitous uncultured Alpha- and Gammaproteobacteria. The results further indicate that distinct microbial community profiles in the Levantine basin are associated with prevailing water masses that have formed due to different physico-chemical parameters and thus might act as physical barriers rather than physiological discriminators, as has been suggested in studies from other ocean regions., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The Afghan population suffers from a long standing armed conflict. We investigated patients' experiences of their access to and use of the health services., Methods: Data were collected in four clinics from different provinces. Mixed methods were applied. The questions focused on access obstacles during the current health problem and health seeking behaviour during a previous illness episode of a household member., Results: To access the health facilities 71.8% (545/759) of patients experienced obstacles. The combination of long distances, high costs and the conflict deprived people of life-saving healthcare. The closest public clinics were underused due to perceptions regarding their lack of availability or quality of staff, services or medicines. For one in five people, a lack of access to health care had resulted in death among family members or close friends within the last year., Conclusions: Violence continues to affect daily life and access to healthcare in Afghanistan. Moreover, healthcare provision is not adequately geared to meet medical and emergency needs. Impartial healthcare tailored to the context will be vital to increase access to basic and life-saving healthcare., (© The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Context: The consequence of elevated body mass index (BMI) at adolescence on early adulthood mortality rate and on predicted life expectancy is unclear., Objective: The objective of the investigation was to study the relationship between BMI at adolescence and mortality rate as well as the mortality trend over the past 4 decades across the entire BMI range., Design and Setting: The study included a nationwide longitudinal cohort., Participants: A total of 2 159 327 adolescents (59.1% males) born between 1950 and 1993, who were medically evaluated for compulsory military service in Israel, participated in the study., Interventions: Height and weight were measured at age 17 years, and BMI was stratified based on the Centers for Disease Control and Prevention-established percentiles for age and sex., Main Outcome Measure: Incident cases of all-cause mortality before age 50 years were recorded. Cox-proportional hazard models were used to assess mortality rates and its trend overtime., Results: During 43 126 211 person-years of follow-up, 18 530 deaths were recorded. As compared with rates observed in the 25th to 50th BMI percentiles, all-cause mortality continuously increased across BMI range, reaching rates of 8.90/10(4) and 2.90/10(4) person-years for men and women with BMI greater than the 97th percentile, respectively. A multivariate analysis adjusted for age, socioeconomic status, education, and ethnicity demonstrated a significant increase in mortality at BMI greater than the 50th percentile (BMI > 20.55 kg/m(2)) for men and the 85th percentile or greater in women (BMI > 24.78 kg/m(2)). During the last 4 decades, a significant decrease in mortality rates was documented in normal-weight participants born between 1970 and 1980 vs those born between 1950 and 1960 (3.60/104 vs 4.99/10(4) person-years, P < .001). However, no improvement in the survival rate was observed among overweight and obese adolescents during the same time interval. Significant interaction between BMI and birth year was observed (P = .007)., Conclusions: BMI at adolescence, within the normal range, is associated with all-cause mortality in adulthood. Mortality rates among overweight and obese adolescents did not improve in the last 40 years, suggesting that preadulthood obesity may attenuate the progressive increase in life expectancy.

Body mass index (BMI) (calculated as weight (kg)/height (m)²) and height are linked to the pathogenesis of low back pain, but evidence-based confirmation is lacking. We examined the prevalence of low back pain in adolescents and its association with BMI and height. Disability clauses (official military limitations related to a person's health status) indicating low back pain severity were divided according to symptoms of low back pain alone and symptoms of low back pain with objective corroborating findings. All 829,791 males and females undergoing mandatory premilitary recruitment examinations since 1998 were included. Logistic regression models assessed the relationships of BMI and height with low back pain. Prevalence of low back pain was 0.2% for both males and females with objective findings and 5.2% for males and 2.7% for females without objective findings. Higher BMI was significantly associated with low back pain in males (for overweight, odds ratio = 1.097, P < 0.001; for obese, odds ratio = 1.163, P < 0.001) and in females (for overweight, odds ratio = 1.174, P < 0.001; for obese, odds ratio = 1.211, P < 0.001). Height was associated with increased risk of low back pain in both genders. Odds ratios for low back pain in the tallest group compared with the shortest group were 1.438 (P < 0.001) for males and 1.224 (P < 0.001) for females. Low back pain with or without objective findings was associated with overweight and obesity as well as with height.

Background: We investigated the prevalence and sociodemographic factors associated with diagnosis of inflammatory bowel diseases (IBD) among Jewish Israeli adolescents., Methods: A total of 953,684 Jewish Israeli adolescents (57.8% men) who underwent a general health examination at mean age 17.3 ± 0.5 years from 1998 to 2010 were included. A definite diagnosis of IBD was based on laboratory, endoscopy, and pathology reports. Covariate data included socioeconomic status (SES) as defined by the Israel Central Bureau Statistics, and origin and number of children in household., Results: A total of 2021 patients with IBD were identified (0.21%) in 13 annual cohorts. The prevalence of IBD increased from 149.4 cases per 100,000 to 301.0 cases per 100,000 in the first and last cohort (Ptrend = 0.003). Independent factors associated with occurrence of IBD were SES (high: odds ratio [OR] = 1.84, 95% confidence interval [CI]: 1.60-2.1, P < 0.001; medium: OR = 1.47, 95% CI: 1.3-1.69, P < 0.001; low: reference), Western origin (OR = 1.71, 95% CI: 1.53-1.90, P < 0.001; Asia Africa: reference), and male gender (OR = 1.21, 95% CI: 1.10-1.33, P < 0.001; female: reference). Four or more children in the household were associated with reduced OR for IBD [N ≥ 4: OR = 0.70, 95% CI: 0.62-0.72, P < 0.001, N = 1-3: reference]. The OR among adolescents of Western origin-high SES was 2.95 times higher compared with adolescents of Asia-African origin with low SES., Conclusions: The prevalence of IBD doubled during the 13 years of the study period. Among this large cohort of Jewish adolescents, for each origin, higher SES was associated with increased occurrence of IBD.

Context: The cholesterol side-chain cleavage enzyme P450scc, encoded by CYP11A1, converts cholesterol to pregnenolone to initiate steroidogenesis. P450scc deficiency can disrupt adrenal and gonadal steroidogenesis, resembling congenital lipoid adrenal hyperplasia clinically and hormonally; only 12 such patients have been reported previously., Objective: We sought to expand clinical and genetic experience with P450scc deficiency., Patients and Methods: We sequenced candidate genes in 7 children with adrenal insufficiency who lacked disordered sexual development. P450scc missense mutations were recreated in the F2 vector, which expresses the fusion protein P450scc-Ferredoxin Reductase-Ferredoxin. COS-1 cells were transfected, production of pregnenolone was assayed, and apparent kinetic parameters were calculated. Previously described P450scc mutants were assayed in parallel., Results: Four of five Bedouin children in one kindred were compound heterozygotes for mutations c.694C>T (Arg232Stop) and c.644T>C (Phe215Ser). Single-nucleotide polymorphism analysis confirmed segregation of these mutations. The fifth kindred member and another Bedouin patient presented in infancy and were homozygous for Arg232Stop. A patient from Fiji presenting in infancy was homozygous for c.358T>C (Arg120Stop). All mutations are novel. As assayed in the F2 fusion protein, P450scc Phe215Ser retained 2.5% of wild-type activity; previously described mutants Leu141Trp and Ala269Val had 2.6% and 12% of wild-type activity, respectively, and Val415Glu and c.835delA lacked detectable activity., Conclusions: Although P450scc is required to produce placental progesterone required to maintain pregnancy, severe mutations in P450scc are compatible with term gestation; milder P450scc mutations may present later without disordered sexual development. Enlarged adrenals usually distinguish steroidogenic acute regulatory protein deficiency from P450scc deficiency, but only DNA sequencing is definitive.

Objective: To investigate the association between occupational risk factors and the incidence of knee disorders in a young adult population., Methods: Israeli recruits to the Israel Defense Forces go through a rigorous medical investigation. Study participants were classified by prior knee condition status and divided into 5 categories of prospective occupational exposure to physical activity according to their assigned military duties, and were then followed for 30 months for the development of severe knee disorders (SKD). Logistic regression analysis was used to estimate the occupational risks for incident SKD, adjusted for any previous mild or moderate disorder, body mass index, and body height at induction., Results: The study population consisted of 76,491 males. SKD developed in 615 (0.8%). Compared to administrative workers as referents, a higher risk of developing SKD was manifest among high intensity combat occupations, (odds ratios [OR] 2.15), those in moderate intensity combat occupations (OR 2.57) and maintenance (OR 1.59). Drivers did not demonstrate increased risk of knee disorders compared to referents., Conclusions: Occupational factors during military service are associated with incident SKD, even when taking into account previous knee disorders, body mass index, and height, which also had independent effects in our study population.

Background: The A(3) adenosine receptor (A(3)AR) is overexpressed in the tumor and in the peripheral blood mononuclear cells of patients with hepatocellular carcinoma (HCC). The orally active drug candidate CF102, an A(3)AR agonist, induces apoptosis of HCC cells via deregulation of the Wnt signaling pathway. In this open label phase I/II trial, the safety and clinical effects of CF102 were assessed in patients with advanced unresectable HCC., Methods: The primary objectives of this trial were to examine the safety and pharmacokinetic (PK) behavior of CF102 given orally (1, 5, and 25 mg BID) in 28-day cycles. Evaluation of anti-tumor effects and the utilization of A(3)AR as a biological predictive marker of response to CF102 were the secondary objectives., Results: Eighteen patients received CF102-six at each dose level. No serious drug-related adverse events or dose-limiting toxicities were observed. CF102 demonstrated good oral bioavailability and linear PK behavior. Median overall survival in the study population, 67% of whom had received prior sorafenib, was 7.8 months, and for Child Pugh B patients (28%) it was 8.1 months. Stable disease by RECIST was observed in four patients for at least 4 months. CF102 maintained liver function over a 6-month period. A correlation between receptor overexpression levels at baseline and patients' overall survival was found. One of the patients who presented with skin nodules that were biopsy-proven to be HCC metastases prior to the trial showed complete metastasis regression during three months of treatment with CF102., Conclusions: CF102 is safe and well-tolerated, showing favorable PK characteristics in Child Pugh A and B HCC patients, justifying further clinical development.

Motivation: High-throughput single nucleotide polymorphism (SNP) arrays have become the standard platform for linkage and association analyses. The high SNP density of these platforms allows high-resolution identification of ancestral recombination events even for distant relatives many generations apart. However, such inference is sensitive to marker mistyping and current error detection methods rely on the genotyping of additional close relatives. Genotyping algorithms provide a confidence score for each marker call that is currently not integrated in existing methods. There is a need for a model that incorporates this prior information within the standard identical by descent (IBD) and association analyses., Results: We propose a novel model that incorporates marker confidence scores within IBD methods based on the Lander-Green Hidden Markov Model. The novel parameter of this model is the joint distribution of confidence scores and error status per array. We estimate this probability distribution by applying a modified expectation-maximization (EM) procedure on data from nuclear families genotyped with Affymetrix 250K SNP arrays. The converged tables from two different genotyping algorithms are shown for a wide range of error rates. We demonstrate the efficacy of our method in refining the detection of IBD signals using nuclear pedigrees and distant relatives., Availability: Plinke, a new version of Plink with an extended pairwise IBD inference model allowing per marker error probabilities is freely available at: http://bioinfo.bgu.ac.il/bsu/software/plinke., Contact: obirk@bgu.ac.il; markusb@bgu.ac.il, Supplementary Information: Supplementary data are available at Bioinformatics online.

Peptide-protein interactions are among the most prevalent and important interactions in the cell, but a large fraction of those interactions lack detailed structural characterization. The Rosetta FlexPepDock web server (http://flexpepdock.furmanlab.cs.huji.ac.il/) provides an interface to a high-resolution peptide docking (refinement) protocol for the modeling of peptide-protein complexes, implemented within the Rosetta framework. Given a protein receptor structure and an approximate, possibly inaccurate model of the peptide within the receptor binding site, the FlexPepDock server refines the peptide to high resolution, allowing full flexibility to the peptide backbone and to all side chains. This protocol was extensively tested and benchmarked on a wide array of non-redundant peptide-protein complexes, and was proven effective when applied to peptide starting conformations within 5.5 Å backbone root mean square deviation from the native conformation. FlexPepDock has been applied to several systems that are mediated and regulated by peptide-protein interactions. This easy to use and general web server interface allows non-expert users to accurately model their specific peptide-protein interaction of interest.

Differentiation of human embryonic stem cells (HESCs) can be studied in vivo through the induction of teratomas in immune-deficient mice. Cells within the teratomas differentiate into all three embryonic germ layers. However, the exact nature of the proliferation and differentiation of HESCs within the teratoma is not fully characterized, and it is not clear whether the differentiation is cell autonomous or affected by neighboring cells. Here, we establish a genetic approach to study the clonality of differentiation in teratomas using a mixture of HESC lines. We first demonstrate, by means of 5-bromo-2'-deoxyuridine incorporation, that cell proliferation occurs throughout the teratoma, and that there are no clusters of undifferentiated-proliferating cells. Using a combination of laser capture microdissection and DNA fingerprinting analysis, we show that different cell lines contribute mutually to the same distinctive tissue structures. Further support for the nonclonal differentiation within the teratoma was achieved by fluorescence in situ hybridization analysis of sex chromosomes. We therefore suggest that in vivo differentiation of HESCs is polyclonal and, thus, may not be cell autonomous, stressing the need for a three-dimensional growth in order to achieve complex differentiation of HESCs. Disclosure of potential conflicts of interest is found at the end of this article.

Monocytes remodel the extracellular matrix (ECM) by secreting proteins composing the ECM such as fibronectin (FN) and degrading proteases such as matrix metalloproteinase-9 (MMP-9), which cleaves FN into fragments. The effects of FN and its fragmented products on the expression of monocyte MMP-9 are controversial and largely unknown. We showed that in human monocytes, the proinflammatory cytokine TNF-alpha induced MMP-9 secretion and increased fragmentation of FN into distinct fragments. When primary monocytes or the U937 monocytic cell line were incubated on a plastic substrate, plastic-coated with native FN, and plastic-coated with fragmented FN (frag-FN), native FN inhibited TNF-alpha-induced proMMP-9 secretion by twofold (P<0.01) compared with plastic or frag-FN. Exploration of the dynamics of inflammation by incubating cells sequentially on the three substrates showed that frag-FN opposed the inhibitory effect of native FN. Inhibition of proMMP-9 by native FN was exerted at the translational level, as no change in MMP-9 mRNA, intracellular protein accumulation, or proteomic degradation was observed, and when degradation was blocked, no de novo translation of MMP-9 could be measured. We also showed that the reduction of MMP-9 secretion by native FN was responsible for attenuated migration of U937 cells (P<0.05). We suggest that in the inflammatory tissue, intact, native FN has a homeostatic role in harnessing MMP-9 activity. However, as fragmented products accumulate locally, they alleviate the inhibition and enable faster migration of the monocytes through the degraded ECM.

Motivation: Secondary structures are key descriptors of a protein fold and its topology. In recent years, they facilitated intensive computational tasks for finding structural homologues, fold prediction and protein design. Their popularity stems from an appealing regularity in patterns of geometry and chemistry. However, the definition of secondary structures is of subjective nature. An unsupervised de-novo discovery of these structures would shed light on their nature, and improve the way we use these structures in algorithms of structural bioinformatics., Methods: We developed a new method for unsupervised partitioning of undirected graphs, based on patterns of small recurring network motifs. Our input was the network of all H-bonds and covalent interactions of protein backbones. This method can be also used for other biological and non-biological networks., Results: In a fully unsupervised manner, and without assuming any explicit prior knowledge, we were able to rediscover the existence of conventional alpha-helices, parallel beta-sheets, anti-parallel sheets and loops, as well as various non-conventional hybrid structures. The relation between connectivity and crystallographic temperature factors establishes the existence of novel secondary structures.

Cellular hypoxia, characterizing tumors, ischemia, and inflammation induce recruitment of monocytes/macrophages, immobilize them at the hypoxic site, and alter their function. To migrate across the extracellular matrix and as part of their inflammatory functions, monocytes and macrophages secrete proteases, including matrix metalloproteinase-9 (MMP-9), whose expression is induced by proinflammatory cytokines [e.g., tumor necrosis factor alpha (TNF-alpha)]. We show that hypoxia (<0.3% O2 for 48 h) reduced the output of TNF-alpha-induced proMMP-9 by threefold (P < 0.01) in the U937 monocytic cell line and in primary human monocytes. TNF-alpha induced MMP-9 transcription by threefold, but no significant difference was observed in MMP-9 mRNA steady-state between normoxia and hypoxia, which inhibited the trafficking of proMMP-9 via secretory vesicles and increased the intracellular accumulation of proMMP-9 in the cells by 47% and 62% compared with normoxia (P < 0.05), as evaluated by zymography of cellular extracts and confocal microscopy, respectively. Secretion of proMMP-9 was reduced by the addition of cytochalazin B or nocodazole, which inhibits the polymerization of actin and tubulin fibers, or by the addition of the Rho kinase inhibitor Y27632, suggesting the involvement of the cytoskeleton and the Rho GTPases in the process of enzyme secretion. Furthermore, attachment of proMMP-9 to the cell membrane increased after hypoxia via its interactions with surface molecules such as CD44. In addition, the reduced migration of monocytes in hypoxia was shown to be mediated, at least partially, by secreted MMP-9. Thus, hypoxia post-translationally reduced the secreted amounts of proMMP-9 by using two mutually nonexclusive mechanisms: mostly, inhibition of cellular trafficking and to a lesser extent, attachment to the membrane.

This paper describes an institutional approach taken to build a primary care reference portal. The objective for the site is to make access to and use of clinical reference faster and easier and to facilitate the use of evidence-based answers in daily practice. Reference objects were selected and metadata applied to a core set of sources. Metadata were used to search, sort, and filter results and to define deep-linked queries and structure the interface. User feedback resulted in an expansion in the scope of reference objects to meet the broad spectrum of information needs, including patient handouts and interactive risk management tools. RESULTS of a user satisfaction survey suggest that a simple interface to customized content makes it faster and easier for primary care clinicians to find information during the clinic day and to improve care to their patients. The PrimeAnswers portal is a first step in creating a fast search of a customized set of reference objects to match a clinician's patient care questions in the clinic. The next step is developing methods to solve the problem of matching a clinician's question to a specific answer through precise retrieval from reference sources; however, lack of internal structure and Web service standards in most clinical reference sources is an unresolved problem.

The computational power of the neocortex arises from interactions of multiple neurons, which display a wide range of electrical properties. The gene expression profiles underlying this phenotypic diversity are unknown. To explore this relationship, we combined whole-cell electrical recordings with single-cell multiplex RT-PCR of rat (p13-16) neocortical neurons to obtain cDNA libraries of 26 ion channels (including voltage activated potassium channels, Kv1.1/2/4/6, Kvbeta1/2, Kv2.1/2, Kv3.1/2/3/4, Kv4.2/3; sodium/potassium permeable hyperpolarization activated channels, HCN1/2/3/4; the calcium activated potassium channel, SK2; voltage activated calcium channels, Caalpha1A/B/G/I, Cabeta1/3/4), three calcium binding proteins (calbindin, parvalbumin and calretinin) and GAPDH. We found a previously unreported clustering of ion channel genes around the three calcium-binding proteins. We further determined that cells similar in their expression patterns were also similar in their electrical properties. Subsequent regression modeling with statistical resampling yielded a set of coefficients that reliably predicted electrical properties from the expression profile of individual neurons. This is the first report of a consistent relationship between the co-expression of a large profile of ion channel and calcium binding protein genes and the electrical phenotype of individual neocortical neurons.