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Toll-like receptors (TLRs) contribute to host resistance to microbial pathogens and can drive the evolution of virulence mechanisms. We have examined the relationship between host resistance and pathogen virulence using mice with a functional allele of the nramp-1 gene and lacking combinations of TLRs. Mice deficient in both TLR2 and TLR4 were highly susceptible to the intracellular bacterial pathogen Salmonella typhimurium, consistent with reduced innate immune function. However, mice lacking additional TLRs involved in S. typhimurium recognition were less susceptible to infection. In these TLR-deficient cells, bacteria failed to upregulate Salmonella pathogenicity island 2 (SPI-2) genes and did not form a replicative compartment. We demonstrate that TLR signaling enhances the rate of acidification of the Salmonella-containing phagosome, and inhibition of this acidification prevents SPI-2 induction. Our results indicate that S. typhimurium requires cues from the innate immune system to regulate virulence genes necessary for intracellular survival, growth, and systemic infection., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The ligand bis(2-isobutyrylamidophenyl)amine has been prepared and used to stabilize both mononuclear and dinuclear cobalt(II) complexes. The nuclearity of the cobalt product is regulated by the deprotonation state of the ligand. Both complexes catalytically oxidize triphenylphosphine to triphenylphosphine oxide in the presence of O(2).

Ecology is a synthetic discipline benefiting from open access to data from the earth, life, and social sciences. Technological challenges exist, however, due to the dispersed and heterogeneous nature of these data. Standardization of methods and development of robust metadata can increase data access but are not sufficient. Reproducibility of analyses is also important, and executable workflows are addressing this issue by capturing data provenance. Sociological challenges, including inadequate rewards for sharing data, must also be resolved. The establishment of well-curated, federated data repositories will provide a means to preserve data while promoting attribution and acknowledgement of its use.

In this work we describe the identification of a copper-inducible regulon in Mycobacterium tuberculosis (Mtb). Among the regulated genes was Rv0190/MT0200, a paralogue of the copper metalloregulatory repressor CsoR. The five-locus regulon, which includes a gene that encodes the copper-protective metallothionein MymT, was highly induced in wild-type Mtb treated with copper, and highly expressed in an Rv0190/MT0200 mutant. Importantly, the Rv0190/MT0200 mutant was hyper-resistant to copper. The promoters of all five loci share a palindromic motif that was recognized by the gene product of Rv0190/MT0200. For this reason we named Rv0190/MT0200 RicR for regulated in copper repressor. Intriguingly, several of the RicR-regulated genes, including MymT, are unique to pathogenic Mycobacteria. The identification of a copper-responsive regulon specific to virulent mycobacterial species suggests copper homeostasis must be maintained during an infection. Alternatively, copper may provide a cue for the expression of genes unrelated to metal homeostasis, but nonetheless necessary for survival in a host., (© 2010 Blackwell Publishing Ltd.)

Human embryonic stem cells (hESC) require a balance of growth factors and signaling molecules to proliferate and retain pluripotency. Conditioned medium (CM) from a human embryonic germ-cell-derived cell culture, SDEC, was observed to support the growth of hESC on type I collagen (COL I) and on Matrigel (MAT) biomatricies. After 1 month, the population doubling of hESC grown in SDEC CM on COL I was equivalent to that of hESC grown in mouse embryonic fibroblast (MEF) CM on MAT. hESC grown in SDEC CM on COL I expressed OCT4, NANOG, SSEA-4, alkaline phosphatase (AP), and TRA-1-60; retained a normal karyotype; and were capable of forming teratomas. DNA microarray analysis was used to compare the transcriptional profiles of SDEC and the less supportive WI38 and Detroit 551 human cell lines. The mRNA level of secreted frizzled-related protein (sFRP-1), a known antagonist of the WNT/β-catenin signaling pathway, was significantly reduced in SDEC as compared with the other 2 cell lines, whereas the mRNA levels of prostaglandin-endoperoxide synthase 2 (PTGS2 or COX-2) and prostaglandin I₂ synthase (PGIS), two prostaglandin biosynthesis genes, were significantly increased in SDEC. The level of sFRP-1 protein was significantly reduced, and levels of 2 prostaglandins that are downstream products of PTGS2 and PGIS, prostaglandin E₂ and 6-keto-prostaglandin F(1α), were significantly elevated in SDEC CM compared with WI38, Detroit 551, and MEF CM. Further, addition of purified sFRP-1 to SDEC CM reduced the proliferation of hESC grown on COL I as well as MAT in a dose-dependent manner.

With increasing oil exploration in Arctic regions, the risk of an accidental oil spill into the environment is inevitably elevated. As a result, concerns have been raised over the potential impact of oil exposure on Arctic organisms. This study assessed the effects of an acute oil exposure (mimicking an accidental spill) on the immune function and oxidative stress status of the Arctic scallop Chlamys islandica. Scallops were exposed to the water accommodated fraction of crude oil over 21 d (maximum SigmaPAH 163 microg l(-1)) and immune endpoints and oxidative stress parameters were measured. Mortalities were recorded during the exposure and reductions in immunocompetence were observed, with significant impairment of phagocytosis and cell membrane stability. Scallops were also subjected to oxidative stress, with a significant reduction in glutathione levels and induction of lipid peroxidation. After the acute oil exposure had subsided, no recovery of immune function was observed indicating potential for prolonged sublethal effects., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Background: Menstrual Toxic Shock Syndrome (mTSS) is thought to be associated with the vaginal colonization with specific strains of Staphylococcus aureus TSST-1 in women who lack sufficient antibody titers to this toxin. There are no published studies that examine the seroconversion in women with various colonization patterns of this organism. Thus, the aim of this study was to evaluate the persistence of Staphylococcus aureus colonization at three body sites (vagina, nares, and anus) and serum antibody to toxic shock syndrome toxin-producing Staphylococcus aureus among a small group of healthy, menstruating women evaluated previously in a larger study., Methods: One year after the completion of that study, 311 subjects were recalled into 5 groups. Four samples were obtained from each participant at several visits over an additional 6-11 month period: 1) an anterior nares swab; 2) an anal swab; 3) a vagina swab; and 4) a blood sample. Gram stain, a catalase test, and a rapid S. aureus-specific latex agglutination test were performed to phenotypically identify S. aureus from sample swabs. A competitive ELISA was used to quantify TSST-1 production. Human TSST-1 IgG antibodies were determined from the blood samples using a sandwich ELISA method., Results: We found only 41% of toxigenic S. aureus and 35.5% of non-toxigenic nasal carriage could be classified as persistent. None of the toxigenic S. aureus vaginal or anal carriage could be classified as persistent. Despite the low persistence of S. aureus colonization, subjects colonized with a toxigenic strain were found to display distributions of antibody titers skewed toward higher titers than other subjects. Seven percent (5/75) of subjects became seropositive during recall, but none experienced toxic shock syndrome-like symptoms., Conclusions: Nasal carriage of S. aureus appears to be persistent and the best predicator of subsequent colonization, whereas vaginal and anal carriage appear to be more transient. From these findings, it appears that antibody titers in women found to be colonized with toxigenic S. aureus remained skewed toward higher titers whether or not the colonies were found to be persistent or transient in nature. This suggests that colonization at some point in time is sufficient to elevate antibody titer levels and those levels appear to be persistent. Results also indicate that women can become seropositive without experiencing signs or symptoms of toxic shock syndrome.

Recent findings have established a role for the ST6Gal-1 sialyltransferase in modulating inflammatory cell production during Th1 and Th2 responses. ST6Gal-1 synthesizes the Sia(alpha2,6) to Gal(beta1,4)GlcNAc linkage on glycoproteins on cell surfaces and in systemic circulation. Engagement of P1, one of six promoter/regulatory regions driving murine ST6Gal-1 gene expression, generates the ST6Gal-1 for myelopoietic regulation. P1 utilization, however, is restricted to the liver and silent in hematopoietic cells. We considered the possibility that myelopoiesis is responsive to the sialylation of liver-derived circulatory glycoproteins, such that reduced alpha2,6-sialylation results in elevated myelopoiesis. However, 2-dimensional differential in gel electrophoresis (2D-DIGE) analysis disclosed only minimal alterations in the sialylation of sera glycoproteins of ST6Gal-1-deficient mice when compared with wild-type controls, either at baseline or during an acute phase response when the demand for sialylation is greatest. Furthermore, sera from ST6Gal-1-deficient animals did not enhance myelopoietic activity in ex vivo colony formation assays. Whereas there was only minimal consequence to the alpha2,6-sialylation of circulatory glycoproteins, ablation of the P1 promoter did result in strikingly depressed levels of ST6Gal-1 released into systemic circulation. Therefore, we considered the alternative possibility that myelopoiesis may be regulated not by the hepatic sialyl glycoproteins, but by the ST6Gal-1 that was released directly into circulation. Supporting this, ex vivo colony formation was notably attenuated upon introduction of physiologic levels of ST6Gal-1 into the culture medium. Our data support the idea that circulatory ST6Gal-1, mostly of hepatic origin, limits myelopoiesis by a mechanism independent of hepatic sialylation of serum glycoproteins.

The utility of some biomarkers in environmental monitoring may be limited due to the lack of knowledge that exists on how they respond to extrinsic abiotic and intrinsic biotic factors. During the present study we investigated the seasonal responses of three biomarkers, Neutral Red Retention, clearance/filtration rate and heart rate in the common blue mussel Mytilus edulis located in the Exe Estuary, UK during September 2006-September 2007. During the current study, a significant decrease in feeding rate was observed in mussels during June, July and August 2007, coinciding with the period following spawning when the mussels lay down nutrient reserves. Heart rate also increased between April and September 2007 and corresponded with times when mussels were spawning and laying down nutrient reserves. By integrating the individual biomarker responses into a Biomarker Response Index (BRI) we were able to identify times of the year when environmental impact was highest and hence when the timing of monitoring programmes using biomarkers should be carried out. For many years the lack of knowledge of normal physiological ranges of biomarkers has impeded their applied use, however by integrating biomarker responses into the BRI and creating an index of health, we have shown that we can limit the natural variability of individual responses; and thus we are better able to make informed judgements on the overall health status of these populations of mussels.

Carbohydrates are attractive candidates for drug development because sugars are involved in many, if not most, complex human diseases including cancer, immune dysfunction, congenital disorders, and infectious diseases. Unfortunately, potential therapeutic benefits of sugar-based drugs are offset by poor pharmacologic properties that include rapid serum clearance, poor cellular uptake, and relatively high concentrations required for efficacy. To address these issues, pilot studies are reported here where 'Bu(4)ManNAc', a short chain fatty acid-monosaccharide hybrid molecule with anti-cancer activities, was encapsulated in polyethylene glycol-sebacic acid (PEG-SA) polymers. Sustained release of biologically active compound was achieved for over a week from drug-laden polymer formulated into microparticles thus offering a dramatic improvement over the twice daily administration currently used for in vivo studies. In a second strategy, a tributanoylated ManNAc analog (3,4,6-O-Bu(3)ManNAc) with anti-cancer activities was covalently linked to PEG-SA and formulated into nanoparticles suitable for drug delivery; once again release of biologically active compound was demonstrated.

Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man(5)GlcNAc(2)-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man(9)GlcNAc(2)-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc(3)Man(9)GlcNAc(2)-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man(5)GlcNAc(2)-PP-Dol through Glc(1)Man(9)GlcNAc(2)-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc(3)Man(9)GlcNAc(2)-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation., (2010 Elsevier Inc. All rights reserved.)

Quorum-sensing (QS), the regulation of bacterial gene expression in response to changes in cell density, involves pathways that synthesize signaling molecules (auto-inducers). The luxS/AI-2-mediated QS system has been identified in both gram-positive and gram-negative bacteria. Bacillus anthracis, the etiological agent of anthrax, possesses genes involved in luxS/AI-2-mediated QS, and deletion of luxS in B. anthracis Sterne strain 34F2 results in inhibition of AI-2 synthesis and a growth defect. In the present study, we created a ΔluxS B. anthracis strain complemented in trans by insertion of a cassette, including luxS and a gene encoding erythromycin resistance, into the truncated plcR regulator locus. The complemented ΔluxS strain has restored AI-2 synthesis and wild-type growth. A B. anthracis microarray study revealed consistent differential gene expression between the wild-type and ΔluxS strain, including downregulation of the B. anthracis S-layer protein gene EA1 and pXO1 virulence genes. These data indicate that B. anthracis may use luxS/AI-2-mediated QS to regulate growth, density-dependent gene expression and virulence factor expression.

Phenanthrene, a major component of crude oil, is one of the most abundant PAHs in aquatic ecosystems, and is readily bioavailable and toxic to a range of marine invertebrates. Within bivalves, the haemolymph acts as a transfer medium for these pollutants and their metabolic products, leaving haemocytes susceptible to deleterious effects. Using a suite of biological endpoints, this study determined the sublethal (7-d exposure to 50, 100 and 200microgL(-1)) effects of phenanthrene on several oxidative stress and immunological parameters in the haemolymph of the commercially-important scallop Pecten maximus. Phenanthrene exposure (200microgL(-1)) resulted in immune modulation with significant reductions in cell membrane stability (P<0.05) and phagocytosis (P<0.05), and a significant increase in the number of total haemocytes (P<0.05). Oxidative stress was also observed with a significant decrease in total glutathione (P<0.05) and significantly increased levels of lipid peroxidation in the haemolymph (P<0.05). Changes in the cellular and biochemical endpoints observed in this study illustrate their potential use in assessing the subtle effects of contaminant exposure. Whilst previous reports have suggested a link between free radical generation and immune suppression in vertebrates, this is the first instance where oxidative stress and immune function have been measured together in the haemolymph of a bivalve mollusc, demonstrating a possible link between PAH-induced oxidative stress and the subsequent inhibition in haemocyte immune function., (2010 Elsevier Ltd. All rights reserved.)

Purpose: Due to variation of outcome among cases, we sought to examine whether overall survival in ovarian cancer was associated with common inherited variants in 227 candidate genes from ovarian cancer-related pathways including angiogenesis, inflammation, detoxification, glycosylation, one-carbon transfer, apoptosis, cell cycle regulation, and cellular senescence., Experimental Design: Blood samples were obtained from 325 women with invasive epithelial ovarian cancer diagnosed at the Mayo Clinic from 1999 to 2006. During a median follow-up of 3.8 years (range, 0.1-8.6 years), 157 deaths were observed. Germline DNA was analyzed at 1,416 single nucleotide polymorphisms (SNP). For all patients, and for 203 with serous subtype, we assessed the overall significance of each gene and pathway, and estimated risk of death via hazard ratios (HR) and 95% confidence intervals (CI), adjusting for known prognostic factors., Results: Variation within angiogenesis was most strongly associated with survival time overall (P = 0.03) and among patients with serous cancer (P = 0.05), particularly for EIF2B5 rs4912474 (all patients HR, 0.69; 95% CI, 0.54-0.89; P = 0.004), VEGFC rs17697305 (serous subtype HR, 2.29; 95% CI, 1.34-3.92; P = 0.003), and four SNPs in VHL. Variation within the inflammation pathway was borderline significant (all patients, P = 0.09), and SNPs in CCR3, IL1B, IL18, CCL2, and ALOX5 which correlated with survival time are worthy of follow-up., Conclusion: An extensive multiple-pathway assessment found evidence that inherited differences may play a role in outcome of ovarian cancer patients, particularly in genes within the angiogenesis and inflammation pathways. Our work supports efforts to target such mediators for therapeutic gain.

Background: Menstrual tampons are available in a range of absorbencies to allow women to use the product most appropriate to their needs. This study assessed the safety of an ultra absorbency (15 g to 18 g) tampon compared with a currently marketed super-plus absorbency (12 g to 15 g) tampon as a control., Methods: Healthy women age 18-45 years (n = 95) were enrolled in this single-blind, crossover study. Subjects used, in random order, the experimental tampon during one menstrual cycle and the control tampon during the other. Subjects were also randomly assigned to receive either vaginal microbial assessments for determination of the presence and density of Staphylococcus aureus (n = 35) or colposcopic examinations for assessment of changes in the vaginal mucosa (n = 60). Data on comfort and acceptability of the tampons were collected by using diaries and questionnaires completed by the subjects in both groups., Results: Twenty-eight women in the microbial assessment group and 43 in the colposcopic examination group completed the study; these subjects made up the primary analysis population. No differences in isolation frequency or cell density of S. aureus or in vaginal mucosal changes were observed with the experimental tampon in comparison with the control tampon. No reported adverse events were attributed to the experimental tampon. Both tampons received positive comfort ratings., Conclusions: Based upon microbial assessments, colposcopic examinations, adverse events, and subject reporting of comfort, the ultra absorbency tampon is similar in safety profile to the currently marketed super plus absorbency tampon.

The synthesis and characterization of nickel complexes supported by a family of open-chain, tetradentate, tris(amidate) ligands, [N(o-PhNC(O)R)(3)](3-) ([L(R)](3-) where R = (i)Pr, (t)Bu, and Ph) is described. The complexes [Ni(L(iPr))](-), [Ni(L(tBu))](-), and [Ni(L(Ph))(CH(3)CN)](-) have been characterized by solution-state spectroscopic methods and single crystal X-ray diffraction. Each ligand gives rise to a different primary coordination sphere about the nickel centre. These studies indicate that the ligands' acyl substituents can be used to regulate the coordination mode of the amidate donors to nickel and the coordination number of the nickel centres. In addition, the ability of these complexes to bind cyanide has been explored. These experiments demonstrate that only one of these complexes, [Ni(L(iPr))](-), is able to irreversibly bind cyanide and can be used to assemble [Et(4)N](3)[Ni(L(iPr))(mu(2)-CN)Co(L(iPr))], a cyanide bridged, heterobimetallic complex. The synthesis and characterization of the cyanide containing complexes, including magnetic susceptibility studies, are described.

Pathogen-associated molecular patterns (PAMPs) enable recognition of structures present in microorganisms such as lipopolysaccharides (LPS). LPS are an essential constituent of the outer membrane of Gram-negative bacteria, stimulating the innate immune system of invertebrates. Here, LPS from Escherichia coli (055:B5) were used to investigate the functional immune response of Pecten maximus after stimulation with a PAMP and to determine the combined effect of a phenanthrene exposure and LPS challenge. Organisms were exposed to 200 mug l(-1) phenanthrene and after 7 d were injected with either physiological saline (injection controls) or LPS solution, and returned to their respective exposure tanks. Haemolymph was sampled from the scallops 48 h post-injection and immune function was assessed using a combination of cellular biological responses. The LPS challenge significantly altered the immune response in P. maximus with increased cell counts and phagocytic activity. An immunosuppressive effect of phenanthrene was also observed in this study; however, exposure to phenanthrene did not significantly impair the organism's ability to respond to a PAMP challenge. The overall level of phagocytosis and cytotoxic capability following the LPS challenge was lower in phenanthrene exposed scallops and may have consequences for disease resistance in this commercially-exploited species., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 microg/ml, 5 microg/ml, 12 microg/ml, 3 microg/ml, and 4 microg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 microg/ml, 8 microg/ml, and 2 to 8 microg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

This paper presents an innovative, quantitative assessment of pollution avoidance attributable to environmental regulation enforced through integrated licensing, using Ireland's pharmaceutical-manufacturing sector as a case study. Emissions data reported by pharmaceutical installations were aggregated into a pollution trend using an Environmental Emissions Index (EEI) based on Lifecycle Assessment methodologies. Complete sectoral emissions data from 2001 to 2007 were extrapolated back to 1995, based on available data. Production volume data were used to derive a sectoral production index, and determine 'no-improvement' emission trends, whilst questionnaire responses from 20 industry representatives were used to quantify the contribution of integrated licensing to emission avoidance relative to these trends. Between 2001 and 2007, there was a 40% absolute reduction in direct pollution from 27 core installations, and 45% pollution avoidance relative to hypothetical 'no-improvement' pollution. It was estimated that environmental regulation avoided 20% of 'no-improvement' pollution, in addition to 25% avoidance under business-as-usual. For specific emissions, avoidance ranged from 14% and 30 kt a(-1) for CO(2) to 88% and 598 t a(-1) for SO(x). Between 1995 and 2007, there was a 59% absolute reduction in direct pollution, and 76% pollution avoidance. Pollution avoidance was dominated by reductions in emissions of VOCs, SO(x) and NO(x) to air, and emissions of heavy metals to water. Pollution avoidance of 35% was attributed to integrated licensing, ranging from between 8% and 2.9 t a(-1) for phosphorus emissions to water to 49% and 3143 t a(-1) for SO(x) emissions to air. Environmental regulation enforced through integrated licensing has been the major driver of substantial pollution avoidance achieved by Ireland's pharmaceutical sector - through emission limit values associated with Best Available Techniques, emissions monitoring and reporting requirements, and performance targets specified in environmental management plans. This compliant sector offers a positive, but not necessarily typical, case study of IPPC effectiveness.

Integrated laboratory studies addressed multiple biomarker responses in the sea star (Asterias rubens) and the blue mussel (Mytilus edulis) exposed to a range of concentrations of direct and indirect acting genotoxins: methyl methane sulfonate (MMS) and cyclophosphamide (CP; an environmentally relevant anti-cancer pharmaceutical), respectively, in order to determine if the expressed genotoxicity has knock-on effects at the higher levels of biological organisation. The experimental design aimed to concurrently evaluate biomarkers of behavioural and physiological conditions (i.e. 'righting time' and 'clearance rate' for sea stars and mussels, respectively) in addition to cytotoxicity (neutral red retention assay), induction of micronuclei (Mn) and DNA strand breaks (as determined by the Comet assay). The protocol also included the determination of the maximum tolerated concentration (MTC), prior to genotoxic evaluation. The 3d MTC, as determined by the survival of the organisms, showed sea stars to be more sensitive than mussels to MMS (18 and 32 mg L(-1), respectively) and CP (56 and 180 mg L(-1), respectively). For both species and chemicals, cytotoxicity was not found to be significantly different compared to controls. Apart from the MMS exposure to sea stars (which showed 100% mortality at higher concentrations after 5d exposure), clear dose-response relationships were observed for both genotoxicity endpoints in each species. Following exposure to CP, good correlations were also found between the behavioural and physiological responses and genetic damage in each species (sea stars-MN vs. RT: R=0.73; Comet vs. RT: R=0.91; mussels-MN vs. CR: R=0.69; Comet vs. CR: R=0.72). This integrated approach, applying non-invasive assays to simultaneously determine the responses at different levels of biological organisation, indicates the potential value of behavioural and physiological measures in determining the toxicity of chemicals to marine organisms and highlights also the relevance of including adult echinoderms in environmental studies.

A series of European Marine Sites has been designated as Special Areas of Conservation (SAC) in England. The aim of this study was to develop a practical methodology to assess the condition of SACs by applying a suite of biomarkers. Biomarkers were applied to the blue mussel Mytilus edulis and the shore crab Carcinus maenas from the Fal and Helford SAC (Cornwall). Individual biomarkers provided useful diagnostic information on the activity of certain classes of contaminants and an integrated Biomarker Response Index (BRI) was used to achieve a more holistic understanding of the condition of the SAC. The BRI indicated that the general health of both organisms was impacted in the upper part of the SAC (Fal Estuary) which correlated well with known chemical hotspots and sources of contamination. The BRI allows a pragmatic way to prioritise SAC sites that may require further investigative studies.

N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide.

With the current expansion of offshore oil activities in Arctic regions, there is an urgent need to establish the potential effects of oil-related compounds on Arctic organisms. As susceptibility to growth, disease and survival is determined partly by the condition of an organism's immune system, measurement of endpoints linked to the latter system provide important early warning signals of the sub-lethal effects of exposure to contaminants. This study assessed the impact of dispersed oil exposure on immune endpoints in the Arctic Scallop Chlamys islandica, using a combination of cellular and humoral biological responses. Laboratory exposures of C. islandica to sub-lethal dispersed oil concentrations (0.06 and 0.25 mg l(-1)) were conducted over 15 days, followed by a 7-day recovery period in clean, filtered seawater. Cellular endpoints were significantly altered following dispersed oil exposure: haemocyte counts (P<0.01) and protein levels (P<0.01) were significantly elevated, whilst cell membrane stability (P<0.001) and phagocytosis (P<0.01) demonstrated a significant reduction. Whilst these results indicate alteration in the immune endpoints measured, this appears to be reversible upon removal of the contaminant stress. However, the impact of long-term continuous exposure and high-level acute exposure to oil is still unknown, and may have consequences for disease resistance and hence survival.

The induction of genetic competence is a strategy used by bacteria to increase their genetic repertoire under stressful environmental conditions. Recently, Streptococcus pneumoniae has been shown to co-ordinate the uptake of transforming DNA with fratricide via increased expression of the peptide pheromone responsible for competence induction. Here, we document that environmental stress-induced expression of the peptide pheromone competence-stimulating peptide (CSP) in the oral pathogen Streptococcus mutans. We showed that CSP is involved in the stress response and determined the CSP-induced regulon in S. mutans by microarray analysis. Contrary to pneumococcus, S. mutans responds to increased concentrations of CSP by cell lysis in only a fraction of the population. We have focused on the mechanism of cell lysis and have identified a novel bacteriocin as the 'death effector'. Most importantly, we showed that this bacteriocin causes cell death via a novel mechanism of action: intracellular action against self. We have also identified the cognate bacteriocin immunity protein, which resides in a separate unlinked genetic locus to allow its differential regulation. The role of the lytic response in S. mutans competence is also discussed. Together, these findings reveal a novel autolytic pathway in S. mutans which may be involved in the dissemination of fitness-enhancing genes in the oral biofilm.

Background: After observing disparate rates of cytoreduction, we initiated efforts to improve outcomes through feedback and education, and we reassessed outcomes., Study Design: Outcomes from group A (2006 and 2007, n=105) were compared with those from the cohort predating quality-improvement efforts (group B, 2000 to 2003, n=132). All stage IIIC ovarian cancer patients at our institution were evaluated for tumor dissemination, age, performance status, surgical complexity, residual disease (RD), morbidity, and mortality. A surgical complexity score previously described was used to categorize extent of operation., Results: No significant differences in age, performance status, or extent of disease were observed between cohorts. Surgical complexity increased after initiation of quality improvement (mean surgical complexity score, 5.5 to 7.1; p < 0.001), rates of optimal RD (< 1 cm) improved from 77% to 85% (p=0.157), and rates of complete resection of all gross disease rose from 31% to 43% (p=0.188). In the subset of patients with carcinomatosis most likely to benefit from extended surgical resection, radical procedures were used more frequently (63% versus 79%; p=0.028), rates of optimal debulking (RD<1 cm) increased (64% to 79%), and the rate of RD=0 increased from 6% to 24% (p=0.006). When disease was noted on the diaphragm, procedures to remove the disease were more frequently used (38% to 64%; p=0.001). The rates of major perioperative morbidity (group B, 21% versus group A, 20%; p=0.819) and 3-month mortality (8% versus 6%; p=0.475) were not affected despite this more aggressive surgical approach., Conclusions: Analysis of outcomes with appropriate feedback and education is a powerful tool for quality improvement. We observed improvements in rates of cytoreduction and use of specific radical procedures, with no increase in morbidity as a result of this process.

Motivation: Gaussian graphical models (GGMs) are a popular tool for representing gene association structures. We propose using estimated partial correlations from these models to attach lengths to the edges of the GGM, where the length of an edge is inversely related to the partial correlation between the gene pair. Graphical lasso is used to fit the GGMs and obtain partial correlations. The shortest paths between pairs of genes are found. Where terminal genes have the same biological function intermediate genes on the path are classified as having the same function. We validate the method using genes of known function using the Rosetta Compendium of yeast (Saccharomyces Cerevisiae) gene expression profiles. We also compare our results with those obtained using a graph constructed using correlations., Results: Using a partial correlation graph, we are able to classify approximately twice as many genes to the same level of accuracy as when using a correlation graph. More importantly when both methods are tuned to classify a similar number of genes, the partial correlation approach can increase the accuracy of the classifications.

Perennial species with the C(4) pathway hold promise for biomass-based energy sources. We have explored the extent that CO(2) uptake of such species may be limited by light in a temperate climate. One energetic cost of the C(4) pathway is the leakiness () of bundle sheath tissues, whereby a variable proportion of the CO(2), concentrated in bundle sheath cells, retrodiffuses back to the mesophyll. In this study, we scale from leaf to canopy level of a Miscanthus crop (Miscanthus x giganteus hybrid) under field conditions and model the likely limitations to CO(2) fixation. At the leaf level, measurements of photosynthesis coupled to online carbon isotope discrimination showed that leaves within a 3.3-m canopy (leaf area index = 8.3) show a progressive increase in both carbon isotope discrimination and as light decreases. A similar increase was observed at the ecosystem scale when we used eddy covariance net ecosystem CO(2) fluxes, together with isotopic profiles, to partition photosynthetic and respiratory isotopic flux densities (isofluxes) and derive canopy carbon isotope discrimination as an integrated proxy for at the canopy level. Modeled values of canopy CO(2) fixation using leaf-level measurements of suggest that around 32% of potential photosynthetic carbon gain is lost due to light limitation, whereas using determined independently from isofluxes at the canopy level the reduction in canopy CO(2) uptake is estimated at 14%. Based on these results, we identify as an important limitation to CO(2) uptake of crops with the C(4) pathway.

This study measured aspects of the physiology of juvenile [(<35mm carapace width (CW)] and adult (>60mm CW) Carcinus maenas to test the hypothesis that these different life-history stages exhibit different sensitivities to environmental contamination. Newly-collected juveniles had significantly (P<0.05) lower immunocompetence (phagocytosis and cellular integrity), lower metabolic energy (haemolymph glucose) and increased scope for growth compared with adults. Seven day exposure to a sub-lethal concentration (200microgL(-1)) of pyrene significantly (P<0.01) reduced immunocompetence, elevated basal heart rate and decreased respiration (at rest) for juveniles but had no overall impact on adult crab physiology. Results confirm that physiological differences exist between juvenile and adult shore crabs, and cause juveniles to be more susceptible to the effects of pyrene exposure than adults. Such differences in sensitivity to contamination between life-cycle stages of the same species have to be taken into account during the risk assessment process.

Using the shore crab Carcinus maenas as a model, this study tested the hypothesis that nutritional status influences susceptibility of adult crabs (>60mm carapace width (CW)) to environmental contamination. In the laboratory, crabs were either starved, given a restricted diet (fed on alternate days) or fully fed (fed each day). In addition, crabs under each feeding regime were exposed to a sublethal concentration (200microgl(-1)) of pyrene (PYR) as a model organic (PAH (polyaromatic hydrocarbon)) contaminant. Various physiological end points were measured after 7 and 14 days. Results indicated that adult shore crab physiology was relatively robust to short-term (7 days) nutritional changes as multivariate analysis (ANOSIM) showed no significant difference in shore crab physiological condition between control and pyrene-exposed crabs, irrespective of dietary feeding regime [Global R=0.018, P (%)=19.2]. After 14 days, however, starved crabs showed significant impacts to physiological condition (as revealed by multivariate analysis) [Global R=0.134, P (%)=0.1], [R=0.209, P (%)=0.1]; starved individuals had significantly lower antioxidant status (F(2,48)=5.35, P<0.01) compared to crabs under both types of feeding regime. Exposure to pyrene resulted in significantly elevated pyrene metabolite concentrations in the urine at 7 and 14 days compared with control individuals (P<0.001), validating contaminant bioavailability, and this was found for all dietary treatments. Also, exposed crabs had significantly increased protein levels (proteinuria) than controls (P<0.001) in their urine after 7 and 14 days, irrespective of dietary regime. After 7 days, pyrene-exposed crabs showed significantly increased antioxidant status (P<0.001) and cellular functioning (increased cellular viability and decreased phagocytosis) (P<0.001) compared to control crabs; however, after 14 days, antioxidant status (P<0.01) and cellular viability (P<0.001) were significantly decreased in pyrene-exposed compared to unexposed crabs. Results indicate that differences in nutritional status of adult crabs result in shore crabs being robust to short-term sublethal (7 days) pyrene exposure. Susceptibility to contaminant exposure, however, was measured after prolonged exposure (14 days) as indicated by reduced ability to combat oxidative stress. These results indicate that ecotoxicological studies need to take into account the nutritional state of the test organism to achieve the full assessment of contaminant impact. In addition, the results highlight that subtle seasonal biotic features of an organism can influence biomarker responses, and these need to be considered when interpreting field data and during the routine application of biological-effects tools in environmental monitoring.

Many cases of neonatal toxic shock syndrome (TSS)-like exanthematous disease but few cases of menstrual TSS (mTSS) have been reported in Japan. We determined the prevalence of mucosal colonization with Staphylococcus aureus and of positive antibodies to TSS toxin 1 (TSST-1) among 209 healthy Japanese women in Tokyo. S. aureus isolates from mucosal sites were characterized with respect to TSST-1 production and resistance genotype. Antibody titers were determined for test subjects and for 133 Japanese and 137 Caucasian control women living in the United States. S. aureus was isolated from at least one site in 108 of 209 women (52%) in Tokyo. Of the 159 S. aureus isolates recovered, 14 (9%) were TSST-1 positive (12 unique strains). Twelve of 209 women (6%) were colonized with a TSST-1-producing strain; two (<1%) had vaginal colonization. Only 2 of 12 unique toxigenic strains (14%) were methicillin resistant. Of the 12 TSST-1-positive strains isolated, 6 (50%) were pulsed-field gel electrophoresis type USA200, multilocus sequence type clonal complex 30. Fewer Japanese women in Tokyo (47%) than Caucasian and Japanese women in the United States (89% and 75%, respectively) had TSST-1 antibodies. The prevalences of colonization with TSST-1-producing S. aureus were comparable in Japan and the United States, despite low seropositivity to TSST-1 in Japan. Environmental factors appear to be important in promoting the development of anti-TSST-1 antibodies, as there was a significant difference in titers between Japanese women living in Tokyo and those living in the United States. Most colonizing TSST-1-producing S. aureus strains in Japan were genotypically similar to mTSS strains found in the United States.

To answer the requirement of the European Commission's Water Framework Directive (WFD) for biological-effects endpoints to classify the ecological health of aquatic ecosystems, we propose the biomarker response index (BRI). The BRI, based on a suite of biomarkers at different levels of biological response at the individual level, provides an integrated relative measure of the general health status of coastal invertebrates. Using the BRI, the health of mussels (Mytilus edulis) from 10 estuaries classified by the Environment Agency of England and Wales under the WFD was compared. Eight sites were healthier than predicted and two showed a similar health status to that of the predicted point-source pollution risk classification. Results indicate that the BRI offers a potential measure of organism health that can be used in monitoring under the WFD as an additional aid to reduce uncertainty in defining risk classification and to provide better evidence of existing impact.

Objective: Tight junction (TJ) proteins claudin-3 and claudin-4 may be differentially expressed in uterine serous papillary carcinoma (USPC), a rare form of endometrial cancer characterized by a particularly poor prognosis. Our aim was to determine the expression pattern and prognostic relevance of claudin-3 and claudin-4 in a large cohort of endometrial cancer patients of diverse histological type and stage., Methods: Claudin-3 and claudin-4 expression was studied in a cohort of 287 patients with endometrial cancer including 137 cases of USPC or clear-cell histology using immunohistochemistry. Patients were completely surgically staged. Outcome data is available on all 287 patients., Results: The rate of claudin-3 and claudin-4 expression was significantly higher in USPC and clear-cell endometrial cancer compared to endometrioid endometrial cancer (claudin-3: 78% and 61% versus 38%, p<.0001; claudin-4: 56% and 44% versus 9%, p<.0001). Furthermore, expression of both TJ proteins was significantly associated with poor clinical outcome (claudin-3, DFS RR 1.70, p=.0087, OS RR 1.62, p=.0247; claudin-4, DFS RR 2.66, p<0.0001, and OS RR 2.50, p<0.0001). However, both markers did not maintain prognostic independence in multivariate analyses, as their expression was tightly associated with more advanced disease stages (p<.0001 for both), and higher nuclear grade (p<.0001 for both)., Conclusion: These clinical observations confirm the hypothesis based on preclinical evidence that increased expression of claudin-3 and claudin-4 may contribute to the aggressive phenotype of endometrial cancer of serous papillary or clear-cell histology and suggest their potential utility as diagnostic biomarkers and possible targets for therapeutic intervention.

Objective: To prospectively assess pelvic and para-aortic lymph node metastases in endometrial cancer with lymphatic dissemination, emphasizing the examination of para-aortic metastases relative to the inferior mesenteric artery (IMA)., Methods: Over 36 months, 422 consecutive patients were managed by predefined surgical guidelines differentiating low-risk patients from patients at risk for dissemination requiring systematic lymphadenectomy. Low risk was defined as grade 1 or 2 endometrioid type with myometrial invasion (MI) < or = 50% and primary tumor diameter (PTD) < or = 2 cm. Pelvic and para-aortic lymph nodes were submitted separately, with nodes identified from all 8 pelvic and 4 para-aortic node-bearing basins. Surgical quality assessments examined median node counts (primary surrogate for quality) and nodes harvested above and below the IMA and excised gonadal veins (secondary surrogates)., Results: Lymphadenectomy was not required in 27% of patients (all low risk) and in 33% (n=112) of endometrioid cases. However, 22 patients (20%) of this latter cohort had lymphadenectomy and all lymph nodes were negative. Sixty-three (22%) of 281 patients undergoing lymphadenectomy had lymph node metastases: both pelvic and para-aortic in 51%, only pelvic in 33%, and isolated to the para-aortic area in 16%. Therefore, 67% of patients with lymphatic dissemination had para-aortic lymph node metastases. Furthermore, 77% of patients with para-aortic node involvement had metastases above the IMA, whereas nodes in the ipsilateral para-aortic area below the IMA and ipsilateral common iliac basin were declared negative in 60% and 71%, respectively. Gonadal veins were excised in 25 patients with para-aortic node metastases; 7 patients (28%) had documented metastatic involvement of gonadal veins or surrounding soft tissue., Conclusions: The high rate of lymphatic metastasis above the IMA indicates the need for systematic pelvic and para-aortic lymphadenectomy (vs sampling) up to the renal vessels. The latter should include consideration of excision of the gonadal veins. Conversely, lymphadenectomy does not benefit patients with grade 1 and 2 endometrioid lesions with MI < or = 50% and PTD < or = 2 cm.

Although the potential use of biomarkers within environmental risk assessment (ERA) has long been recognised their routine use is less advanced compared with clinical human health risk assessment, where a number of familiar biomarkers (such as blood pressure and serum cholesterol) are in common usage. We have examined how biomarkers are incorporated into human health risk assessment and have identified several 'required elements'. These include identification of the (clinical) assessment endpoint at the outset, rational selection of the biomarker(s) (the measurement endpoint), biomarker 'validation' (e.g. QA/QC) and biomarker 'qualification' (evidence linking the measurement and assessment endpoints). We discuss these elements in detail and propose that their adoption will facilitate the routine use of biomarkers in environmental risk assessment. Furthermore, our analysis highlights the need for cooperation between those working with biomarkers within human and environmental risk assessment to exchange best practice between common disciplines for mutual advantage.

Here, we identify and characterise cholinesterase (ChE) and carboxylesterase (CbE) activities in the body tissues of the sediment dwelling worm Arenicola marina. Exposure to the organophosphorus pesticide azamethiphos yielded an in vitro IC50 of 5 microg l(-1) for propionylcholinesterase (PChE). PChE was significantly inhibited in vivo after a 10 day exposure to 100 microg l(-1) azamethiphos, equivalent to the recommended aquatic application rate (ANOVA; F=2.75, P=0.033). To determine sensitivity to environmental conditions, A. marina were exposed for 10 days to field collected sediments. PChE activity was significantly lower in worms exposed to sediments from an estuary classified to be at high risk from point source pollution by the UK Environment Agency (ANOVA; F=15.33, P<0.001). Whilst causality cannot be directly attributed from these latter exposures, they provide an important illustration of the potential utility of esterase activity as a biomarker of environmental quality in this ecologically relevant sentinel species.

Ecology is inherently cross-disciplinary, drawing together many types of information to address questions about the natural world. Finding and integrating relevant data to assist in these analyses is crucial, but is difficult owing to ambiguous terminology and the lack of sufficient information about datasets. Ontologies provide a formal mechanism for defining terms and their relationships, and can improve the location, interpretation and integration of data based on its inherent meaning. Ontologies have assisted other disciplines (e.g. molecular biology) in unifying and enriching descriptions of data, and ecology can benefit from similar approaches. We review ontology efforts in ecology, and describe how these can benefit research by enhancing the location and interpretation of relevant data for confronting crucial ecological questions.

This paper presents an elastohydrodynamic model of the human eyelid wiper. Standard lubrication theory is applied to the fluid layer between the eyelid wiper and ocular surface. The role of the lubrication film is to reduce the shear stresses by preventing solid to solid contact between the eyelid wiper and ocular surface. For the lubrication film to be effective, it is required that the orientation of the eyelid wiper changes between the opening and closing phases of a blink. In order to model this, the hydrodynamic model is coupled with an elastic mattress model for the soft tissue of the eyelid wiper and ocular surface. This leads to a one-dimensional non-linear partial differential equation governing the fluid pressure in the lubrication film. In order to solve the differential equation, a loading condition or constraint equation must be specified. The resulting system is then solved numerically. The model allows predictions of the tear film flux from under the upper eyelid, as well as normal and shear stresses acting on the ocular surface. These factors are important in relation to dry eye syndrome, deformation of the cornea and contact lens design. It is found that the pressure and shear stress under the eyelid act across a length of approximately 0.1 mm which is consistent with clinical observations. It order to achieve a flow of tears from under the upper eyelid during a blink, the model requires that the normal force the eyelid applies to the ocular surface during the closing phase of the blink is significantly higher than during the opening phase of the blink.

The objective of this article is to decompose the level of functioning phenotype in autism to see if it can be conceptualized as two simpler, but still familial, dimensional phenotypes of language and non-verbal IQ. We assembled 80 sibpairs with either autism, Asperger syndrome or atypical autism. To see whether the familial correlation on language scores was accounted for by the familial correlation on non-verbal IQ, residual language scores were calculated for each member of the sibpair based on a multiple regression equation using their IQ score as an explanatory or independent variable and controlling for the age and gender of the affected individual. These residual scores were then used to calculate intraclass correlations between affected sibs. This process was repeated using IQ as the dependent variable and language as a covariate. Within affected individuals there was a strong relation between non-verbal IQ (as measured by the Leiter performance scale) and language (as measured by the Vineland Communication Scale). In addition, there was familial correlation between sibs on both measures. Evidence of familial aggregation on both non-verbal IQ and language remained even after partialling out the effect of the covariates by regression analysis and by generalized estimating equation. These findings suggest that non-verbal IQ and language in PDD may arise from independent genetic mechanisms. The implications of this finding for linkage analysis and for identifying genetically informative phenotypes are discussed., ((c) 2007 Wiley-Liss, Inc.)

The syntheses of two phenylamine-based ligand systems, N(o-PhNH(2))(3) and N(o-PhNHC(O)(i)Pr)(3), are reported. These ligands readily coordinate to Co(II) to form monomeric complexes. X-ray diffraction studies establish that the [N(o-PhNC(O)(i)Pr)(3)](3-) ligand stabilizes the Co(II) ion in a trigonal-monopyramidal coordination environment. The axial coordination site in this complex is accessible and, upon cyanide coordination, generates an electrochemically active species.

Background: Bacillus anthracis, the etiologic agent of inhalational anthrax, is a facultative intracellular pathogen. Despite appropriate antimicrobial therapy, the mortality from inhalational anthrax approaches 45%, underscoring the need for better adjuvant therapies. The variable latency between exposure and development of disease suggests an important role for the host's innate immune response. Type I and Type II Interferons (IFN) are prominent members of the host innate immune response and are required for control of intracellular pathogens. We have previously described a protective role for exogenous Type I and Type II IFNs in attenuating intracellular B.anthracis germination and macrophage cell death in vitro., Methodology and Principal Findings: We sought to extend these findings in an in vivo model of inhalational anthrax, utilizing the Sterne strain (34F2) of B.anthracis. Mice devoid of STAT1, a component of IFN-alpha and IFN-gamma signaling, had a trend towards increased mortality, bacterial germination and extrapulmonary spread of B.anthracis at 24 hrs. This was associated with impaired IL-6, IL-10 and IL-12 production. However, administration of exogenous IFN-gamma, and to a lesser extent IFN-alpha, at the time of infection, markedly increased lethality. While IFNs were able to reduce the fraction of germinated spores within the lung, they increased both the local and systemic inflammatory response manifest by increases in IL-12 and reductions in IL-10. This was associated with an increase in extrapulmonary dissemination. The mechanism of IFN mediated inflammation appears to be in part due to STAT1 independent signaling., Conclusions: In conclusion, while endogenous IFNs are essential for control of B.anthracis germination and lethality, administration of exogenous IFNs appear to increase the local inflammatory response, thereby increasing mortality.

Objectives: Assess the pattern of groin node metastases in squamous cell carcinoma (SCC) of the vulva in relation to the site of the primary lesion. Assess whether the identified pattern of lymphatic spread supports the current surgical practice of assessing contralateral nodes for lateral lesions with ipsilateral nodal involvement., Methods: A retrospective study of surgically staged patients with primary SCC of the vulva between 1955 and 1990 was conducted. This cohort of patients was divided in 4 subgroups by location of primary lesion: unilateral, bilateral, midline, and patients with mediolateral lesions. All clinical and pathological data were reviewed and updated to the 1988 TNM vulvar classification., Results: 320 patients met the inclusion criteria, and almost all of them (>95%) underwent bilateral groin assessment. Of the 108 patients with positive groin lymph-node (LN) involvement, 77 presented with unilateral and 24 with bilateral inguinofemoral involvement. Of the 163 patients presenting with only unilateral vulvar lesions, 48 had inguinofemoral node involvement: 37 with ipsilateral-only nodal metastases, 8 with bilateral LN invasion, and only 3 (1.8%) had isolated contralateral nodal metastases. None of these patients with unilateral vulvar lesion that was either < or = 2 cm in biggest diameter or with invasion < or = 5 mm had bilateral groin LN involvement at diagnosis., Conclusions: Ipsilateral lymphadenectomy is suitable for patients with unilateral lesions, distant from the midline, and either negative ipsilateral nodes, or with positive ipsilateral LN with lesions smaller than 2 cm.

Multi-drug tolerance is a key phenotypic property that complicates the sterilization of mammals infected with Mycobacterium tuberculosis. Previous studies have established that iniBAC, an operon that confers multi-drug tolerance to M. bovis BCG through an associated pump-like activity, is induced by the antibiotics isoniazid (INH) and ethambutol (EMB). An improved understanding of the functional role of antibiotic-induced genes and the regulation of drug tolerance may be gained by studying the factors that regulate antibiotic-mediated gene expression. An M. smegmatis strain containing a lacZ gene fused to the promoter of M. tuberculosis iniBAC (PiniBAC) was subjected to transposon mutagenesis. Mutants with constitutive expression and increased EMB-mediated induction of PiniBAC::lacZ mapped to the lsr2 gene (MSMEG6065), a small basic protein of unknown function that is highly conserved among mycobacteria. These mutants had a marked change in colony morphology and generated a new polar lipid. Complementation with multi-copy M. tuberculosis lsr2 (Rv3597c) returned PiniBAC expression to baseline, reversed the observed morphological and lipid changes, and repressed PiniBAC induction by EMB to below that of the control M. smegmatis strain. Microarray analysis of an lsr2 knockout confirmed upregulation of M. smegmatis iniA and demonstrated upregulation of genes involved in cell wall and metabolic functions. Fully 121 of 584 genes induced by EMB treatment in wild-type M. smegmatis were upregulated ("hyperinduced") to even higher levels by EMB in the M. smegmatis lsr2 knockout. The most highly upregulated genes and gene clusters had adenine-thymine (AT)-rich 5-prime untranslated regions. In M. tuberculosis, overexpression of lsr2 repressed INH-mediated induction of all three iniBAC genes, as well as another annotated pump, efpA. The low molecular weight and basic properties of Lsr2 (pI 10.69) suggested that it was a histone-like protein, although it did not exhibit sequence homology with other proteins in this class. Consistent with other histone-like proteins, Lsr2 bound DNA with a preference for circular DNA, forming large oligomers, inhibited DNase I activity, and introduced a modest degree of supercoiling into relaxed plasmids. Lsr2 also inhibited in vitro transcription and topoisomerase I activity. Lsr2 represents a novel class of histone-like proteins that inhibit a wide variety of DNA-interacting enzymes. Lsr2 appears to regulate several important pathways in mycobacteria by preferentially binding to AT-rich sequences, including genes induced by antibiotics and those associated with inducible multi-drug tolerance. An improved understanding of the role of lsr2 may provide important insights into the mechanisms of action of antibiotics and the way that mycobacteria adapt to stresses such as antibiotic treatment.

The granulin-epithelin precursor (GEP/PCDGF), a 68-88 kDa secreted glycoprotein, has been shown to be an important growth and survival factor for ovarian cancer cells. Furthermore, GEP expression is a predictor of patient survival in metastatic ovarian cancer cells. Up to this point, however, the molecular mechanisms and clinical relevance of a GEP-mediated prosurvival phenotype remain poorly characterized. We hypothesize that the prosurvival function of GEP is important in ovarian cancer tumor progression and chemoresponse. To explore this hypothesis, we examined the effects of GEP overexpression on migration, invasion and cisplatin (CDDP) chemosensitivity in the ovarian cancer cell line A2780. Full length GEP transfectants demonstrated an increased capacity to migrate and invade their substratum when compared to empty vector controls. In addition, GEP overexpression was associated with CDDP chemoresistance. Finally, GEP overexpression increased tumor formation and protected cells from tumor regression in response to CDDP treatment in vivo. Taken together, these data support a role for GEP in tumor progression and development of drug resistance.

Objective: To characterize the molecular pathways involved in apoptosis following administration of histone deacetylase inhibitors to Type I and II endometrial cancer cells., Methods: Ark2, Ishikawa, and AN3 cell lines representing both Type I and II endometrial cancers were treated with various concentrations of oxamflatin and HDAC inhibitor-1. Cell apoptosis was determined by flow cytometry, nuclear staining, Western blotting, and mitochondrial membrane potential assays., Results: Compared to controls, there was a 95% reduction in the growth of Ark2 cells following administration of histone deacetylase inhibitors and this response was dose-dependent. These agents also caused profound morphologic changes and loss of mitochondrial membrane potentials consistent with the induction of apoptosis. Cleavage of PARP, caspase-9, and caspase-8 was detected, confirming the activation of apoptotic cascades in endometrial carcinoma cells. This effect was present in both serous and endometrioid cell types., Conclusion: Our results suggest that oxamflatin and HDAC inhibitor-1 have potent cytotoxicity in endometrial cancer cells by inducing cell apoptosis. Histone deacetylase inhibitors are promising agents for the treatment of both Type I and II endometrial carcinoma.

Autism spectrum disorders (ASDs) are common, heritable neurodevelopmental conditions. The genetic architecture of ASDs is complex, requiring large samples to overcome heterogeneity. Here we broaden coverage and sample size relative to other studies of ASDs by using Affymetrix 10K SNP arrays and 1,181 [corrected] families with at least two affected individuals, performing the largest linkage scan to date while also analyzing copy number variation in these families. Linkage and copy number variation analyses implicate chromosome 11p12-p13 and neurexins, respectively, among other candidate loci. Neurexins team with previously implicated neuroligins for glutamatergic synaptogenesis, highlighting glutamate-related genes as promising candidates for contributing to ASDs.