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Parasitic worms are amongst the most common pathogens to infect humans and have a long-established history of inflicting disease in their hosts. There is a large body of evidence that states intestine-dwelling helminths ensure their survival by influencing the host immune response against them. In recent years, it has become apparent that the large and diverse microbial communities that exist in the gastrointestinal (GI) tract of the host and within the parasite itself have a pivotal role in worm survival and persistence. Using a variety of mouse models (including laboratory, germ-free and rewilded mice), there have been new insights into how bacteria and worms interact with each other; this includes the discovery that Trichuris is unable to hatch and/or infect their host in the absence of bacteria, and that these worms contain a Trichuris-specific gut microbiota. These interactions are determined in part by the capacity of the host, gut microbiota and worms to communicate via metabolites such as butyrate, which are microbially derived and have known immunoregulatory properties. By exploring the contribution of gut bacteria to worm infections and the intricate relationship that exists between them, an exciting and emerging field in whipworm parasitology is established.

BackgroundTubulointerstitial fibrosis is a powerful predictor of future progression inimmunoglobulin A (IgA) nephropathy (IgAN). Proximal tubular epithelial cells (PTECs), in concert with infiltrating macrophages, are regarded as the agents provocateurs for driving this fibrotic process. However, evidence is now emerging for a contributory role of the distal nephron. The aim of this study was to examine the potential influence of macrophages on collecting duct epithelial cells (CDECs) and their combined role in the progression of IgAN.MethodsCDECs were cultured with macrophage-conditioned media (MCM) generated from human monocyte cell lines U937 and THP-1 stimulated with or without 100 μg/mL galactose-deficient IgA1. CDECs were analysed for evidence of inflammation and fibrosis.ResultsStaining of IgAN biopsies for CD68+ macrophages revealed the presence of macrophages juxtaposed to collecting ducts and within their lumina. CDEC exposed to MCM from IgA1-stimulated THP-1 cells (THP-1-IgA-MCM) exhibited markedly increased expression of neutrophil-associated gelatinase (NGAL) and proinflammatory cytokinesinterleukin (IL)-1β, tumour necrosis factor-α, IL-6 and IL-8 compared with MCM from non-IgA-stimulated THP-1 cells (THP-1-MCM). U937-IgA-MCM increased fibronectin levels and reduced E-cadherinmRNA expression. THP-1-IgA-MCM-derived exosomes induced similar increases in NGAL and cytokine expression while in cross-over experiments exosomes extracted from IL-1β-exposed CDEC induced IL-1β and IL-6 mRNA expression in both sets of macrophages. MiRnome analysis revealed that microRNA (miR)-146a, -155 and -200b exhibited a >2-fold increase in expression in CDEC treated with THP-1-IgA-MCM compared with THP-1-MCM. Enforced miR-146a suppression further enhanced NGAL expression, while ectopic miR-146a over-expression downregulated it. NGAL mRNA and miR-146a were upregulated in the biopsies of patients with progressive IgAN compared with non-progressive IgAN.ConclusionsTaken together, these data suggest that CDEC–macrophage interactions potentially contribute to the tubulointerstitial fibrosis characteristic of progressive IgAN.

The majority of chronic infections are caused by biofilms, which have higher levels of antibiotic resistance than planktonic growth. Violet-blue 405 nm light has recently emerged as a novel bactericide, but limited studies have been conducted on its effectiveness against biofilms. We found that in response to 405 nm light both Pseudomonas aeruginosa and Bacillus subtilis biofilms exhibited cell dispersal and membrane potential hyperpolarisations. The response to 405 nm light depended on the stage of biofilm growth. The use of reactive oxygen species scavengers reduced membrane hyperpolarisation and biofilm dispersal in response to 405 nm light. This is the first time that membrane potential hyperpolarisations have been linked with photooxidative stress in bacteria and with biofilm dispersal. These results provide a new insight into the role of membrane potentials in the bacterial stress response and could be used in the development of 405 nm light based biofilm treatments.

Introducción: The exoU gene, a marker for highly virulent strains of Pseudomonas aeruginosa, is the major contributor to a wide varietyof healthcare-associated infections. Methods: In this study, the antibiotic susceptibility profile, prevalence and genotyping of exoU+ P.aeruginosa were demonstrated. A total of 101 isolates of P. aeruginosa were analysed from different clinical and environmental sources. Results: The antibiotic susceptibility profile classified these isolates as extensively drug resistant (35.6%), multidrug resistant (40.5%) and non-multidrug resistant (23.7%). The prevalence of exoU gene was screened by PCR and 23 exoU+ genotypes were detected which all were clinical isolates. Multilocus sequence typing (MLST) analysis of seven loci assigned these exoU+ genotypes to 21 sequence types (STs) from which 16 new STs were identified. The prevalent STs were ST-308 and ST-235. Phylogenetic analysis using the concatenated nucleotide sequences of the seven housekeeping genes, exoU and the ITS region differentiated these exoU+ strains into five main groups. However, distinct evolutionary origins for some new sequence types were also indicated. Conclusions: The studied isolates showed the coexistence of exoU- and exoU+ genotypes of clinical P. aeruginosa in Kurdistan with a majority of MDR and XDR pattern. The prevalent STs found in other hospitals worldwide and at the international level.

Quorum sensing (QS), where bacteria secrete and respond to chemical signals to coordinate population-wide behaviors, has revealed that bacteria are highly social. Here, we investigate how diversity in QS signals and receptors can modify social interactions controlled by the QS system regulating bacteriocin secretion in Streptococcus pneumoniae, encoded by the blp operon (bacteriocin-like peptide). Analysis of 4 096 pneumococcal genomes detected nine blp QS signals (BlpC) and five QS receptor groups (BlpH). Imperfect concordance between signals and receptors suggested widespread social interactions between cells, specifically eavesdropping (where cells respond to signals that they do not produce) and crosstalk (where cells produce signals that non-clones detect). This was confirmed in vitro by measuring the response of reporter strains containing each of six different blp QS receptors to cognate and non-cognate peptides. Assays between pneumococcal colonies grown adjacent to one another provided further evidence that crosstalk and eavesdropping occur at endogenous levels of signal secretion. Finally, simulations of QS strains producing bacteriocins revealed that eavesdropping can be evolutionarily beneficial even when the affinity for non-cognate signals is very weak. Our results highlight that social interactions can mediate intraspecific competition among bacteria and reveal that competitive interactions can be modified by polymorphic QS systems.

Introduction Mast cells (MCs) are tissue‐resident immune cells implicated in antibacterial responses. These include chemokine secretion, degranulation, and the release of mast cell‐extracellular traps, which are primarily dependent on reactive oxygen species (ROS) production. Our study investigated whether human mast cells (hMCs) develop individual response patterns to bacteria located at different tissue sites: Escherichia coli (gut commensal), Listeria monocytogenes (foodborne intracellular pathogen), Staphylococcus aureus (skin commensal and opportunistic pathogen), and Streptococcus pneumoniae (upper respiratory tract commensal and lung pathogen). Methods After live bacteria exposure, hMCs were analyzed by a combined flow cytometry assay for degranulation, ROS production, DNA externalization, and for β‐hexosaminidase, chemokine, and prostaglandin release. Results L. monocytogenes induced hMC degranulation, IL‐8 and MCP‐1 release coupled with DNA externalization in a novel hMC ROS independent manner. In contrast, S. pneumoniae caused ROS production without DNA release and degranulation. E. coli induced low levels of hMC degranulation combined with interleukin 8 and MCP‐1 secretion and in the absence of ROS and DNA externalization. Finally, S. aureus induced hMCs prostaglandin D2 release and DNA release selectively. Our findings demonstrate a novel hMC phenomenon of DNA externalization independent of ROS production. We also showed that ROS production, degranulation, DNA externalization, and mediator secretion occur as independent immune reactions in hMCs upon bacterial encounter and that hMCs contribute to bacterial clearance. Conclusions Thus, hMCs exhibit a highly individualized pattern of immune response possibly to meet tissue requirements and regulate bacteria coexistence vs defense., In our study we have assessed human mast cell responses after their encounter with bacteria typically localized at tissue interfaces: Escherichia coli (gut commensal), Listeria monocytogenes (gut pathogen), Staphylococcus aureus (skin commensal), and Streptococcus pneumoniae (lung commensal). The human mast cell immune reactions included extracellular DNA secretion, reactive oxygen species (ROS) production and degranulation measured in a novel flow cytometric assay, as well cytokine/chemokine and prostaglandin D2 secretion. Our findings demonstrate that DNA externalization, ROS production, and mediator secretion occur as independent immune reactions in human mast cells upon bacterial encounter. Thus, these results suggest that human mast cells exhibit a highly individualized pattern of antimicrobial responses.

SUMMARYArterial stiffening is a hallmark of premature aging in Hutchinson-Gilford Progeria Syndrome (HGPS), but the key molecular regulators initiating arterial stiffening in HGPS remain unknown. To identify these early events, we compared arterial mechanics and ECM remodeling in very young HGPS (LMNAG609G/G609G) mice to those of age-matched and much older wild-type (WT) mice. Biaxial inflation-extension tests of carotid arteries of 2-month mice showed that circumferential stiffness of HGPS arteries was comparable to that of 24-month WT controls whereas axial arterial stiffening, an additional hallmark of normal aging, was mostly spared in HGPS. In an effort to identify underlying mechanisms, we examined expression levels of the major stiffness-regulatory molecules in WT and HGPS arteries. Transmission electron microscopy revealed slightly increased amounts of collagen within the elastin folds of HGPS carotid arteries, but this change was barely detectable by immunostaining carotid cross sections or qPCR of isolated aortas for collagens I, III, or V. Elastin integrity was also similar in the WT and HGPS arteries. In contrast, immunostaining readily revealed an increased expression of Lysyl oxidase (LOX) protein in young HGPS carotid arteries relative to aged-matched WT controls. Further analysis showed that HGPS arteries express increased amounts of LOX mRNA, and this effect extends to each of the arterial LOX family members. Remarkably, treatment of HGPS mice with the pan-LOX inhibitor β-aminopropionitrile (BAPN) restored near-normal circumferential arterial mechanics to HGPS carotid arteries, mechanistically and causally linking LOX upregulation to premature arterial stiffening in HGPS. Finally, we show that this premature increase in arterial LOX expression in HGPS foreshadows the increased expression of LOX that accompanies circumferential arterial stiffening during normal aging.

In eukaryotes, microRNAs (miRNAs) have roles in development, homeostasis, disease and the immune response. Recent work has shown that plant and mammalian miRNAs also mediate cross-kingdom and cross-domain communications. However, these studies remain controversial and are lacking critical mechanistic explanations. Bacteria do not produce miRNAs themselves, and therefore it is unclear how these eukaryotic RNA molecules could function in the bacterial recipient. In this review, we compare and contrast the biogenesis and functions of regulatory RNAs in eukaryotes and bacteria. As a result, we discovered several conserved features and homologous components in these distinct pathways. These findings enabled us to propose novel mechanisms to explain how eukaryotic miRNAs could function in bacteria. Further understanding in this area is necessary to validate the findings of existing studies and could facilitate the use of miRNAs as novel tools for the directed remodelling of the human microbiota.

The purpose of this chapter is to propose a unified analysis of a range of “second-position” phenomena that have been attested in various languages, and in so doing to motivate a more elaborated theory of Nominative Case assignment. Because our focus is on the interaction of clause structure with structural Case assignment, the authors concentrate almost exclusively on the processes and properties of S-structure. It is a classic tenet of generative grammar that inflectional affixes may be separate syntactic entities at pre-phonological levels of representation; see the analysis of the English auxiliary system in Chomsky. At the turn of the 1990s, this idea received new impetus, beginning with Pollock. Finally, the appendix is devoted to the discussion of Stylistic Fronting. The authors also added a postscript to this version of the paper, explaining its rather unusual and protracted prepublication history.

The emergence of high-throughput DNA sequencing methods provides unprecedented opportunities to further unravel bacterial biodiversity and its worldwide role from human health to ecosystem functioning. However, despite the abundance of sequencing studies, combining data from multiple individual studies to address macroecological questions of bacterial diversity remains methodically challenging and plagued with biases. Here, using a machine-learning approach that accounts for differences among studies and complex interactions among taxa, we merge 30 independent bacterial data sets comprising 1,998 soil samples from 21 countries. Whereas previous meta-analysis efforts have focused on bacterial diversity measures or abundances of major taxa, we show that disparate amplicon sequence data can be combined at the taxonomy-based level to assess bacterial community structure. We find that rarer taxa are more important for structuring soil communities than abundant taxa, and that these rarer taxa are better predictors of community structure than environmental factors, which are often confounded across studies. We conclude that combining data from independent studies can be used to explore bacterial community dynamics, identify potential ‘indicator’ taxa with an important role in structuring communities, and propose hypotheses on the factors that shape bacterial biogeography that have been overlooked in the past.

The human fungal microbiota known as mycobiota is increasingly recognized as a critical factor in human gut health and disease. Non-pathogenic commensal yeasts such as Saccharomyces cerevisiae promote homeostasis in the gut, whereas dysbiosis of the gut mycobiota is associated with inflammation. Glycan-binding receptors (lectins) are key host factors in host–mycobiota interaction in the gut. They are expressed on immune cells such as dendritic cells (DCs) and recognize fungal polysaccharides. This interaction is imperative to mount appropriate immune responses for immune homeostasis in the gut as well as clearance of fungal pathogens. Recent studies demonstrate that microtubule-associated protein light-chain 3 (LC3)-associated phagocytosis (LAP) is involved in lectin–fungi interactions. Yet, the biological impact of LAP on the lectin function remains largely elusive. In this report, we demonstrate that in mouse LAP is linked to dendritic cell-associated lectin 2 (Dectin-2), a C-type lectin specific to fungal α-mannan polysaccharide. We found that mouse Dectin-2 recognizes commensal yeast S. cerevisiae and Kazachstania unispora. Mouse bone marrow-derived DCs (BMDCs) produced inflammatory cytokines TNFα and IL-1β in response to the yeasts in a Dectin-2 and spleen tyrosine kinase (Syk)-dependent manner. We found that S. cerevisiae and K. unispora induced LAP in mouse BMDCs upon internalization. Furthermore, LC3 was activated by stimulation of BMDCs with the yeasts in a Dectin-2 and Syk-dependent manner. To address the biological impact of LAP on Dectin-2 yeast interaction, we established a knock-in mouse strain (Atg16L1E230, thereafter called E230), which BMDCs exhibit autophagy-active and LAP-negative phenotypes. When stimulated with yeasts, E230 BMDCs produced significantly less amounts of TNFα and IL-1β. Taken together, we revealed a novel link between Dectin-2 and LAP that enables host immune cells to respond to mycobiota.

This case illustrates heterotopic bone formation in a patient with cystic nephroma (CN) of the kidney. Renal CN may be difficult to discern clinically and radiologically from ‘Multilocular Cystic Renal Cell Neoplasm of Low Malignant Potential (MCRCNLMP)' or a clear cell renal cell carcinoma with extensive cystic change. In both of the latter, dystrophic calcification or ossification is not uncommon. The formation of heterotopic bone in CN/MEST is rare and should not be confused with sarcomatous transformation on histology. Radiologically, the presence of central calcification increases the suspicion of a cystic renal cell carcinoma, a potential diagnostic pitfall.

The study of mutations causing the steroid-resistant nephrotic syndrome in children has greatly advanced our understanding of the kidney filtration barrier. In particular, these genetic variants have illuminated the roles of the podocyte, glomerular basement membrane and endothelial cell in glomerular filtration. However, in a significant number of familial and early onset cases, an underlying mutation cannot be identified, indicating that there are likely to be multiple unknown genes with roles in glomerular permeability. We now show how the combination of N-ethyl-N-nitrosourea mutagenesis and next-generation sequencing could be used to identify the range of mutations affecting these pathways. Using this approach, we isolated a novel mouse strain with a viable nephrotic phenotype and used whole-genome sequencing to isolate a causative hypomorphic mutation in Lamb2. This discovery generated a model for one part of the spectrum of human Pierson’s syndrome and provides a powerful proof of principle for accelerating gene discovery and improving our understanding of inherited forms of renal disease. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

VHL is mutated in the majority of patients with clear cell renal cell carcinoma (ccRCC), with conflicting clinical relevance. Recent studies have identified recurrent mutations in histone modifying and chromatin remodeling genes, including BAP1, PBRM1, SETD2, KDM6A, and JARID1c. Current evidence suggests that BAP1 mutations are associated with aggressive disease. The clinical significance of the remaining genes is unknown. In this study, targeted sequencing of VHL and JARID1c (entire genes) and coding regions of BAP1, PBRM1, SETD2, and KDM6A was performed on 132 ccRCCs and matched normal tissues. Associations between mutations and clinical and pathological outcomes were interrogated. Inactivation of VHL (coding mutation or promoter methylation) was seen in 75% of ccRCCs. Somatic noncoding VHL alterations were identified in 29% of ccRCCs and may be associated with improved overall survival. BAP1 (11%), PBRM1 (33%), SETD2 (16%), JARID1c (4%), and KDM6A (3%) mutations were identified. BAP1-mutated tumors were associated with metastatic disease at presentation (P = 0.023), advanced clinical stage (P = 0.042) and a trend towards shorter recurrence free survival (P = 0.059) when compared with tumors exclusively mutated for PBRM1. Our results support those of recent publications pointing towards a role for BAP1 and PBRM1 mutations in risk stratifying ccRCCs. Further investigation of noncoding alterations in VHL is warranted. © 2013 Wiley Periodicals, Inc.

Glomeruli are highly sophisticated filters and glomerular disease is the leading cause of kidney failure. Morphological change in glomerular podocytes and the underlying basement membrane are frequently observed in disease, irrespective of the underlying molecular etiology. Standard electron microscopy techniques have enabled the identification and classification of glomerular diseases based on two-dimensional information, however complex three-dimensional ultrastructural relationships between cells and their extracellular matrix cannot be easily resolved with this approach. We employed serial block face-scanning electron microscopy to investigate Alport syndrome, the commonest monogenic glomerular disease, and compared findings to other genetic mouse models of glomerular disease (Myo1e−/−, Ptpro−/−). These analyses revealed the evolution of basement membrane and cellular defects through the progression of glomerular injury. Specifically we identified sub-podocyte expansions of the basement membrane with both cellular and matrix gene defects and found a corresponding reduction in podocyte foot process number. Furthermore, we discovered novel podocyte protrusions invading into the glomerular basement membrane in disease and these occurred frequently in expanded regions of basement membrane. These findings provide new insights into mechanisms of glomerular barrier dysfunction and suggest that common cell-matrix-adhesion pathways are involved in the progression of disease regardless of the primary insult.

What you need to know Worldwide at least 200 per 100 000 people are killed or admitted to hospital each year after traumatic brain injury.1 This results in more than 10 million deaths or hospital admissions.2 In the UK, around one million people attend emergency departments every year with a traumatic brain injury.3 Intracranial bleeding is common after traumatic brain injury, and the larger the bleed the greater the risk of death and disability.4 5 Bleeding continues after hospital admission in 84% of patients with moderate or severe injuries,6 7 and can continue for up to 24 hours.8 About one third of patients have laboratory evidence of abnormal coagulation.9 High levels of fibrin degradation products are seen within the first three hours.10 Such patients have a higher risk of intracranial haemorrhage and mortality. Tranexamic acid reduces bleeding by inhibiting the enzymatic breakdown of fibrin blood clots (fig 1⇓). Fig 1 A Normal fibrinolysis. B: Fibrinolysis inhibited by tranexamic acid. Plasmin binds to fibrin via lysine binding sites and then splits fibrin into fibrin degradation products. Tranexamic acid is a molecular analogue of lysine that inhibits fibrinolysis by reducing the binding of plasmin to fibrin Tranexamic acid is used routinely in some cases of trauma and in surgery. For example, it reduces the need for blood transfusion in surgical patients.11 12 In trauma patients with extracranial haemorrhage:13

Natural transformation in the Gram-positive pathogenStreptococcus pneumoniaeoccurs when cells become “competent”, a state that is induced in response to high extracellular concentrations of a secreted peptide signal called CSP (Competence Stimulating Peptide) encoded by thecomClocus. Two main CSP signal types (pherotypes) are known to dominate the pherotype diversity across strains. Using thousands of fully sequenced pneumococcal genomes, we confirm that pneumococcal populations are highly genetically structured and that there is significant variation among diverged populations in pherotype frequencies; most carry only a single pherotype. Moreover, we find that the relative frequencies of the two dominant pherotypes significantly vary within a small range across geographical sites. It has been variously proposed that pherotypes either promote genetic exchange among cells expressing the same pherotype, or conversely that they promote recombination between strains bearing different pherotypes. We distinguish these hypotheses using a bioinformatics approach by estimating recombination frequencies within and between pherotypes across 4,089 full genomes. Despite underlying population structure, we observe extensive recombination between populations; additionally, we found significantly higher rates of genetic exchange between strains expressing different pherotypes than among isolates carrying the same pherotype. Our results indicate that pherotypes do not restrict, and marginally facilitate, recombination between strains. Furthermore, our results suggest that the CSP balanced polymorphism does not causally underlie population differentiation. Therefore, when strains carrying different pherotypes encounter one another during co-colonization, genetic exchange can freely occur.

Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra-species competition within the human host. However, the triggers of pneumocin expression are poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39. Furthermore, by analogy with pneumococcal competence, we show that several antibiotics activate the blp-genes. Using real-time gene expression measurements we show that while the promoter driving expression of the two-component regulatory system blpR/H is constitutive, the remaining blp-promoters that control pneumocin expression, immunity and the inducer peptide BlpC, are pH-dependent and induced in the late exponential phase. Intriguingly, competence for genetic transformation, mediated by the paralogous ComD/E two-component quorum system, is induced by the same environmental cues. To test for interplay between these regulatory systems, we quantified the regulatory response to the addition of synthetic BlpC and competence-stimulating peptide (CSP). Supporting the idea of such interplay, we found that immediately upon addition of CSP, the blp-promoters were activated in a comD/E-dependent manner. After a delay, blp-expression was highly induced and was strictly dependent on blpRH and blpC. This raised the question of the mechanism of BlpC export, since bioinformatic analysis showed that the genes encoding the putative exporter for BlpC, blpAB, are not intact in strain D39 and most other strains. By contrast, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. Consistent with the idea that comAB mediate BlpC export, we finally show that high-level expression of the blp-genes requires comAB. Together, our results demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might therefore have unpredictable consequences on pneumococcal colonization dynamics by activating genes that mediate intra-specific interference competition., Author Summary Streptococcus pneumoniae is an opportunistic pathogen with high carriage rates in children. Pneumococci express pneumocins that kill competing bacteria. Pneumocin expression is controlled by a pheromone-induced two-component system (BlpR/H) but the triggers for the system are poorly understood. We show that the pheromone-induced two-component system driving competence for genetic transformation, ComD/E, also controls expression of BlpC, the peptide pheromone activating BlpR/H-dependent gene expression. Importantly, we show that the competence pheromone exporter, ComAB, also exports BlpC. Since antibiotics that disrupt protein quality control or DNA replication trigger competence, it follows that the same antibiotics activate pneumocin expression. Our experiments show that this dual-quorum sensing system ensures that pneumocins are expressed at the end of exponential growth when nutrients become limiting. Pneumocin expression might thus be used to liberate nutrients by lysing competing bacteria. Antibiotic-induced pneumocin production might also aid in clearing the niche after antibiotic stress. Any free DNA can then be used for transformation to acquire antibiotic-resistance.

Background: We studied the biological significance of genes involved in a novel t(8;12)(p21.3;p13.31) reciprocal translocation identified in cervical squamous cell carcinoma (SCC) cells. Methods: The rearranged genes were identified by breakpoint mapping, long-range PCR and sequencing. We investigated gene expression in vivo using reverse-transcription PCR and tissue microarrays, and studied the phenotypic consequences of forced gene overexpression. Results: The rearrangement involved lipoprotein lipase (LPL) and peroxisome biogenesis factor-5 (PEX5). Whereas LPL–PEX5 was expressed at low levels and contained a premature stop codon, PEX5–LPL was highly expressed and encoded a full-length chimeric protein (including the majority of the LPL coding region). Consistent with these findings, PEX5 was constitutively expressed in normal cervical squamous cells, whereas LPL expression was negligible. The LPL gene was rearranged in 1 out of 151 cervical SCCs, whereas wild-type LPL overexpression was common, being detected in 10 out of 28 tissue samples and 4 out of 10 cell lines. Forced overexpression of wild-type LPL and PEX5–LPL fusion transcripts resulted in increased invasiveness in cervical SCC cells, attributable to the C-terminal non-catalytic domain of LPL, which was retained in the fusion transcripts. Conclusion: This is the first demonstration of an expressed fusion gene in cervical SCC. Overexpressed wild-type or translocated LPL is a candidate for targeted therapy.

We report here the identification and characterization of two zinc uptake systems, ZurAM and ZinABC, in the intracellular pathogen Listeria monocytogenes . Transcription of both operons was zinc responsive and regulated by the zinc-sensing repressor Zur. Deletion of either zurAM or zinA had no detectable effect on growth in defined media, but a double zurAM zinA mutant was unable to grow in the absence of zinc supplementation. Deletion of zinA had no detectable effect on intracellular growth in HeLa epithelial cells. In contrast, growth of the zurAM mutant was significantly impaired in these cells, indicating the importance of the ZurAM system during intracellular growth. Notably, the deletion of both zinA and zurAM severely attenuated intracellular growth, with the double mutant being defective in actin-based motility and unable to spread from cell to cell. Deletion of either zurAM or zinA had a significant effect on virulence in an oral mouse model, indicating that both zinc uptake systems are important in vivo and establishing the importance of zinc acquisition during infection by L. monocytogenes . The presence of two zinc uptake systems may offer a mechanism by which L. monocytogenes can respond to zinc deficiency within a variety of environments and during different stages of infection, with each system making distinct contributions under different stress conditions.

Oncostatin M receptor (OSMR) shows frequent copy number gain and overexpression in advanced cervical squamous cell carcinoma (SCC). We used cell-based in vitro assays, RNA interference, and integrative gene expression profiling to investigate the functional significance of this observation. CaSki and SW756 were selected as representative cervical SCC cells that overexpressed OSMR, and ME180 and MS751 as cells that did not. The STAT-dependent pro-angiogenic factors VEGF-A and ID1 were rapidly induced by OSM in CaSki/SW756 but not in ME180/MS751. However, rapid induction did occur in MS751 following forced OSMR overexpression, while depleting OSMR in CaSki abrogated VEGF-A expression. Conditioned medium from both CaSki and SW756 stimulated endothelial tube formation in vitro, effects that were inhibited by depleting OSMR in the SCC cells. For both CaSki and SW756, migration in a wound healing assay and invasion through Matrigel were stimulated by OSM and consistently inhibited by OSMR depletion. The phenotype was rescued by transfection with OSMR containing a silent mutation that provided specific siRNA resistance. Overall, there was a positive correlation between OSMR levels and invasiveness. We used gene expression profiling to identify genes induced by OSM in CaSki/SW756 but not in ME180/MS751. The most prominent gene ontology category groups for the differentially expressed genes were cell motility/invasion, angiogenesis, signal transduction, and apoptosis. We also profiled 23 cervical SCC samples, identifying genes that were differentially expressed in cases with OSMR overexpression versus those without. Integration of the datasets identified 15 genes that showed consistent differential expression in association with OSMR levels in vitro and in vivo. We conclude that OSMR overexpression in cervical SCC cells provides increased sensitivity to OSM, which induces pro-malignant changes. OSMR is a potential prognostic and therapeutic target in cervical SCC. The genes that mediate OSM:OSMR effects will be valuable indicators of the effectiveness of antibody blockade in pre-clinical systems.

Germline mutations in the FH gene encoding the Krebs cycle enzyme fumarate hydratase predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. FH-deficient cells and tissues accumulate high levels of fumarate, which may act as an oncometabolite and contribute to tumourigenesis. A recently proposed role for fumarate in the covalent modification of cysteine residues to S-(2-succinyl) cysteine (2SC) (termed protein succination) prompted us to assess 2SC levels in our existing models of HLRCC. Herein, using a previously characterized antibody against 2SC, we show that genetic ablation of FH causes high levels of protein succination. We next hypothesized that immunohistochemistry for 2SC would serve as a metabolic biomarker for the in situ detection of FH-deficient tissues. Robust detection of 2SC was observed in Fh1 (murine FH)-deficient renal cysts and in a retrospective series of HLRCC tumours (n = 16) with established FH mutations. Importantly, 2SC was undetectable in normal tissues (n = 200) and tumour types not associated with HLRCC (n = 1342). In a prospective evaluation of cases referred for genetic testing for HLRCC, the presence of 2SC-modified proteins (2SCP) correctly predicted genetic alterations in FH in every case. In two series of unselected type II papillary renal cancer (PRCC), prospectively analysed by 2SCP staining followed by genetic analysis, the biomarker accurately identified previously unsuspected FH mutations (2/33 and 1/36). The investigation of whether metabolites in other tumour types produce protein modification signature(s) that can be assayed using similar strategies will be of interest in future studies of cancer.

Although gain of chromosome 5p is one of the most frequent DNA copy-number imbalances in cervical squamous cell carcinoma (SCC), the genes that drive its selection remain poorly understood. In a previous cross-sectional clinical study, we showed that the microRNA processor Drosha (located on chromosome 5p) demonstrates frequent copy-number gain and overexpression in cervical SCC, associated with altered microRNA profiles. Here, we have conducted gene depletion/overexpression experiments to demonstrate the functional significance of up-regulated Drosha in cervical SCC cells. Drosha depletion by RNA interference (RNAi) produced significant, specific reductions in cell motility/invasiveness in vitro, with a silent RNAi-resistant Drosha mutation providing phenotype rescue. Unsupervised hierarchical clustering following global profiling of 319 microRNAs in 18 cervical SCC cell line specimens generated two groups according to Drosha expression levels. Altering Drosha levels in individual SCC lines changed the group into which the cells clustered, with gene depletion effects being rescued by the RNAi-resistant mutation. Forty-five microRNAs showed significant differential expression between the groups, including four of 14 that were differentially expressed in association with Drosha levels in clinical samples. miR-31 up-regulation in Drosha-overexpressing samples/cell lines was the highest-ranked change (by adjusted p value) in both analyses, an observation validated by northern blotting. These functional data support the role of Drosha as an oncogene in cervical SCC, by affecting expression of cancer-associated microRNAs that have the potential to regulate numerous protein-coding genes.

K5 lyase A (KflA) is a tail spike protein (TSP) encoded by a K5A coliphage, which cleaves K5 capsular polysaccharide, a glycosaminoglycan with the repeat unit [-4)-betaGlcA-(1,4)- alphaGlcNAc(1-], displayed on the surface of Escherichia coli K5 strains. The crystal structure of KflA reveals a trimeric arrangement, with each monomer containing a right-handed, single-stranded parallel beta-helix domain. Stable trimer formation by the intertwining of strands in the C-terminal domain, followed by proteolytic maturation, is likely to be catalyzed by an autochaperone as described for K1F endosialidase. The structure of KflA represents the first bacteriophage tail spike protein combining polysaccharide lyase activity with a single-stranded parallel beta-helix fold. We propose a catalytic site and mechanism representing convergence with the syn-beta-elimination site of heparinase II from Pedobacter heparinus.

Escherichia coli strain Nissle 1917, which has been widely used as a probiotic for the treatment of inflammatory bowel disorders, expresses a K5 capsule, the expression of which is often associated with extraintestinal and urinary tract isolates of E. coli . Previously, it had been shown that the expression of a K5 capsule by Nissle 1917 was important in mediating interactions with epithelial cells and the extent of chemokine expression. In this paper, we show that infection with Nissle 1917 induces expression of Toll-like receptor 4 (TLR4) and TLR5 in Caco-2 cells and that maximal induction of TLR5 required the K5 capsule. In addition, purified K5 polysaccharide was capable of inducing expression of TLR5 and mCD14 and potentiated the activity of both TLR4 and TLR5 agonists to increase the proinflammatory response. Infection with Nissle 1917 also increased the expression of the adaptor molecules MyD88 and TRIF, which was K5 capsule dependent. By Western blot analysis, it was possible to show that induction of interleukin-8 by Nissle 1917 was predominantly through the mitogen-activated protein (MAP) kinase pathway and that expression of the K5 capsule was important for activation of the MAP kinase pathway. This paper provides new information on the function of the K5 capsule in mediating interactions between Nissle 1917 and epithelial cells and the mechanisms that underlie the probiotic properties of Nissle 1917.

The enzyme CMP-Kdo synthetase (KdsB) catalyzes the addition of 2-keto-3-deoxymanno-octulonic acid (Kdo) to CTP to form CMP-Kdo, a key reaction in the biosynthesis of lipopolysaccharide. The reaction catalyzed by KdsB and the related CMP-acylneuraminate synthase is unique among the sugar-activating enzymes in that the respective sugars are directly coupled to a cytosine monophosphate. Using inhibition studies, in combination with isothermal calorimetry, we show the substrate analogue 2β-deoxy-Kdo to be a potent competitive inhibitor. The ligand-free Escherichia coli KdsB and ternary complex KdsB-CTP-2β-deoxy-Kdo crystal structures reveal that Kdo binding leads to active site closure and repositioning of the CTP phosphates and associated Mg2+ ion (Mg-B). Both ligands occupy conformations compatible with an Sn2-type attack on the α-phosphate by the Kdo 2-hydroxyl group. Based on strong similarity with DNA/RNA polymerases, both in terms of overall chemistry catalyzed as well as active site configuration, we postulate a second Mg2+ ion (Mg-A) is bound by the catalytically competent KdsB-CTP-Kdo ternary complex. Modeling of this complex reveals the Mg-A coordinated to the conserved Asp100 and Asp235 in addition to the CTP α-phosphate and both the Kdo carboxylic and 2-hydroxyl groups. EPR measurements on the Mn2+-substituted ternary complex support this model. We propose the KdsB/CNS sugar-activating enzymes catalyze the formation of activated sugars, such as the abundant CMP-5-N-acetylneuraminic acid, by recruitment of two Mg2+ to the active site. Although each metal ion assists in correct positioning of the substrates and activation of the α-phosphate, Mg-A is responsible for activation of the sugar-hydroxyl group.

Escherichia coli strain Nissle 1917 has been widely used as a probiotic for the treatment of inflammatory bowel disorders and shown to have immunomodulatory effects. Nissle 1917 expresses a K5 capsule, the expression of which often is associated with extraintestinal and urinary tract isolates of E. coli . In this paper, we investigate the role of the K5 capsule in mediating interactions between Nissle 1917 and intestinal epithelial cells. We show that the loss of capsule significantly reduced the level of monocyte chemoattractant protein 1 (MCP-1), RANTES, macrophage inflammatory protein 2α (MIP-2α), MIP-2β, interleukin-8, and gamma interferon-inducible protein 10 induction by Nissle 1917 in both Caco-2 cells and MCP-1 induction in ex vivo mouse small intestine. The complementation of the capsule-minus mutation confirmed that the effects on chemokine induction were capsule specific. The addition of purified K5, but not K1, capsular polysaccharide to the capsule-minus Nissle 1917 at least in part restored chemokine induction to wild-type levels. The purified K5 capsular polysaccharide alone was unable to stimulate chemokine production, indicating that the K5 polysaccharide was acting to mediate interactions between Nissle 1917 and intestinal epithelial cells. The induction of chemokine by Nissle 1917 was generated predominantly by interaction with the basolateral surface of Caco-2 cells, suggesting that Nissle 1917 will be most effective in inducing chemokine expression where the epithelial barrier is disrupted.

Escherichia coli group 2 capsule gene clusters are temperature regulated, being expressed at 37°C but not at 20°C. Expression is regulated at the level of transcription by two convergent promoters, PR1 and PR3. In this paper, we show that regulation of transcription from PR3 involves a number of novel features including H-NS, SlyA, and a large 741-bp 5′ untranslated region (UTR). H-NS represses transcription from PR3 at 20°C and binds both 5′ and 3′ of the transcription start site. The 3′ downstream regulatory element (DRE) was essential for temperature-dependent H-NS repression. At 37°C, SlyA activates transcription independent of H-NS but maximal transcription requires H-NS. The UTR is present between the transcription start site and the first gene in the operon, kpsM . We demonstrate that the UTR, as well as containing the H-NS DRE, functions to moderate the extent of transcription that reaches kpsM and allows the binding of antitermination factor RfaH.

As the high-risk human papillomavirus (HPV) integrants seen in anogenital carcinomas represent the end-point of a clonal selection process, we used the W12 model to study the naturally occurring integration events that exist in HPV16-infected cervical keratinocytes before integrant selection. We performed limiting dilution cloning to identify integrants present in cells that also maintain episomes. Such integrants arise in a natural context and exist in a noncompetitive environment, as they are transcriptionally repressed by episome-derived E2. We found that integration can occur at any time during episome maintenance, providing biological support for epidemiologic observations that persistent HPV infection is a major risk factor in cervical carcinogenesis. Of 24 different integration sites isolated from a single nonclonal population of W12, 12 (50%) occurred within chromosome bands containing a common fragile site (CFS), similar to observations for selected integrants in vivo. This suggests that such regions represent relatively accessible sites for insertion of foreign DNA, rather than conferring a selective advantage when disrupted. Interestingly, however, integrants and CFSs did not accurately colocalize. We further observed that local DNA rearrangements occur frequently and rapidly after the integration event. The majority of integrants were in chromosome bands containing a cancer-associated coding gene or microRNA, indicating that integration occurs commonly in these regions, regardless of selective pressure. The cancer-associated genes were generally a considerable distance from the integration site, and there was no evidence for altered expression of nine strong candidate genes. These latter observations do not support an important role for HPV16 integration in causing insertional mutagenesis. [Cancer Res 2008;68(20):8249–59]

Glomerular deposition of fibrillar collagen is a characteristic finding of genetically distinct conditions, including nail-patella syndrome and collagen type III glomerulopathy. A case of familial nephropathy in which steroid-sensitive nephrotic syndrome and glomerular deposits of fibrillar collagen are associated with multiple exostoses due to mutation of the EXT1 gene is described. This gene encodes a glycosyltransferase required for synthesis of heparan sulfate glycosaminoglycans. There is deficiency of heparan sulfate and perlecan, together with accumulation of collagens, in the matrix of EXT1-associated osteochondromas. Similar glomerular basement membrane abnormalities could offer an explanation for both the renal ultrastructural changes and steroid-sensitive nephrotic syndrome.

All bacteria are bound by at least one membrane that acts as a barrier between the cell’s interior and the outside environment. Surface components within and attached to the cell membrane are essential for ensuring that the overall homeostasis of the cell is maintained. However, many surface components of the bacterial cell also have an indispensable role mediating interactions of the bacteria with their immediate environment and as such are essential to the pathogenesis of infectious disease. During the course of an infection, bacterial pathogens will encounter many different ecological niches where environmental conditions such as salinity, temperature, pH, and the availability of nutrients fluctuate. It is the bacterial cell surface that is at the front-line of these host–pathogen interactions often protecting the bacterium from hostile surroundings but at the same time playing a critical role in the adherence to host tissues promoting colonization and subsequent infection. To deal effectively with the changing environments that pathogens may encounter in different ecological niches within the host many of the surface components of the bacterial cell are subject to phenotypic variation resulting in heterogeneous subpopulations of bacteria within the clonal population. This dynamic phenotypic heterogeneity ensures that at least a small fraction of the population will be adapted for a particular circumstance should it arise. Diversity within the clonal population has often been masked by studies on entire bacterial populations where it was often assumed genes were expressed in a uniform manner. This chapter, therefore, aims to highlight the non-uniformity in certain cell surface structures and will discuss the implication of this heterogeneity in bacterial–host interaction. Some of the recent advances in studying bacterial surface structures at the single cell level will also be reviewed.

We have previously reported the large-scale synthesis of neolactotetraose (Galbeta-4GlcNAcbeta-3Galbeta-4Glc) from lactose in engineered Escherichia coli cells (Priem B, Gilbert M, Wakarchuk WW, Heyraud A and Samain E. 2002. A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. Glycobiology. 12:235-240). In the present study we analyzed the adaptation of this system to glucuronylated oligosaccharides. The catalytic domain of mouse glucuronyl transferase GlcAT-P was cloned and expressed in an engineered strain which performed the in vivo synthesis of neolactotetraose. Under these conditions, efficient glucuronylation of neolactotetraose was achieved, but some residual neolactotetraose was still present. Although E. coli K-12 has an indigenous UDP-glucose dehydrogenase, the yield of glucuronylated oligosaccharides was greatly improved by the additional expression of the orthologous gene kfiD from E. coli K5. Glucuronylation of neolactohexaose and lactose was also observed. The final glucuronylated oligosaccharides are precursors of the brain carbohydrate motif HNK-1, involved in neural cell adhesion.

In this paper, we present the first evidence of a role for the transcriptional regulator SlyA in the regulation of transcription of the Escherichia coli K5 capsule gene cluster and demonstrate, using a combination of reporter gene fusions, DNase I footprinting, and electrophoretic mobility shift assays, the dependence of transcription on the functional interplay between H-NS and SlyA. Both SlyA and H-NS bind to multiple overlapping sites within the promoter in vitro, but their binding is not mutually exclusive, resulting in a remodeled nucleoprotein complex. In addition, we show that expression of the E. coli slyA gene is temperature-regulated, positively autoregulated, and independent of H-NS.

For many oncogenes, increased expression resulting from copy number gain confers a selective advantage to cells that consequently make up the tumour bulk. To identify oncogenes of potential biological significance in cervical squamous cell carcinoma (SCC), 36 primary samples and ten cell lines were screened by array comparative genomic hybridization (CGH). The most commonly occurring regions of copy number gain that also showed amplification were 5p15.2–14.3 (59%), 5p13.3 (65%), and 5p13.2–13.1 (63%). Gene copy numbers were significantly associated with expression levels for three candidate oncogenes at these loci: OSMR (oncostatin M receptor) (p = 0.03), PDZK3 (PDZ domain containing protein 3) (p = 0.04), and TRIO (triple functional domain) (p = 0.03). Further examination by fluorescence in situ hybridization on a tissue microarray of 110 primary cervical SCC samples revealed copy number gain frequencies of 60.9%, 57.3%, and 54.5% for OSMR, PDZK3, and TRIO, respectively, with OSMR adversely influencing overall patient survival independently of tumour stage (p = 0.046). By array CGH, copy number gain of OSMR was not seen in any of 40 microdissected precursor cervical squamous intraepithelial lesions (SILs). Moreover, global mRNA expression analysis, using Affymetrix U133A 2.0 Arrays, showed no overexpression of OSMR in SILs, suggesting that OSMR gain and overexpression are relatively late steps in cervical carcinogenesis. In the cervical SCC cell lines CaSki and SW756, exogenous OSM activated downstream-signalling elements of OSMR including STAT3, p44/42 MAPK, and S6 ribosomal protein, and induced transcription of the angiogenic factor VEGF, effects that were reduced by OSMR depletion using RNA interference. We conclude that copy number gain of OSMR is frequently found in cervical SCC and is associated with adverse clinical outcome. As well as being a potential prognostic marker, OSMR is a candidate cell surface therapeutic target. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Listeria monocytogenes is a Gram-positive intracellular parasite and the causative organism of human listeriosis. In this article we demonstrate that L. monocytogenes encodes a functional member of the CodY family of global regulatory proteins that is responsive to both GTP and branched chain amino acids. By transcript analyses we identified the CodY regulon in L. monocytogenes and demonstrated that it comprises genes involved in amino acid metabolism, nitrogen assimilation as well as genes involved in sugar uptake and incorporation, indicating a role for CodY in L. monocytogenes in both carbon and nitrogen assimilation. A DeltarelA mutation reduced expression of the CodY regulon in early stationary phase and introduction of a DeltacodY mutation into a DeltarelA strain restored virulence. These data indicate that the avirulence of the DeltarelA mutant can in part be explained by the continued repression of the CodY regulon. The phenotypes of DeltarelA and DeltacodY mutants were studied in J774.A1 and Caco-2 cells and the DeltarelA mutation shown to effect intracellular growth. These results provide the first direct evidence that the activity of a CodY-type protein influences pathogenesis and provides new information on the physiological adaptation of L. monocytogenes to post-exponential phase growth and virulence.

Malignant germ cell tumours (MGCTs) of childhood are a rare group of neoplasms that comprise many histological subtypes and arise at numerous different sites. Genomic imbalances have been described in these tumours but, largely because of the paucity of cases reported in the literature, it is unclear how they relate to abnormalities in adult MGCTs and impact on potential systems for classifying GCTs. We have used metaphase-based comparative genomic hybridisation to analyse the largest series of paediatric MGCTs reported to date, representing 34 primary tumours (22 yolk sac tumours (YSTs), 11 germinomatous tumours and one metastatic embryonal carcinoma) occurring in children from birth to age 16, including 17 ovarian MGCTs. The large dataset enabled us to undertake statistical analysis, with the aim of identifying associations worthy of further investigation between patterns of genomic imbalance and clinicopathological parameters. The YSTs showed an increased frequency of 1p- (P=0.003), 3p+ (P=0.02), 4q− (P=0.07) and 6q− (P=0.004) compared to germinomatous tumours. Gain of 12p, which is invariably seen in adult MGCTs, was present in 53% of primary MGCTs of children aged 5–16 and was also observed in four of 14 YSTs affecting children less than 5. Two of these cases (14% of MGCTs in children less than 5) showed gain of the 12p11 locus considered to be particularly relevant in adult MGCTs. Gain of 12p showed a significant association with gain of 12q. Conversely, MGCTs without 12p gain displayed a significantly increased frequency of loss on 16p (P=0.04), suggesting that this imbalance may contribute to tumour development in such cases. This data provides new insight into the biology of this under-investigated tumour group and will direct future studies on the significance of specific genetic abnormalities.

Changes in metabolite levels of the kynurenine pathway have been observed in patients with CKD, suggesting involvement of this pathway in disease pathogenesis. Our recent genetic analysis in the mouse identified the kynurenine 3-mono-oxygenase (KMO) gene (Kmo) as a candidate gene associated with albuminuria. This study investigated this association in more detail. We compared KMO abundance in the glomeruli of mice and humans under normal and diabetic conditions, observing a decrease in glomerular KMO expression with diabetes. Knockdown of kmo expression in zebrafish and genetic deletion of Kmo in mice each led to a proteinuria phenotype. We observed pronounced podocyte foot process effacement on long stretches of the filtration barrier in the zebrafish knockdown model and mild podocyte foot process effacement in the mouse model, whereas all other structures within the kidney remained unremarkable. These data establish the candidacy of KMO as a causal factor for changes in the kidney leading to proteinuria and indicate a functional role for KMO and metabolites of the tryptophan pathway in podocytes.

We analysed 148 primary breast cancers using BAC-arrays containing 287 clones representing cancer-related gene/loci to obtain genomic molecular portraits. Gains were detected in 136 tumors (91.9%) and losses in 123 tumors (83.1%). Eight tumors (5.4%) did not have any genomic aberrations in the 281 clones analysed. Common (more than 15% of the samples) gains were observed at 8q11-qtel, 1q21-qtel, 17q11-q12 and 11q13, whereas common losses were observed at 16q12-qtel, 11ptel-p15.5, 1p36-ptel, 17p11.2-p12 and 8ptel-p22. Patients with tumors registering either less than 5% (median value) or less than 11% (third quartile) total copy number changes had a better overall survival (log-rank test: P=0.0417 and P=0.0375, respectively). Unsupervised hierarchical clustering based on copy number changes identified four clusters. Women with tumors from the cluster with amplification of three regions containing known breast oncogenes (11q13, 17q12 and 20q13) had a worse prognosis. The good prognosis group (Nottingham Prognostic Index (NPI)

Array comparative genomic hybridization (CGH) is being widely used to screen for recurrent genomic copy number alterations in neoplasms, with imbalances typically detected through the application of gain and loss thresholds. Review of array CGH publications for the year 2005 showed that a wide range of thresholds are used. However, the effect of sample ploidy on the sensitivity of these thresholds for single copy alterations (SCAs) has not been evaluated. Here, we describe a method to evaluate the detection accuracy of thresholds for detecting SCAs in cell line array CGH data. By applying a hidden Markov model-based method, we segmented array CGH data from well-karyotyped cell lines and generated ploidy-specific sensitivity-specificity plots, from which we identified optimum thresholds relevant to sample ploidy. We demonstrate that commonly used nonploidy-specific thresholds are suboptimal in their ability to call SCAs, particularly when applied to hypertriploid or tetraploid cell lines. We conclude that the use of ploidy-specific thresholds improves the sensitivity of thres-hold-based array CGH for detecting SCAs in cell lines. Because polyploidy is a common feature of cancer cells, the application of ploidy-specific thresholds to cell lines (and potentially to clinical samples) may improve the detection sensitivity of SCAs of biological significance.

On the basis of increased resistance to K5 capsule-specific bacteriophage, a waaR transposon mutant defective in the biosynthesis of lipopolysaccharide outer core was isolated. In a K1-expressing strain the mutation equally affected sensitivity to K1 capsule-specific bacteriophage, indicating a general effect on group 2 capsules. The waaR mutation affected retention on the cell surface of the K5 polysaccharide, with increased polysaccharide accumulating in the culture supernatant. This indicates that interactions between the outer core of lipopolysaccharide and group 2 capsular polysaccharides are important for the stabilization of group 2 capsular polysaccharides on the cell surface.

The export of large negatively charged capsular polysaccharides across the outer membrane represents a significant challenge to Gram negative bacteria. In the case of Escherichia coli group 2 capsular polysaccharides, the mechanism of export across the outer membrane was unknown, with no identified candidate outer membrane proteins. In this paper we demonstrate that the KpsD protein, previously believed to be a periplasmic protein, is an outer membrane protein involved in the export of group 2 capsular polysaccharides across the outer membrane. We demonstrate that KpsD and KpsE are located at the poles of the cell and that polysaccharide biosynthesis and export occurs at these polar sites. By in vivo chemical cross-linking and MALDI-TOF-MS analysis we demonstrate the presence of a multi-protein biosynthetic/export complex in which cytoplasmic proteins involved in polysaccharide biosynthesis could be cross-linked to proteins involved in export across the inner and outer membranes. In addition, we show that the RhsA protein, of previously unknown function, could be cross-linked to the complex and that a rhsA mutation reduces K5 biosynthesis suggesting a role for RhsA in coupling biosynthesis and export.

A common aim in cancer research is to investigate mechanisms of malignant progression by genetic analysis of key stages, including pre-malignancy, microinvasion, and micrometastases. As such lesions are small and require microdissection from clinical samples, the amount of DNA that can be recovered is limited and frequently inadequate for commonly used techniques of genomic analysis, such as comparative genomic hybridization (CGH). There is a critical requirement for techniques of whole-genome amplification that minimize representation bias in the amplified sample. Several techniques have been described, although their relative suitability for CGH has not been examined adequately. Here we compare the abilities of degenerate oligonucleotide-primed PCR (DOP-PCR), multiple-strand displacement amplification (MDA), and balanced PCR accurately to amplify limited amounts of template DNA for use in CGH. Amplification by DOP-PCR and MDA, but not balanced PCR faithfully preserved the original genomic content following amplification, as evidenced by generally concordant CGH copy number karyograms. Whereas the amplification products of DOP-PCR were immediately available for labeling and hybridization, the products of MDA required a further digestion step to produce optimal-sized probes for CGH. Moreover, MDA was less reliable overall than DOP-PCR at the lowest starting amount of 10 pg of template DNA. We conclude that DOP-PCR is the method of choice for whole-genome amplification of minute quantities of DNA to enable global genomic analysis to be performed on limited clinical samples.

Hypoxia-inducible genes have been linked to the aggressive phenotype of cancer. However, nearly all work on hypoxia-regulated genes has been conducted in vitro on cell lines. We investigated the hypoxia transcriptome in primary human bladder cancer using cDNA microarrays to compare genes induced by hypoxia in vitro in bladder cancer cell line EJ28 with genes upregulated in 39 bladder tumour specimens (27 superficial and 12 invasive). We correlated array mRNA fold changes with carbonic anhydrase 9 (CA IX) staining of tumours as a surrogate marker of hypoxia. Of 6000 genes, 32 were hypoxia inducible in vitro more than two-fold, five of which were novel, including lactate transporter SLC16A3 and RNAse 4. Eight of 32 hypoxia-inducible genes in vitro were also upregulated on the vivo array. Vascular endothelial growth factor mRNA was upregulated two-fold by hypoxia and 2-18-fold in 31 out of 39 tumours. Glucose transporter 1 was also upregulated on both arrays mRNA, and fold changes on the in vivo array significantly correlated with CA IX staining of tumours (P=0.008). However, insulin-like growth factor binding protein 3 mRNA was the most strongly differentially expressed gene in both arrays and this confirmed its upregulation in urine of bladder cancer patients (n=157, P

A novel sialic acid-specific lectin has been isolated from Helicobacter pylori lysate using fetuin-agarose affinity chromatography followed by cleavage of the alpha(2,3) and alpha(2,6) linkages of sialic acids using neuraminidase. The protein had a molecular weight of 17.5 kDa on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and was identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to be protein of unknown function with gene number HP0721. Recombinant HP0721 was shown to bind to fetuin-agarose and sialic acid-containing glycosphingolipids on thin-layer plates suggesting this protein may represent another sialic acid-specific adhesin of H. pylori. A H. pylori mutant defective for HP0721 was generated and its ability to bind to human AGS cells assayed.

The Escherichia coli K5 capsular polysaccharide (glycosaminoglycan) chains are composed of the repeated disaccharide structure: -GlcAβ1,4-GlcNAcα1,4-(where GlcA is glucuronic acid and GlcNAc is N-acetyl-d-glucosamine). The GlcA, present in most glycosaminoglycans, is donated from UDP-GlcA, which, in turn, is generated from UDP-glucose by the enzyme UDP-glucose dehydrogenase (UDPGDH). The formation of UDP-GlcA is critical for the biosynthesis of glycosaminoglycans. To investigate the role of UDPGDH in glycosaminoglycan biosynthesis, we used K5 polysaccharide biosynthesis as a model. E. coli was transformed with the complete gene cluster for K5 polysaccharide production. Additional transformation with an extra copy of UDPGDH resulted in an approx. 15-fold increase in the in vitro UDPGDH enzyme activity compared with the strain lacking extra UDPGDH. UDP-GlcA levels were increased 3-fold in overexpressing strains. However, metabolic labelling with [14C]glucose showed, unexpectedly, that overexpression of UDPGDH lead to decreased formation of K5 polysaccharide. No significant difference in the K5 polysaccharide chain length was observed between control and overexpressing strains, indicating that the decrease in K5-polysaccharide production most probably was due to synthesis of fewer chains. Our results suggest that K5-polysaccharide biosynthesis is strictly regulated such that increasing the amount of available UDP-GlcA results in diminished K5-polysaccharide production.

The acute phase of chronic myeloid leukemia (CML) is accompanied by secondary chromosomal changes. The additional changes have a non-random pattern; however, highly abnormal (marker) chromosomes are reported in some 20% of abnormal karyotypes. These marker chromosomes have proved to be beyond the resolution of conventional G-banding analysis. We used molecular cytogenetic techniques to determine the structure of complex chromosome markers in 10 CML-derived cell lines after our investigations of CML patients in blast crisis. Multicolor fluorescence in situ hybridization identified a multitude of structural chromosome aberrations. In addition, genomic gains identified by comparative genomic hybridization (CGH) were mapped to highly complex marker chromosomes in more than one cell line. The most common genomic loss detected by CGH affected chromosome 9, whereas the most common genomic gains affected, in order of frequency, the sequences of 8q,6, and 13q. The smallest discrete amplification on 8q was identified in cell line MEG-01. This amplicon contains sequences represented by the marker D8S263/RMC08P029 but did not contain the proximal MYC gene or a more distal marker, D8S256/RMC08P025. We determined the size of the amplicon to be less than the chromosome segment 8q24.12-q24.13. The use of region- and locus-specific probes to analyze the organization of highly complex marker structures aided the identification of preferentially amplified genomic regions. The resultant amplifications could harbor gene(s) driving disease progression. (C) 2003 Wiley-Liss, Inc.

Gout, which is commonly associated with hyperuricemia, affects 0.2% of the population. Hyperuricemia has a heterogeneous etiology that may be due to either over production and/or reduced renal clearance, of urate. In order to identify the mechanisms underlying reduced excretion of urate, we undertook positional cloning studies of familial juvenile hyperuricaemic nephropathy (FJHN), which is an autosomal dominant disorder characterized by hyperuricaemia, a low fractional renal excretion of urate, and chronic renal failure that is associated with interstitial fibrosis. The FJHN locus has been previously localized to a 22 centiMorgan interval flanked centromerically by D16S401 and telomerically by D16S3069, on chromosome 16p11-p13. This interval contains over 120 genes and we selected 13 renal expressed sequences to search for mutations in 5 unrelated FJHN families that contained 21 affected and 24 unaffected members. This revealed 5 heterozygous missense mutations (Cys77Tyr, Cys126Arg, Asn128Ser, Cys255Tyr and Cys300Gly) that altered evolutionary conserved residues in the gene encoding UROMODULIN. UROMODULIN, which is an 85 Kda glycoprotein, has roles in renal stone formation, the modulation of immune responses, and urothelial cytoprotection. The results of our studies, which have identified the gene causing FJHN, now indicate a further, novel role for UROMODULIN in urate metabolism.

Smad6 and Smad7 are inhibitory SMADs with putative functional roles at the intersection of major intracellular signaling networks, including TGF-beta, receptor tyrosine kinase (RTK), JAK/STAT, and NF-kappaB pathways. This study reports differential functional roles and regulation of Smad6 and Smad7 in TGF-beta signaling in renal cells, in murine models of renal disease and in human glomerular diseases. Smad7 is upregulated in podocytes in all examined glomerular diseases (focal segmental glomerulosclerosis [FSGS], minimal-change disease [MCD], membranous nephropathy [MNP], lupus nephritis [LN], and diabetic nephropathy [DN]) with a statistically significant upregulation in "classical" podocyte-diseases such as FSGS and MCD. TGF-beta induces Smad7 synthesis in cultured podocytes and Smad6 synthesis in cultured mesangial cells. Although Smad7 expression inhibited both Smad2- and Smad3-mediated TGF-beta signaling in podocytes, it inhibited only Smad3 but not Smad2 signaling in mesangial cells. In contrast, Smad6 had no effect on TGF-beta/Smad signaling in podocytes and enhanced Smad3 signaling in mesangial cells. These data suggest that Smad7 is activated in injured podocytes in vitro and in human glomerular disease and participates in negative control of TGF-beta/Smad signaling in addition to its pro-apoptotic activity, whereas Smad6 has no role in TGF-beta response and injury in podocytes. In contrast, Smad6 is upregulated in the mesangium in human glomerular diseases and may be involved in functions independent of TGF-beta/Smad signaling. These data indicate an important role for Smad6 and Smad7 in glomerular cells in vivo that could be important for the cell homeostasis in physiologic and pathologic conditions.

Helicobacter pylori NCTC11637 expresses a lipopolysaccharide (LPS) that comprises an O antigen side-chain with structural homology to the human blood group antigen Lewis X (Le(x)). The role of this molecule in adhesion of H. pylori to gastric epithelial cells was investigated. Mutants expressing truncated LPS structures were generated through insertional mutagenesis of rfbM and galE; genes encode GDP mannose pyrophosphorylase and galactose epimerase respectively. Compositional and structural analysis revealed that the galE mutant expressed a rough LPS that lacked an O antigen side-chain. In contrast, an O antigen side-chain was still synthesized by the rfbM mutant, but it lacked fucose and no longer reacted with anti-Le(x) monoclonal antibodies (Mabs). The ability of these mutants to bind to paraffin-embedded sections from the antrum region of a human stomach was assessed. Adhesion of the wild type was characterized by tropic binding to the apical surface of mucosal epithelial cells and cells lining gastric pits. In contrast, both the rfbM and galE mutants failed to demonstrate tropic binding and adhered to the tissue surface in a haphazard manner. These results indicate that LPS and, more specifically, Le(x) structures in the O antigen side-chain play an important role in targeting H. pylori to specific cell lineages within the gastric mucosa. The role of Le(x) in this interaction was confirmed by the tropic binding of synthetic Le(x), conjugated to latex beads, to gastric tissue. The observed pattern of adhesion was indistinguishable from that of wild-type H. pylori.

Calcification and fibrointimal proliferation are associated with advanced complicated atherosclerosis in large arteries but may also occur in smaller vessels, resulting in ischaemic tissue necrosis. This study investigates whether the mechanisms of calcification and intimal fibrosis are similar in vessels of different sizes. The localization of osteopontin (OPN), matrix Gla protein (MGP), thrombospondin-1 (TSP-1), and cartilage oligomeric matrix protein (COMP) was investigated in three types of human vascular lesions: atherosclerosis, chronic vascular rejection (CVR) in renal allografts, and calcific uraemic arteriolopathy (calciphylaxis). These lesions were chosen as they affect different sized blood vessels and they exhibit a fibroproliferative intimal reaction, with or without calcification, resulting in luminal obliteration and ischaemic complications. OPN, MGP, TSP-1, and COMP were not detected in normal blood vessels. However, OPN and MGP were expressed at sites of calcification within atherosclerotic lesions and in microvessels in calciphylaxis, suggesting that calcification in different sized vessels may occur by a common mechanism. These proteins were not detected in areas of fibrointimal proliferation. In contrast, TSP-1 was localized primarily within the fibrous tissue of atherosclerotic lesions and was also expressed in the expanded fibrous intima of arteries showing CVR. COMP was localized primarily within the fibrous tissue under the lipid core of the majority of advanced atherosclerotic lesions. TSP-1 and COMP were also detected in areas of microcalcification in atherosclerotic lesions and TSP-1 was detected adjacent to areas of calcification in calciphylaxis. However, neither TSP-1 nor COMP was localized to calcific foci within these lesions. The localization of OPN, MGP, TSP-1, and COMP to pathological, but not normal arterial intima supports a pathogenetic role for these proteins in the development of vascular fibrosis and calcification. Modulation of their production and activity may offer a novel approach to the therapy of a number of vascular diseases.