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p38 kinases are key elements of the cellular stress response in animals. They mediate the cell response to a multitude of stress stimuli, from osmotic shock to inflammation and oncogenes. However, it is unknown how such diversity of function in stress evolved in this kinase subfamily. Here, we show that the p38 kinase was already present in a common ancestor of animals and fungi. Later, in animals, it diversified into three JNK kinases and four p38 kinases. Moreover, we identified a fifth p38 paralog in fishes and amphibians. Our analysis shows that each p38 paralog has specific amino acid substitutions around the hinge point, a region between the N-terminal and C-terminal protein domains. We showed that this region can be used to distinguish between individual paralogs and predict their specificity. Finally, we showed that the response to hyperosmotic stress in Capsaspora owczarzaki , a close unicellular relative of animals, follows a phosphorylation-dephosphorylation pattern typical of p38 kinases. At the same time, Capsaspora 's cells upregulate the expression of GPD1 protein resembling an osmotic stress response in yeasts. Overall, our results show that the ancestral p38 stress pathway originated in the root of opisthokonts, most likely as a cell's reaction to salinity change in the environment. In animals, the pathway became more complex and incorporated more stimuli and downstream targets due to the p38 sequence evolution in the docking and substrate binding sites around the hinge region. This study improves our understanding of p38 evolution and opens new perspectives for p38 research.

Aim : To assess the quality of life of menopausal women with genitourinary syndrome receiving local drugs (prasterone, estriol or promestriene). Methods : Prospective, longitudinal, randomized study in which quality of life was assessed using the 16-item Cervantes scale (EC16) before and after treatment. Results : A total of 45 women were assessed (35.6% received prasterone, 33.3% estriol and 31.1% promestriene). After treatment, statistically significant mean score differences were observed in EC16, mainly with prasterone. Improvement in EC16 score only shows a statistically significant relationship with age and drug use. Conclusion : The EC16 is an affordable and quick-to-apply tool that allows physicians and patients to know patients' self-perceived quality of life. Local treatment has been shown to improve the quality of life of menopausal patients with genitourinary syndrome.

The epidemiological importance of mycobacterial species is indisputable, and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important bacterial metabolites, represents a set of targets for inhibitors of Mycobacterium tuberculosis growth. The aroA -encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene disruption, gene knockdown, point mutations (D61W, R134A, E321N), and kinetic analysis to evaluate aroA gene essentiality and vulnerability of its protein product, EPSPS, from Mycolicibacterium ( Mycobacterium ) smegmatis ( Ms EPSPS). We demonstrate that aroA -deficient cells are auxotrophic for aromatic amino acids (AroAAs) and that the growth impairment observed for aroA -knockdown cells grown on defined medium can be rescued by AroAA supplementation. We also evaluated the essentiality of selected Ms EPSPS residues in bacterial cells grown without AroAA supplementation. We found that the catalytic residues R134 and E321 are essential, while D61, presumably important for protein dynamics and suggested to have an indirect role in catalysis, is not essential under the growth conditions evaluated. We have also determined the catalytic efficiencies ( K cat / K m ) of recombinant wild-type (WT) and mutated versions of Ms EPSPS (D61W, R134A, E321N). Our results suggest that drug development efforts toward EPSPS inhibition may be ineffective if bacilli have access to external sources of AroAAs in the context of infection, which should be evaluated further. In the absence of AroAA supplementation, aroA from M. smegmatis is essential, its essentiality is dependent on Ms EPSPS activity, and Ms EPSPS is vulnerable. IMPORTANCE We found that cells from Mycobacterium smegmatis, a model organism safer and easier to study than the disease-causing mycobacterial species, when depleted of an enzyme from the shikimate pathway, are auxotrophic for the three aromatic amino acids (AroAAs) that serve as building blocks of cellular proteins: l-tryptophan, l-phenylalanine, and l-tyrosine. That supplementation with only AroAAs is sufficient to rescue viable cells with the shikimate pathway inactivated was unexpected, since this pathway produces an end product, chorismate, that is the starting compound of essential pathways other than the ones that produce AroAAs. The depleted enzyme, the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), catalyzes the sixth step of shikimate pathway. Depletion of this enzyme inside cells was performed by disrupting or silencing the EPSPS-encoding aroA gene. Finally, we evaluated the essentiality of specific residues from EPSPS that are important for its catalytic activity, determined with experiments of enzyme kinetics using recombinant EPSPS mutants.

Latin America (LATAM) children offer special insight into Severe Acute Respiratory Syndrome Coronavirus 2 (SARS COV2) due to high-risk race/ethnicity, variability in medical resources, diverse socioeconomic background, and numerous involved organ systems. This multinational study of LATAM youth examined the distinguishing features of acute or late multisystem SARS COV2 with versus without cardiac involvement. A consecutive sample of youth 0-18 years old (N = 98;50% male) presenting with multisystem SARS COV2 to 32 centers in 10 Latin American countries participating in a pediatric cardiac multi-imaging society were grouped as with versus without cardiac involvement, defined as abnormal echocardiographic findings or arrhythmia. Collected clinical data were analyzed by Student's t-test or Fisher's exact test. Cardiac (N = 48, 50% male) versus no cardiac (N = 50, 50% male) were similar in age; weight; nonrespiratory symptoms; and medical history. The cardiac group had 1 death and symptoms including coronary artery dilation, ejection fraction <50%, pericardial effusion, peripheral edema, arrhythmia, and pulmonary artery thrombus. The cardiac group had higher risk of ICU admission (77% vs 54%, p = 0.02); invasive ventilation (23% vs 4%,p = 0.007); vasoactive infusions (27% vs 4%, p = 0.002); prominent respiratory symptoms (60% vs 36%, p < 0.03); abnormal chest imaging (69% vs 34%, p = 0.001); troponin (33% vs 12%, p = 0.01); alanine aminotransferase (33% vs 12%, p = 0.02); and thrombocytopenia (46% vs 22%, p = 0.02). Receiver operating curve analysis showed that abnormal laboratories had 94% sensitivity and 98% negative predictive value on the need for ICU interventions.Conclusion: In LATAM children with multisystem SARS COV2, cardiac involvement was prevalent. Cardiac involvement was more likely to require ICU interventions, certain abnormal labs, and respiratory involvement. What is Known: • SARS COV2 can be asymptomatic in children but in some cases can have serious multisystemic involvement. • Hispanic ethnicity is purportedly at high risk of SARS COV2 in nations where they are often disadvantaged minority populations. What is New: • Latin American children presenting with multisystem SARS COV2 frequently have cardiac involvement which was associated with ICU interventions; prominent respiratory symptoms; abnormal chest X-ray; elevated troponin, ALT, and thrombocytopenia. • Elevated troponin, ALT or thrombocytopenia had high sensitivity and negative predictive value on the need for intensive care interventions., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background and Aims: Lipid goals have become more stringent in high risk patients. However, no studies have analyzed lipid control defined as the composite achievement of goals in low-density lipoprotein cholesterol (LDL-C), non-high-density lipoprotein cholesterol (Non-HDL-C) and apolipoproteinB-100 (ApoB-100), in patients with premature coronary artery disease (CAD). We aimed to analyze lipid control rates, and the associated factors with its poor achievement in patients with premature CAD., Methods and Results: The study included 1196 patients with CAD diagnosed before 55 and 65 years old in men and women, respectively. The American Heart Association/American College of Cardiology (non-strict) and the American Association of Clinical Endocrinologists (strict) criteria were used to analyze lipid control rates. Sociodemographic, dietary-healthy and clinical characteristics of the patients were collected. Participants were 54 ± 8 years old, 19.7% were women, and median CAD evolution was 2.4 years. Non-strict and strict lipid control was achieved in 23.0% and 8.9% of the patients, respectively. Moreover, 46.5% and 62.8% of the patients did not achieve any lipid goal using both criteria. Sociodemographic data were not different among patients who achieved or not lipid control. Treatment adherence<85%, prescription of low- and moderate-intensity statins, and obesity were consistently associated with poor lipid control., Conclusions: Lipid control is suboptimal in patients with premature CAD. Low lipid-lowering treatment adherence, low prescription of high-intensity statins, and obesity were independently associated with poor lipid control. Novel preventive programs and more aggressive pharmacological intervention should be implemented in order to reduce the burden of premature CAD., Competing Interests: Declaration of Competing Interest The authors have nothing to disclose., (Copyright © 2020 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Objective: Positive psychological constructs, such as optimism, are associated with cardiovascular health, and changes in biological measures associated with heart health have been proposed as potential mediators of these relationships. In this analysis of data from a randomized controlled trial, we examined the impact of an optimism training intervention on biological measures associated with cardiac health in patients with coronary artery disease., Methods: We analyzed data from an 8-week, randomized, controlled trial of a group-based optimism training intervention in 61 patients with coronary artery disease. High-sensitivity C-reactive protein (hs-CRP), interleukin 6, irisin, and fibrinogen were measured at baseline, 8 weeks, and 16 weeks. Mixed-effects regression analyses were performed to examine the effects of the intervention on changes in biological measures at 8 and 16 weeks., Results: At 8 weeks, the intervention led to significantly greater reductions in hs-CRP (B = -0.851 [standard error {SE} = 0.273, p = .002) and fibrinogen (B = -0.148 [SE = 0.062], p = .016), and a greater increase in irisin (B = 0.252 [SE = 0.114], p = .027) compared with the control condition. These changes persisted at 16 weeks (hs-CRP: B = -1.078 [SE = 0.276], p < .001; fibrinogen: B = -0.270 [SE = 0.062], p < .001; irisin: B = 0.525 [SE = 0.116], p < .001), and interleukin 6 additionally was impacted at this time point (B = -0.214 [SE = 0.064], p = .001). Exploratory mediation analyses failed to identify significant psychological or health behavior mediators of these relationships., Conclusions: A group-based optimism training intervention resulted in significant, robust, and sustained changes in biological measures associated with cardiac health. Further studies are needed to confirm these findings in a larger sample and identify potential mediating variables., Trial Registration: Iran Registry of Clinical Trials No. 2016070328769 N1.

The Biological Magnetic Resonance Data Bank (BioMagResBank or BMRB), founded in 1988, serves as the archive for data generated by nuclear magnetic resonance (NMR) spectroscopy of biological systems. NMR spectroscopy is unique among biophysical approaches in its ability to provide a broad range of atomic and higher-level information relevant to the structural, dynamic, and chemical properties of biological macromolecules, as well as report on metabolite and natural product concentrations in complex mixtures and their chemical structures. BMRB became a core member of the Worldwide Protein Data Bank (wwPDB) in 2007, and the BMRB archive is now a core archive of the wwPDB. Currently, about 10% of the structures deposited into the PDB archive are based on NMR spectroscopy. BMRB stores experimental and derived data from biomolecular NMR studies. Newer BMRB biopolymer depositions are divided about evenly between those associated with structure determinations (atomic coordinates and supporting information archived in the PDB) and those reporting experimental information on molecular dynamics, conformational transitions, ligand binding, assigned chemical shifts, or other results from NMR spectroscopy. BMRB also provides resources for NMR studies of metabolites and other small molecules that are often macromolecular ligands and/or nonstandard residues. This chapter is directed to the structural biology community rather than the metabolomics and natural products community. Our goal is to describe various BMRB services offered to structural biology researchers and how they can be accessed and utilized. These services can be classified into four main groups: (1) data deposition, (2) data retrieval, (3) data analysis, and (4) services for NMR spectroscopists and software developers. The chapter also describes the NMR-STAR data format used by BMRB and the tools provided to facilitate its use. For programmers, BMRB offers an application programming interface (API) and libraries in the Python and R languages that enable users to develop their own BMRB-based tools for data analysis, visualization, and manipulation of NMR-STAR formatted files. BMRB also provides users with direct access tools through the NMRbox platform.

The growth of the biological nuclear magnetic resonance (NMR) field and the development of new experimental technology have mandated the revision and enlargement of the NMR-STAR ontology used to represent experiments, spectral and derived data, and supporting metadata. We present here a brief description of the NMR-STAR ontology and software tools for manipulating NMR-STAR data files, editing the files, extracting selected data, and creating data visualizations. Detailed information on these is accessible from the links provided.

Off-leash dog parks may enhance human health, but may also lead to health risk through infection or canine aggression. Published evidence was reviewed to examine positive and negative public health impacts of off-leash dog parks, as well as strategies for enhancing benefits and mitigating risks. Evidence suggests that off-leash dog parks can benefit physical and social health, as well as community connectedness. While studies have documented shedding of zoonotic agents in dog parks, the risk of transmission to humans is relatively unknown. Evidence on the risk of dog bites in off-leash dog parks is also limited. Case-examples from North American off-leash dog parks highlight the importance of park location/design, public adherence to safe and hygienic practices, and effective regulatory strategies for mitigating potential risks and maximizing the benefits of off-leash dog parks.

Hepatocellular Carcinoma (HCC) is an aggressive tumor entity, with the only curative options being surgical resection or orthotopic liver transplantation (OLT). The presence of one single tumor nodule of less than 5 centimeters diameter or a maximum of 3 nodules, with the largest of these not exceeding 3 centimeters (Milan criteria) constitute the clinical situation in which the best results for OLT in patients with HCC have been achieved. The survival of patients fulfilling the Milan criteria after transplantation is comparable to patients with similar tumor stages without cirrhosis, undergoing hepatic resection. The application of PET in oncology has become increasingly common in the last decade as it is a non-invasive tool that also gathers information about the degree of the biological aggressiveness of the tumor. The objective of this study was to perform a review of the literature, identifying the strengths and weaknesses of the PET as a prognostic tool in patients with HCC after OLT., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Advances in computation have been enabling many recent advances in biomolecular applications of NMR. Due to the wide diversity of applications of NMR, the number and variety of software packages for processing and analyzing NMR data is quite large, with labs relying on dozens, if not hundreds of software packages. Discovery, acquisition, installation, and maintenance of all these packages is a burdensome task. Because the majority of software packages originate in academic labs, persistence of the software is compromised when developers graduate, funding ceases, or investigators turn to other projects. To simplify access to and use of biomolecular NMR software, foster persistence, and enhance reproducibility of computational workflows, we have developed NMRbox, a shared resource for NMR software and computation. NMRbox employs virtualization to provide a comprehensive software environment preconfigured with hundreds of software packages, available as a downloadable virtual machine or as a Platform-as-a-Service supported by a dedicated compute cloud. Ongoing development includes a metadata harvester to regularize, annotate, and preserve workflows and facilitate and enhance data depositions to BioMagResBank, and tools for Bayesian inference to enhance the robustness and extensibility of computational analyses. In addition to facilitating use and preservation of the rich and dynamic software environment for biomolecular NMR, NMRbox fosters the development and deployment of a new class of metasoftware packages. NMRbox is freely available to not-for-profit users., (Copyright © 2017 Biophysical Society. All rights reserved.)

The metabolome, the collection of small molecules associated with an organism, is a growing subject of inquiry, with the data utilized for data-intensive systems biology, disease diagnostics, biomarker discovery, and the broader characterization of small molecules in mixtures. Owing to their close proximity to the functional endpoints that govern an organism's phenotype, metabolites are highly informative about functional states. The field of metabolomics identifies and quantifies endogenous and exogenous metabolites in biological samples. Information acquired from nuclear magnetic spectroscopy (NMR), mass spectrometry (MS), and the published literature, as processed by statistical approaches, are driving increasingly wider applications of metabolomics. This review focuses on the role of databases and software tools in advancing the rigor, robustness, reproducibility, and validation of metabolomics studies., (Copyright © 2016. Published by Elsevier Ltd.)

Heterologous production of isoprene (C 5 H 8 ) hydrocarbons in cyanobacteria, emanating from sunlight, CO 2 , and water, is now attracting increasing attention. The concept entails application of an isoprene synthase transgene from terrestrial plants, heterologously expressed in cyanobacteria, aiming to reprogram carbon flux in the terpenoid biosynthetic pathway toward formation and spontaneous release of this volatile chemical from the cell and liquid culture. However, flux manipulations and carbon-partitioning reactions between isoprene (the product) and native terpenoid biosynthesis for cellular needs are not yet optimized for isoprene yield. The primary reactant for isoprene biosynthesis is dimethylallyl diphosphate (DMAPP), whereas both DMAPP and its isopentenyl diphosphate (IPP) isomer are needed for cellular terpenoid biosynthesis. The present work addressed the function of an isopentenyl diphosphate (IPP) isomerase in cyanobacteria and its role in carbon partitioning between IPP and DMAPP, both of which serve, in variable ratios, as reactants for the synthesis of different cellular terpenoids. The work was approached upon the heterologous expression in Synechocystis of the "isopentenyl diphosphate isomerase" gene (FNI) from Streptococcus pneumoniae, using isoprene production as a "reporter process" for substrate partitioning between DMAPP and IPP. It is shown that transgenic expression of the FNI gene in Synechocystis resulted in a 250 % increase in the "reporter isoprene" rate and yield, suggesting that the FNI isomerase shifted the endogenous DMAPP-IPP steady-state pool size toward DMAPP, thereby enhancing rates and yield of isoprene production. The work provides insight into the significance and functional role of the IPP isomerase in these photosynthetic microorganisms.

Age remains the main risk factor for developing Alzheimer's disease (AD) although certain metabolic alterations, including prediabetes and hyperinsulinemia, also increase this risk. We present a mouse model of AD (APPswe/PS1dE9 mouse) with severe hyperinsulinemia induced by long-term high fat diet (HFD) treatment. After 23 weeks on HFD learning and memory processes were compromised. We observed a significant increase in tau hyperphosphorylation and Aβ pathology, including Aβ levels and amyloid burden. Microglia activation was also significantly increased in HFD-treated mice, both in close proximity to and far from senile plaques. Insulin degrading enzyme and neprilysin levels were not affected, suggesting that Aβ degradation pathways were preserved, whereas we detected an increase in spontaneous cortical bleeding that could underlay an impairment of Aβ interstitial fluid drainage, contributing to the increase in Aβ deposition in APP/PS1-HFD mice. Altogether our data suggest that early hyperinsulinemia is enough to exacerbate AD pathology observed in APP/PS1 mice, and supports the role of insulin-resistance therapies to stop or delay central complications associated., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The axis inhibition (Axin) scaffold protein colocalizes β-catenin, casein kinase Iα, and glycogen synthetase kinase 3β by their binding to Axin's long intrinsically disordered region, thereby yielding structured domains with flexible linkers. This complex leads to the phosphorylation of β-catenin, marking it for destruction. Fusing proteins with flexible linkers vastly accelerates chemical interactions between them by their colocalization. Here we propose that the complex works by random movements of a "stochastic machine," not by coordinated conformational changes. This non-covalent, modular assembly process allows the various molecular machine components to be used in multiple processes., (Copyright © 2013. Published by Elsevier B.V.)

Intrinsically disordered proteins (IDPs) do not adopt stable three-dimensional structures in physiological conditions, yet these proteins play crucial roles in biological phenomena. In most cases, intrinsic disorder manifests itself in segments or domains of an IDP, called intrinsically disordered regions (IDRs), but fully disordered IDPs also exist. Although IDRs can be detected as missing residues in protein structures determined by X-ray crystallography, no protocol has been developed to identify IDRs from structures obtained by Nuclear Magnetic Resonance (NMR). Here, we propose a computational method to assign IDRs based on NMR structures. We compared missing residues of X-ray structures with residue-wise deviations of NMR structures for identical proteins, and derived a threshold deviation that gives the best correlation of ordered and disordered regions of both structures. The obtained threshold of 3.2Å was applied to proteins whose structures were only determined by NMR, and the resulting IDRs were analyzed and compared to those of X-ray structures with no NMR counterpart in terms of sequence length, IDR fraction, protein function, cellular location, and amino acid composition, all of which suggest distinct characteristics. The structural knowledge of IDPs is still inadequate compared with that of structured proteins. Our method can collect and utilize IDRs from structures determined by NMR, potentially enhancing the understanding of IDPs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In 2006 and 2007 beef and pork carcass swabs from provincially inspected abattoirs in Alberta, Canada were tested to determine the levels of total aerobic bacteria, coliform bacteria, and generic Escherichia coli, and the prevalence of Salmonella spp., Campylobacter spp., and Shiga toxin-producing E. coli (STEC). Swabs from beef and pork carcasses from 48 and 34 facilities, respectively, were analyzed. All samples tested were positive for aerobic bacteria with 99.8% of beef and 96.0% of pork samples, having total counts of ≤ 100 000 CFU/cm(2). Coliform bacteria were isolated from 22.4% and 42.0% of beef and pork carcass samples, respectively. Generic E. coli were recovered from 14.6% of beef and 33.7% of pork carcass samples. For beef carcasses, positive tests were obtained for 0.1% of 1036 samples tested for Salmonella spp., 1.5% of 1022 samples tested for Campylobacter spp. and 5.4% of 1018 samples tested for STEC. For pork carcasses, positive tests were obtained for 1.6 % of 1076 samples tested for Salmonella spp., 8.8% of 1070 samples tested for Campylobacter spp. and 4.8% of 1067 samples tested for STEC.

The best treatment for hepatocellular carcinoma (HCC) associated with liver cirrhosis is liver transplantation and the best results are obtained when the tumors fulfill the Milan criteria. However, although the number of transplants is increasing, the organ deficit is growing, which lengthens time on the waiting list, increasing the risk of tumor progression of and exclusion from the list. The use of elderly donors is a valid option for patients on the transplant waiting list with HCC, reducing time on the waiting list. We report our experience with patients transplanted for HCC associated with hepatic cirrhosis using livers from donors >75 years of age. Our preliminary results supported the use of elderly suboptimal donors making it possible to give priority to these patients. All patients in the series achieved good graft function after a follow-up of 2 years with a 100% disease-free survival rate. More extensive long-term studies are needed to confirm these findings., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

It is widely assumed that new proteins are created by duplication, fusion, or fission of existing coding sequences. Another mechanism of protein birth is provided by overlapping genes. They are created de novo by mutations within a coding sequence that lead to the expression of a novel protein in another reading frame, a process called "overprinting." To investigate this mechanism, we have analyzed the sequences of the protein products of manually curated overlapping genes from 43 genera of unspliced RNA viruses infecting eukaryotes. Overlapping proteins have a sequence composition globally biased toward disorder-promoting amino acids and are predicted to contain significantly more structural disorder than nonoverlapping proteins. By analyzing the phylogenetic distribution of overlapping proteins, we were able to confirm that 17 of these had been created de novo and to study them individually. Most proteins created de novo are orphans (i.e., restricted to one species or genus). Almost all are accessory proteins that play a role in viral pathogenicity or spread, rather than proteins central to viral replication or structure. Most proteins created de novo are predicted to be fully disordered and have a highly unusual sequence composition. This suggests that some viral overlapping reading frames encoding hypothetical proteins with highly biased composition, often discarded as noncoding, might in fact encode proteins. Some proteins created de novo are predicted to be ordered, however, and whenever a three-dimensional structure of such a protein has been solved, it corresponds to a fold previously unobserved, suggesting that the study of these proteins could enhance our knowledge of protein space.

Despite a growing body of evidence demonstrating the importance of cities as sources of many local, regional, and global impacts on the atmosphere, ecosystems, and human populations, most theories on the relationship between society and the environment have focused on the global or national level. A variety of theories exist on human-environment interactions; for example, ecological modernization, urban transitions, and human ecology. However, with the exception of urban transitions, these theories have been mainly concerned with nation states and have ignored the subnational and local (city) levels. This article aims at filling this gap by employing ordinary least squares regression to examine these theories at the city level using the STIRPAT formula. It finds that with the exception of population (which shows an unstable relationship with the impacts indicators applied in the analysis) a remarkable level of variation exists in the importance of drivers across the three exercises. This led us to conclude that urban atmospheric pollutants result from diverse activities (e.g., transportation, industrial), are formed through different processes (vehicle combustion, biomass burning), have a residence time ranging from hours to years, and are the outcome of diverse sets of societal and environmental drivers.

Studies that isolated Listeria spp. from the environment of two meat processing facilities were conducted. Samples were collected in the processing environment of the facilities with three different sampling methods (cotton swab, sterile sponge, and composite-ply tissues) to evaluate their ability to recover Listeria spp. A total of 240 samples for each sampling method were collected and tested. The cotton swab method of sampling was significantly (P < 0.01) less efficient in recovery of Listeria spp. than the sterile-sponge and composite-ply tissue methods, which were similar (P > 0.05) in their ability to recover Listeria spp. The specificity and sensitivity of four detection methods (conventional culture, Petrifilm Environmental Listeria Plates [ELP], lateral-flow immunoprecipitation [LFI], and automated PCR) were evaluated for identification of Listeria spp. Facilities were visited until a minimum of 100 positive and 100 negative samples per detection method were collected. The LFI and PCR methods were highly sensitive (95.5 and 99.1%, respectively) and specific (100%) relative to the culture method. The ELP method was significantly less efficient (P < 0.01) than LFI and PCR in detection of Listeria spp., with lower sensitivity (50.6%) and specificity (91.5%). Kappa values indicated excellent agreement of the LFI and PCR assays and moderate agreement of the ELP method to the culture method. Overall, ELP was easy to use but less efficient in detection of Listeria spp. from environmental samples, while the LFI and PCR methods were found to be excellent alternatives to culture, considering performance and time and labor inputs.

Studies to determine baseline levels of microbial contaminants and foodborne bacterial pathogens are needed to evaluate the effectiveness of Hazard Analysis Critical Control Point (HACCP) programs, Good Manufacturing/Production Practices, and various interventions. In 2004 and 2005 poultry carcass rinses from provincially inspected abattoirs in Alberta, Canada, were tested to determine the levels of aerobic plate count bacteria, coliform bacteria, and generic Escherichia coli, the prevalence and levels of Campylobacter spp., and the prevalence of Salmonella spp. and Shiga toxin-producing E. coli (STEC). Samples were collected from 3 high volume and 62 low volume abbatoirs. All samples (1296) were positive for aerobic plate count bacteria, with 98.8% of samples having counts of 100 000 or less colony forming units (CFU)/cm2. Coliform bacteria were isolated from 99.7% of the 1296 carcasses and were recovered at levels of < or = 1000 CFU/cm2 for 98.3% of the samples. Generic E. coli were recovered from 99.1% of the 1296 carcasses at levels of < or = 1000 CFU/cm2 for 98.6% of the samples. Seventy five percent of 1234 samples that were tested for Campylobacter were positive; 37.5% of 1295 samples that were tested for Salmonella were positive; and only 2 of 1296 samples tested for STEC were positive (0.15%).

The synergistic effect of ultraviolet radiation (UVR) and a polycyclic aromatic hydrocarbon (PAH) was tested on the coral Porites divaricata. Small branches were incubated in different concentrations of fluoranthene followed by exposure to ecologically relevant levels of natural solar radiation, with and without UVR. Exposure to the highest concentration of fluoranthene (60 microg L(- 1)) resulted in decreased photochemical efficiency of corals exposed concomitantly to UVR but not in corals exposed to 60 microg L(- 1) fluoranthene without UVR. After 6 days, most coral samples that were exposed to fluoranthene and UVR bleached or died (78%) on the upper side of the branches. At 60 microg L(- 1) fluoranthene with UVR, 11 out of 12 samples died and the remaining sample bleached, much greater than the number of samples that bleached in manipulation or solvent controls. On the under side of the same samples, where the coral polyps are naturally protected from UVR, 11 out of 12 samples remained healthy and intact. The high UVR doses that shallow water dwelling corals receive combined with the results of this study show that photoinduced toxicity of PAHs is a stress factor that needs to be studied in more detail in coral reef ecosystems.

A new material for matrix solid phase dispersion (MSPD) was synthesized -- p-nitro-N-propylaniline/silica (pNNPASi) by grafting reactions, characterized by elemental analysis and N(2)-adsorption-desorption isotherms, and tested for multiclass multiresidue analysis of pesticides in wet and freeze-dried carrots. Results obtained applying this new solid phase sorbent to MSPD extraction of ten pesticides (trichlorphon, trifluralin, dicloran, chlorothalonil, prometryn, linuron, captan, procymidone, prochloraz, and deltametrin) in wet carrots showed better results than the ones obtained for freeze-dried samples. Recoveries were in the range of 48-106% and precisions varied from 6 to 20% when wet samples were employed. Comparison between pNNPASi sorbent and C(18) showed better performance of pNNPASi for eight out of ten pesticides tested. The LOQs show that the developed method can be used to detect the pesticides investigated in carrots at concentrations below the maximum residue levels (MRL) established by EU, USEPA, and National Sanitary Surveillance Agency (ANVISA). Linuron, captan, prochloraz, and deltamethrin were found in at least one of the two commercial samples studied in concentrations above the LOQ of this method. Concentrations of the last three pesticides were above the European MRL in one of the commercial samples.

The Protein Data Bank (PDB) is the preeminent source of protein structural information. PDB contains over 32,500 experimentally determined 3-D structures solved using X-ray crystallography or nuclear magnetic resonance spectroscopy. Intrinsically disordered regions fail to form a fixed 3-D structure under physiological conditions. In this study, we compare the amino-acid sequences of proteins whose structures are determined by X-ray crystallography with the corresponding sequences from the Swiss-Prot database. The analyzed dataset includes 16,370 structures, which represent 18,101 PDB chains and 5,434 different proteins from 910 different organisms (2,793 eukaryotic, 2,109 bacterial, 288 viral, and 244 archaeal). In this dataset, on average, each Swiss-Prot protein is represented by 7 PDB chains with 76% of the crystallized regions being represented by more than one structure. Intriguingly, the complete sequences of only approximately 7% of proteins are observed in the corresponding PDB structures, and only approximately 25% of the total dataset have >95% of their lengths observed in the corresponding PDB structures. This suggests that the vast majority of PDB proteins is shorter than their corresponding Swiss-Prot sequences and/or contain numerous residues, which are not observed in maps of electron density. To determine the prevalence of disordered regions in PDB, the residues in the Swiss-Prot sequences were grouped into four general categories, "Observed" (which correspond to structured regions), "Not observed" (regions with missing electron density, potentially disordered), "Uncharacterized," and "Ambiguous," depending on their appearance in the corresponding PDB entries. This non-redundant set of residues can be viewed as a 'fragment' or empirical domain database that contains a set of experimentally determined structured regions or domains and a set of experimentally verified disordered regions or domains. We studied the propensities and properties of residues in these four categories and analyzed their relations to the predictions of disorder using several algorithms. "Non-observed," "Ambiguous," and "Uncharacterized" regions were shown to possess the amino acid compositional biases typical of intrinsically disordered proteins. The application of four different disorder predictors (PONDR(R) VL-XT, VL3-BA, VSL1P, and IUPred) revealed that the vast majority of residues in the "Observed" dataset are ordered, and that the "Not observed" regions are mostly disordered. The "Uncharacterized" regions possess some tendency toward order, whereas the predictions for the short "Ambiguous" regions are really ambiguous. Long "Ambiguous" regions (>70 amino acid residues) are mostly predicted to be ordered, suggesting that they are likely to be "wobbly" domains. Overall, we showed that completely ordered proteins are not highly abundant in PDB and many PDB sequences have disordered regions. In fact, in the analyzed dataset approximately 10% of the PDB proteins contain regions of consecutive missing or ambiguous residues longer than 30 amino-acids and approximately 40% of the proteins possess short regions (> or =10 and < 30 amino-acid long) of missing and ambiguous residues.

We described the clinical diagnostic process utilized during the 2001 epidemic of foot-and-mouth-disease in the United Kingdom (UK), and considered it as a series of diagnostic tests. Premises were classified according to these diagnostic-test results and actual disease status, determined by the reference test, which in this case was one or more internationally accepted laboratory tests. The herd-level sensitivity (HSe) and herd-level specificity (HSp) of the clinical diagnostic process were calculated directly, relative to these internationally accepted reference tests. In this process, the first diagnostic test was 'routine monitoring', which resulted in the identification of suspect cases based solely on the clinical observations of farmers or veterinarians. 6762 suspect cases were identified, and the test had a HSe of 97.6% (95% C.I.: 96.7, 98.3) and a HSp of 95.2% (95% C.I.: 95.0, 95.3). Suspect cases were then subject to the second diagnostic test, termed 'declaration', which consisted of a review of a description of the clinical signs by government veterinarians. Premises that tested positive became 'clinical cases'. The HSe of this test was 97.1% (95% C.I.: 96.2, 97.9), and the HSp was 90.9% (95% C.I.: 90.1, 91.6). During the epidemic, these tests were combined and applied in series, with an overall HSe of 94.7% (95% C.I.: 93.5, 95.7) and an overall HSp of 99.6% (95% C.I.: 99.5, 99.6). We also examined the effect of a policy shift that prohibited delaying the diagnosis pending laboratory testing where the animals exhibited equivocal clinical signs.

The expected number of new infections per day per infectious person during an epidemic has been found to exhibit power-law scaling with respect to the susceptible fraction of the population. This is in contrast to the linear scaling assumed in traditional epidemiologic modeling. Based on simulated epidemic dynamics in synthetic populations representing Los Angeles, Chicago, and Portland, we find city-dependent scaling exponents in the range of 1.7-2.06. This scaling arises from variations in the strength, duration, and number of contacts per person. Implementation of power-law scaling of the new infection rate is quite simple for SIR, SEIR, and histogram-based epidemic models. Treatment of the effects of the social contact structure through this power-law formulation leads to significantly lower predictions of final epidemic size than the traditional linear formulation.

We sampled 1,091 Icelandic broiler flocks at slaughter from May 2001 to December 2003 to determine the prevalence of, and investigate risk factors for the presence of, Campylobacter spp. at the flock level. Approximately 15% of the flocks were positive for Campylobacter spp.; most (95%) of the infected flocks being raised during the months of April-September. Based on the data from the latter months, and using multivariable logistic regression with random effects for herd, we found that the odds of a flock being positive for Campylobacter spp. increased with age and flock size. Additionally, vertical ventilation systems were strongly associated with positive flocks (OR=5.3). After controlling for these variables, we found no evidence of an effect of: year; company; Campylobacter being carried over from one flock to the next; time interval between flocks; using (at the hatcheries) eggs laid on the floor; density of bird housing, or the number of catch lots a flock was divided into for slaughtering purposes on the risk of a Campylobacter-positive flock.

Alternative splicing of pre-mRNA generates two or more protein isoforms from a single gene, thereby contributing to protein diversity. Despite intensive efforts, an understanding of the protein structure-function implications of alternative splicing is still lacking. Intrinsic disorder, which is a lack of equilibrium 3D structure under physiological conditions, may provide this understanding. Intrinsic disorder is a common phenomenon, particularly in multicellular eukaryotes, and is responsible for important protein functions including regulation and signaling. We hypothesize that polypeptide segments affected by alternative splicing are most often intrinsically disordered such that alternative splicing enables functional and regulatory diversity while avoiding structural complications. We analyzed a set of 46 differentially spliced genes encoding experimentally characterized human proteins containing both structured and intrinsically disordered amino acid segments. We show that 81% of 75 alternatively spliced fragments in these proteins were associated with fully (57%) or partially (24%) disordered protein regions. Regions affected by alternative splicing were significantly biased toward encoding disordered residues, with a vanishingly small P value. A larger data set composed of 558 SwissProt proteins with known isoforms produced by 1,266 alternatively spliced fragments was characterized by applying the pondr vsl1 disorder predictor. Results from prediction data are consistent with those obtained from experimental data, further supporting the proposed hypothesis. Associating alternative splicing with protein disorder enables the time- and tissue-specific modulation of protein function needed for cell differentiation and the evolution of multicellular organisms.

Vibrio cholerae, the etiological agent of the diarrheal illness cholera, can kill an infected adult in 24 h. V.cholerae lives as an autochthonous microbe in estuaries, rivers and coastal waters. A better understanding of its metabolic pathways will assist the development of more effective treatments and will provide a deeper understanding of how this bacterium persists in natural aquatic habitats. Using the completed V.cholerae genome sequence and PathoLogic software, we created VchoCyc, a pathway-genome database that predicted 171 likely metabolic pathways in the bacterium. We report here experimental evidence supporting the computationally predicted pathways. The evidence comes from microarray gene expression studies of V.cholerae in the stools of three cholera patients [D. S. Merrell, S. M. Butler, F. Qadri, N. A. Dolganov, A. Alam, M. B. Cohen, S. B. Calderwood, G. K. Schoolnik and A. Camilli (2002) Nature, 417, 642-645.], from gene expression studies in minimal growth conditions and LB rich medium, and from clinical tests that identify V.cholerae. Expression data provide evidence supporting 92 (53%) of the 171 pathways. The clinical tests provide evidence supporting seven pathways, with six pathways supported by both methods. VchoCyc provides biologists with a useful tool for analyzing this organism's metabolic and genomic information, which could lead to potential insights into new anti-bacterial agents. VchoCyc is available in the BioCyc database collection (http://BioCyc.org).

Several computational and experimental methods exist for identifying disordered residues within proteins. Computational algorithms can now identify these disordered sequences and predict their occurrence within genomes with relatively high accuracy. Recent advances in NMR and mass spectroscopy permit faster and more detailed studies of disordered states at atomic resolutions. Combining prediction, computation and experimentation is proposed to accelerate and enhance the characterization of intrinsically disordered protein., (Copyright 2004 Elsevier Ltd.)

Objective: The epidemiological risk factors for the different forms of diabetic macular edema (focal or diffuse) are not differentiated in the literature. In the present study, the objective was to determine which risk factors influence the appearance of focal or diffuse macular edema. RESEARCH, DESIGN, AND METHODS: A 4-year prospective study in a population of Type II diabetic patients (NIDDM) in three groups, the first with 29 patients with diffuse diabetic macular edema (Group A), the second with 32 patients with focal macular edema (Group B), and the third with 32 patients with diabetic retinopathy but without diabetic macular edema (Group C); the three groups were selected on the basis of sex, age, and duration of diabetes with similar patterns. We studied the following risk factors: treatment of diabetes mellitus by insulin, presence of diastolic blood hypertension, presence of microalbuminuria or diabetic nephropathy, levels of HbA1c, type of diabetic retinopathy, presence of previous panretinal photocoagulation, and levels of triglycerides and fractions of cholesterol., Results: In discriminant analysis and in order of importance, the epidemiological risk factors for Group A were the following: presence of panretinal laser, diastolic blood hypertension, diabetic nephropathy, and insulin treatment; for group B, the risk factors were the following: insulin treatment, presence of diastolic blood hypertension, and high levels of glycated haemoglobin (HbA1c); and for group, C the risk factors were the following: diastolic blood hypertension, insulin treatment of diabetes, and high levels of HbA1c., Conclusions: In the present study, the group of patients with focal macular edema were epidemiologically similar to the group of patients with diabetic retinopathy without macular edema, the group of patients with diffuse macular edema were epidemiologically different.

Motivation: The prediction of transcription units (TUs, which are similar to operons) is an important problem that has been tackled using many different approaches. The availability of complete microbial genomes has made genome-wide TU predictions possible. Pathway-genome databases (PGDBs) add metabolic and other organizational (i.e. protein complexes) information to the annotated genome, and are able to capture TU organization information. These characteristics of PGDBs make them a suitable framework for the development and implementation of TU predictors., Results: We implemented a TU predictor that uses only intergenic distance and functional classification of genes to predict TU boundaries, and applied it to EcoCyc, our PGDB of Escherichia coli. To this original predictor, we added information on metabolic pathways, protein complexes and transporters, all readily available in EcoCyc, in order to generate an enhanced predictor. The enhanced predictor correctly predicted 80% of the known E.coli TUs (69% of the known operons), a moderate improvement over the original predictor's performance (75% of TUs and 65% of operons correctly predicted), demonstrating that the extra information available in the PGDB does indeed improve prediction performance. Performance of this E.coli-based predictor on a genome other than that of E.coli was tested on BsubCyc, our computationally generated PGDB for Bacillus subtilis, for which a set of 100 known operons is available. Prediction accuracy decreased substantially (46% of the known operons correctly predicted). This was due in part to missing information in BsubCyc, which prevented full use of the predictor's features. The augmented predictor has been implemented as part of our Pathway Tools software suite, and can be used to populate a PGDB with predicted TUs., Availability: The TU predictor is included in version 7.0 of the Pathway Tools software suite. Pathway Tools 7.0 is available free of charge to academic institutions and for a fee to commercial enterprises. It runs on Sun Solaris 8, Linux and Windows. TUs predicted on the Caulobacter crescentus and Mycobacterium tuberculosis (H37Rv) genomes are available in our CauloCyc and MtbrvCyc databases, available at the BioCyc web site (http://biocyc.org). To obtain version 7.0 of Pathway Tools, follow the directions in our web site, http://biocyc.org/download.shtml.

We present an algorithm that solves two related problems in the analysis of metabolic networks stored within a pathway/genome database. (1) The Forward Propagation Problem: given a set of nutrients that are inputs to the metabolic network, what compounds will be produced by the metabolic network? (2) The Backtracking Problem: given the results of a forward propagation, and given a set of essential compounds that are not produced as a result of the forward propagation, what precursors must be supplied to produce those essential compounds? A program based on this algorithm is applied to the EcoCyc database, which is a pathway/genome database for E. coli that consists of annotated genomes and the metabolic reactions and pathways associated with the known gene products. The inputs to the program are a description of the metabolic network of an organism (EcoCyc), a set of nutrients corresponding to a known minimal growth medium, and a list of essential compounds to be produced. The program "fires" the microorganism's metabolism contained in the database and predicts all synthesized and nonsynthesized essential compounds, along with the missing precursors required to produce the latter. When applied to the EcoCyc database, the program identifies a number of missing precursors that indicate incomplete regions of the database. Thus the program results can be used to evaluate existing pathway databases like EcoCyc.

Background: Numerous prognostic factors have been studied for survival in patients with papillary thyroid carcinoma (PTC), although there are few multivariate studies that include the histological variety of PTC., Hypothesis: There are prognostic factors that influence survival in a series of patients with PTC, including the histological variety, and a new prognostic index (PI) for survival can be formulated by accounting for these factors., Design: A retrospective study., Setting: A university hospital department of surgery., Patients: Between January 1970 and December 1995, 200 patients undergoing surgery for PTC were observed (mean follow-up, 8 years)., Main Outcome Measures: A univariate analysis was done for survival rates using the Kaplan-Meier estimation method. The possible prognostic factors were evaluated using a multivariate analysis according to the Cox model. We formulated a PI and defined 3 risk groups (low, medium, and high) for mortality., Results: Of the 200 patients, 175 (87.5%) are still alive. Of the 25 deaths, 19 (9.5%) were due to the tumor. The survival was 97.5% at 1 year, 92.8% at 5, 89.5% at 10, and 83.9% at 15 and 20 years. The prognostic factors obtained after the multivariate analysis were age, tumor size, extrathyroid spread, and histological variant of the PTC. The PI is calculated as follows: PI = (2 x size) + (6 x spread) + (2 x variant) + (3 x age). As for the risk groups, the low-risk group showed a mortality of 0%; the medium-risk group, 17.1%; and the high-risk group, 76.5%., Conclusions: The histological variety of PTC has prognostic value for survival in patients with PTC. As risk factors for PTC mortality, we consider an age of 50 years or older, a tumor larger than 4 cm, the existence of extrathyroid spread, and a certain histological subtype of PTC. With these risk factors, it is possible to formulate a PI and classify patients into low-, medium-, and high-risk groups for mortality.

The amounts of time spent by females in the sector of an open field close to the cage housing a normal male or a castrated male were measured in order to quantitate the tendency of the female to reach physical proximity to a sexually active male (androtropism). Intact proestrous or ovariectomized females primed with 100 micrograms of estradiol benzoate/kg b. wt. (EB) or EB plus 2 mg progesterone/kg b. wt. (P) spent significantly more time close to the sexually active (intact) male than in the proximity of the orchidectomized male. In order to determine whether olfactory clues were sufficient for female rats to distinguish between intact and castrated males, the males were removed from the stimulus cages, leaving the soiled bedding in place. Ovariectomized rats primed with EB or EB plus P clearly preferred proximity to the cage where the intact male had been living. No preference was evident after transection of olfactory nerves in proestrous rats or in ovariectomized rats primed with EB plus P. Resection of the vomeronasal organ also suppressed preference. These results indicate that olfactory input is necessary and sufficient for androtropism to occur, and suggest that the accessory olfactory system is involved in the analysis of olfactory signals used by female rats to identify the endocrine status of prospective sexual partners. In a different group of animals, it was demonstrated that destruction of the posteromedial cortical amygdaloid nucleus also suppressed preference for the intact male. It is proposed that this structure serves as a relay station for the analysis and integration of olfactory input significant for the motivational control of sexual behavior in the female rat.(ABSTRACT TRUNCATED AT 250 WORDS)