Spectroscopic, biochemical and computational studies of bioactive DNA minor groove binders targeting 5'-WGWWCW-3' motif.

Spectroscopic, biochemical, and computational modelling studies have been used to assess the binding capability of a set of minor groove binding (MGB) ligands against the self-complementary DNA sequences 5'-d(CGCACTAGTGCG)-3' and 5'-d(CGCAGTACTGCG)-3'. The ligands were carefully designed to target the DNA response element, 5'-WGWWCW-3', the binding site for several nuclear receptors. Basic 1D 1H NMR spectra of the DNA samples prepared with three MGB ligands show subtle variations suggestive of how each ligand associates with the double helical structure of both DNA sequences. The variations among the investigated ligands were reflected in the line shape and intensity of 1D 1H and 31P-{1H} NMR spectra. Rapid visual inspection of these 1D NMR spectra proves to be beneficial in providing valuable insights on MGB binding molecules. The NMR results were consistent with the findings from both UV DNA denaturation and molecular modelling studies. Both the NMR spectroscopic and computational analyses indicate that the investigated ligands bind to the minor grooves as antiparallel side-by-side dimers in a head-to-tail fashion. Moreover, comparisons with results from biochemical studies offered valuable insights into the mechanism of action, and antitumor activity of MGBs in relation to their structures, essential pre-requisites for future optimization of MGBs as therapeutic agents.

Scary in the eye of the beholder: Attentional bias and attentional retraining for social anxiety.

Consistent with cognitive models of social anxiety, socially anxious individuals show cognitive biases that magnify their perceived level of threat in the environment. OBJECTIVES: The first objective was to determine whether attentional bias for socially threatening stimuli occurs after concomitant depression has been controlled. The second objective was to test the effectiveness of the Attention Control Training Program for Social Anxiety (ACTP-SA) for reducing social anxiety attentional bias and improving therapeutic indices in people with social anxiety. METHOD: In the first study, socially anxious (N = 30) and non-anxious individuals (N = 30) completed the Beck Depression Inventory-II, Spielberger's State-Trait Anxiety Inventory, Conner's Social Phobia Inventory, a social-anxiety Stroop test, and a clinical interview. In the second study, individuals with social anxiety (N = 30) were randomly assigned to an experimental group that received 4 sessions of ACTP-SA, or to a sham-intervention control condition. At the post-test and a 3-month follow-up, both groups completed the same measures as in Study 1. RESULTS: In Study 1, socially anxious individuals showed higher attentional bias for threatening stimuli than the controls, after depression had been controlled for. In Study 2, participants in the experimental group, compared with the controls, showed greater reductions in attentional bias, social anxiety, and trait anxiety at post-test and follow-up. CONCLUSIONS: The results underscore the importance of information processing biases in social anxiety and the benefits of attentional bias training as a complementary intervention for modifying symptoms of social anxiety.

Structural signatures of Escherichia coli chemoreceptor signaling states revealed by cellular crosslinking.

The chemotaxis machinery of Escherichia coli has served as a model for exploring the molecular signaling mechanisms of transmembrane chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). Yet, fundamental questions about signal transmission through MCP molecules remain unanswered. Our work with the E. coli serine chemoreceptor Tsr has developed in vivo reporters that distinguish kinase-OFF and kinase-ON structures in the cytoplasmic methylation helix (MH) cap, which receives stimulus signals from an adjoining, membrane-proximal histidine kinase, adenylyl cyclases, MCPs, and phosphatases (HAMP) domain. The cytoplasmic helices of the Tsr homodimer interact mainly through packing interactions of hydrophobic residues at a and d heptad positions. We investigated the in vivo crosslinking properties of Tsr molecules bearing cysteine replacements at functionally tolerant g heptad positions in the N-terminal and C-terminal cap helices. Upon treatment of cells with bismaleimidoethane (BMOE), a bifunctional thiol-reagent, Tsr-G273C/Q504C readily formed a doubly crosslinked product in the presence of serine but not in its absence. Moreover, a serine stimulus combined with BMOE treatment during in vivo Förster resonance energy transfer-based kinase assays locked Tsr-G273C/Q504C in kinase-OFF output. An OFF-shifting lesion in MH1 (D269P) promoted the formation of the doubly crosslinked species in the absence of serine, whereas an ON-shifting lesion (G268P) suppressed the formation of the doubly crosslinked species. Tsr-G273C/Q504C also showed output-dependent crosslinking patterns in combination with ON-shifting and OFF-shifting adaptational modifications. Our results are consistent with a helix breathing-axial rotation-bundle repacking signaling mechanism and imply that in vivo crosslinking tools could serve to probe helix-packing transitions and their output consequences in other regions of the receptor molecule.

Hexameric rings of the scaffolding protein CheW enhance response sensitivity and cooperativity in Escherichia coli chemoreceptor arrays.

The Escherichia coli chemoreceptor array is a supramolecular assembly that enables cells to respond to extracellular cues dynamically and with great precision and sensitivity. In the array, transmembrane receptors organized as trimers of dimers are connected at their cytoplasmic tips by hexameric rings of alternating subunits of the kinase CheA and the scaffolding protein CheW (CheA-CheW rings). Interactions of CheW molecules with the members of receptor trimers not directly bound to CheA-CheW rings may lead to the formation of hexameric CheW rings in the chemoreceptor array. Here, we detected such CheW rings with a cellular cysteine-directed cross-linking assay and explored the requirements for their formation and their participation in array assembly. We found that CheW ring formation varied with cellular CheW abundance, depended on the presence of receptors capable of a trimer-of-dimers arrangement, and did not require CheA. Cross-linking studies of a CheA~CheW fusion protein incapable of forming homomeric CheW oligomers demonstrated that CheW rings were not essential for the assembly of CheA-containing arrays. Förster resonance energy transfer (FRET)-based kinase assays of arrays containing variable amounts of CheW rings revealed that CheW rings enhanced the cooperativity and the sensitivity of the responses to attractants. We propose that six-membered CheW rings provide the additional interconnectivity required for optimal signaling and gradient tracking performance by chemosensory arrays.

A new platform for high-throughput therapy testing on iPSC-derived lung progenitor cells from cystic fibrosis patients.

For those people with cystic fibrosis carrying rare CFTR mutations not responding to currently available therapies, there is an unmet need for relevant tissue models for therapy development. Here, we describe a new testing platform that employs patient-specific induced pluripotent stem cells (iPSCs) differentiated to lung progenitor cells that can be studied using a dynamic, high-throughput fluorescence-based assay of CFTR channel activity. Our proof-of-concept studies support the potential use of this platform, together with a Canadian bioresource that contains iPSC lines and matched nasal cultures from people with rare mutations, to advance patient-oriented therapy development. Interventions identified in the high-throughput, stem cell-based model and validated in primary nasal cultures from the same person have the potential to be advanced as therapies.

Metabolomic Profiling of the Immune Stimulatory Effect of Eicosenoids on PMA-Differentiated THP-1 Cells.

Honey bee venom has been established to have significant effect in immunotherapy. In the present study, (Z)-11-eicosenol-a major constituent of bee venom, along with its derivations methyl cis-11-eicosenoate and cis-11-eicosenoic acid, were synthesised to investigate their immune stimulatory effect and possible use as vaccine adjuvants. Stimuli that prime and activate the immune system have exerted profound effects on immune cells, particularly macrophages; however, the effectiveness of bee venom constituents as immune stimulants has not yet been established. Here, the abilities of these compounds to act as pro-inflammatory stimuli were assessed, either alone or in combination with lipopolysaccharide (LPS), by examining the secretion of tumour necrosis factor-α (TNF-α) and the cytokines interleukin-1ß (IL-1ß), IL-6 and IL-10 by THP-1 macrophages. The compounds clearly increased the levels of IL-1ß and decreased IL-10, whereas a decrease in IL-6 levels suggested a complex mechanism of action. A more in-depth profile of macrophage behaviour was therefore obtained by comprehensive untargeted metabolic profiling of the cells using liquid chromatography mass spectrometry (LC-MS) to confirm the ability of the eicosanoids to trigger the immune system. The level of 358 polar and 315 non-polar metabolites were changed significantly (p < 0.05) by all treatments. The LPS-stimulated production of most of the inflammatory metabolite biomarkers in glycolysis, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, purine, pyrimidine and fatty acids metabolism were significantly enhanced by all three compounds, and particularly by methyl cis-11-eicosenoate and cis-11-eicosenoic acid. These findings support the proposed actions of (Z)-11-eicosenol, methyl cis-11-eicosenoate and cis-11-eicosenoic acid as immune system stimulators.

Identification of a Kinase-Active CheA Conformation in Escherichia coli Chemoreceptor Signaling Complexes.

Escherichia coli chemotaxis relies on control of the autophosphorylation activity of the histidine kinase CheA by transmembrane chemoreceptors. Core signaling units contain two receptor trimers of dimers, one CheA homodimer, and two monomeric CheW proteins that couple CheA activity to receptor control. Core signaling units appear to operate as two-state devices, with distinct kinase-on and kinase-off CheA output states whose structural nature is poorly understood. A recent all-atom molecular dynamic simulation of a receptor core unit revealed two alternative conformations, "dipped" and "undipped," for the ATP-binding CheA.P4 domain that could be related to kinase activity states. To explore possible signaling roles for the dipped CheA.P4 conformation, we created CheA mutants with amino acid replacements at residues (R265, E368, and D372) implicated in promoting the dipped conformation and examined their signaling consequences with in vivo Förster resonance energy transfer (FRET)-based kinase assays. We used cysteine-directed in vivo cross-linking reporters for the dipped and undipped conformations to assess mutant proteins for these distinct CheA.P4 domain configurations. Phenotypic suppression analyses revealed functional interactions among the conformation-controlling residues. We found that structural interactions between R265, located at the N terminus of the CheA.P3 dimerization domain, and E368/D372 in the CheA.P4 domain played a critical role in stabilizing the dipped conformation and in producing kinase-on output. Charge reversal replacements at any of these residues abrogated the dipped cross-linking signal, CheA kinase activity, and chemotactic ability. We conclude that the dipped conformation of the CheA.P4 domain is critical to the kinase-active state in core signaling units.IMPORTANCE Regulation of CheA kinase in chemoreceptor arrays is critical for Escherichia coli chemotaxis. However, to date, little is known about the CheA conformations that lead to the kinase-on or kinase-off states. Here, we explore the signaling roles of a distinct conformation of the ATP-binding CheA.P4 domain identified by all-atom molecular dynamics simulation. Amino acid replacements at residues predicted to stabilize the so-called "dipped" CheA.P4 conformation abolished the kinase activity of CheA and its ability to support chemotaxis. Our findings indicate that the dipped conformation of the CheA.P4 domain is critical for reaching the kinase-active state in chemoreceptor signaling arrays.

Inter-partner specificity limits the acquisition of thermotolerant symbionts in a model cnidarian-dinoflagellate symbiosis.

The ability of corals and other cnidarians to survive climate change depends partly on the composition of their endosymbiont communities. The dinoflagellate family Symbiodiniaceae is genetically and physiologically diverse, and one proposed mechanism for cnidarians to acclimate to rising temperatures is to acquire more thermally tolerant symbionts. However, cnidarian-dinoflagellate associations vary in their degree of specificity, which may limit their capacity to alter symbiont communities. Here, we inoculated symbiont-free polyps of the sea anemone Exaiptasia pallida (commonly referred to as 'Aiptasia'), a model system for the cnidarian-dinoflagellate symbiosis, with simultaneous or sequential mixtures of thermally tolerant and thermally sensitive species of Symbiodiniaceae. We then monitored symbiont success (relative proportional abundance) at normal and elevated temperatures across two to four weeks. All anemones showed signs of bleaching at high temperature. During simultaneous inoculations, the native, thermally sensitive Breviolum minutum colonized polyps most successfully regardless of temperature when paired against the non-native but more thermally tolerant Symbiodinium microadriaticum or Durusdinium trenchii. Furthermore, anemones initially colonized with B. minutum and subsequently exposed to S. microadriaticum failed to acquire the new symbiont. These results highlight how partner specificity may place strong limitations on the ability of certain cnidarians to acquire more thermally tolerant symbionts, and hence their adaptive potential under climate change.

Effect of Melittin on Metabolomic Profile and Cytokine Production in PMA-Differentiated THP-1 Cells.

Melittin, the major active peptide of honeybee venom (BV), has potential for use in adjuvant immunotherapy. The immune system response to different stimuli depends on the secretion of different metabolites from macrophages. One potent stimulus is lipopolysaccharide (LPS), a component isolated from gram-negative bacteria, which induces the secretion of pro-inflammatory cytokines in macrophage cell cultures. This secretion is amplified when LPS is combined with melittin. In the present study, pure melittin was isolated from whole BV by flash chromatography to obtain pure melittin. The ability of melittin to enhance the release of tumour necrosis factor-α (TNF-α), Interleukin (IL-1ß, IL-6, and IL-10) cytokines from a macrophage cell line (THP-1) was then assessed. The response to melittin and LPS, applied alone or in combination, was characterised by metabolic profiling, and the metabolomics results were used to evaluate the potential of melittin as an immune adjuvant therapy. The addition of melittin enhanced the release of inflammatory cytokines induced by LPS. Effective chromatographic separation of metabolites was obtained by liquid chromatography-mass spectrometry (LC-MS) using a ZIC-pHILIC column and an ACE C4 column. The levels of 108 polar and non-polar metabolites were significantly changed (p ˂ 0.05) following cell activation by the combination of LPS and melittin when compared to untreated control cells. Overall, the findings of this study suggested that melittin might have a potential application as a vaccine adjuvant.

Subtle Differences in Symbiont Cell Surface Glycan Profiles Do Not Explain Species-Specific Colonization Rates in a Model Cnidarian-Algal Symbiosis.

Mutualisms between cnidarian hosts and dinoflagellate endosymbionts are foundational to coral reef ecosystems. These symbioses are often re-established every generation with high specificity, but gaps remain in our understanding of the cellular mechanisms that control symbiont recognition and uptake dynamics. Here, we tested whether differences in glycan profiles among different symbiont species account for the different rates at which they initially colonize aposymbiotic polyps of the model sea anemone Aiptasia (Exaiptasia pallida). First, we used a lectin array to characterize the glycan profiles of colonizing Symbiodinium minutum (ITS2 type B1) and noncolonizing Symbiodinium pilosum (ITS2 type A2), finding subtle differences in the binding of lectins Euonymus europaeus lectin (EEL) and Urtica dioica agglutinin lectin (UDA) that distinguish between high-mannoside and hybrid-type protein linked glycans. Next, we enzymatically cleaved glycans from the surfaces of S. minutum cultures and followed their recovery using flow cytometry, establishing a 48-72 h glycan turnover rate for this species. Finally, we exposed aposymbiotic host polyps to cultured S. minutum cells masked by EEL or UDA lectins for 48 h, then measured cell densities the following day. We found no effect of glycan masking on symbiont density, providing further support to the hypothesis that glycan-lectin interactions are more important for post-phagocytic persistence of specific symbionts than they are for initial uptake. We also identified several methodological and biological factors that may limit the utility of studying glycan masking in the Aiptasia system.

Regression of prostate tumors after intravenous administration of lactoferrin-bearing polypropylenimine dendriplexes encoding TNF-α, TRAIL, and interleukin-12.

The possibility of using gene therapy for the treatment of prostate cancer is limited by the lack of intravenously administered delivery systems able to safely and selectively deliver therapeutic genes to tumors. Given that lactoferrin (Lf) receptors are overexpressed on prostate cancer cells, we hypothesized that the conjugation of Lf to generation 3-diaminobutyric polypropylenimine dendrimer would improve its transfection and therapeutic efficacy in prostate cancer cells. In this study, we demonstrated that the intravenous administration of Lf-bearing DAB dendriplexes encoding TNFα resulted in the complete suppression of 70% of PC-3 and 50% of DU145 tumors over one month. Treatment with DAB-Lf dendriplex encoding TRAIL led to tumor suppression of 40% of PC-3 tumors and 20% of DU145 tumors. The treatment was well tolerated by the animals. Lf-bearing generation 3-polypropylenimine dendrimer is therefore a highly promising delivery system for non-viral gene therapy of prostate cancer.

Global landscape of cell envelope protein complexes in Escherichia coli.

Bacterial cell envelope protein (CEP) complexes mediate a range of processes, including membrane assembly, antibiotic resistance and metabolic coordination. However, only limited characterization of relevant macromolecules has been reported to date. Here we present a proteomic survey of 1,347 CEPs encompassing 90% inner- and outer-membrane and periplasmic proteins of Escherichia coli. After extraction with non-denaturing detergents, we affinity-purified 785 endogenously tagged CEPs and identified stably associated polypeptides by precision mass spectrometry. The resulting high-quality physical interaction network, comprising 77% of targeted CEPs, revealed many previously uncharacterized heteromeric complexes. We found that the secretion of autotransporters requires translocation and the assembly module TamB to nucleate proper folding from periplasm to cell surface through a cooperative mechanism involving the ß-barrel assembly machinery. We also establish that an ABC transporter of unknown function, YadH, together with the Mla system preserves outer membrane lipid asymmetry. This E. coli CEP 'interactome' provides insights into the functional landscape governing CE systems essential to bacterial growth, metabolism and drug resistance.

Metabolomic Profiling of the Synergistic Effects of Melittin in Combination with Cisplatin on Ovarian Cancer Cells.

Melittin, the main peptide present in bee venom, has been proposed as having potential for anticancer therapy; the addition of melittin to cisplatin, a first line treatment for ovarian cancer, may increase the therapeutic response in cancer treatment via synergy, resulting in improved tolerability, reduced relapse, and decreased drug resistance. Thus, this study was designed to compare the metabolomic effects of melittin in combination with cisplatin in cisplatin-sensitive (A2780) and resistant (A2780CR) ovarian cancer cells. Liquid chromatography (LC) coupled with mass spectrometry (MS) was applied to identify metabolic changes in A2780 (combination treatment 5 µg/mL melittin + 2 µg/mL cisplatin) and A2780CR (combination treatment 2 µg/mL melittin + 10 µg/mL cisplatin) cells. Principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) multivariate data analysis models were produced using SIMCA-P software. All models displayed good separation between experimental groups and high-quality goodness of fit (R²) and goodness of prediction (Q²), respectively. The combination treatment induced significant changes in both cell lines involving reduction in the levels of metabolites in the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, purine and pyrimidine metabolism, and the arginine/proline pathway. The combination of melittin with cisplatin that targets these pathways had a synergistic effect. The melittin-cisplatin combination had a stronger effect on the A2780 cell line in comparison with the A2780CR cell line. The metabolic effects of melittin and cisplatin in combination were very different from those of each agent alone.

National Registry Data and Record Linkage to Inform Postmarket Surveillance of Prosthetic Aortic Valve Models Over 15 Years.

Importance: Postmarket evidence generation for medical devices is important yet limited for prosthetic aortic valve devices in the United Kingdom. Objective: To identify prosthetic aortic valve models that display unexpected patterns of mortality or reintervention using routinely collected national registry data and record linkage. Design, Setting, and Participants: This observational study used data from all National Health Service and private hospitals in England and Wales that submit data to the National Adult Cardiac Surgery Audit (NACSA). All patients undergoing first-time elective and urgent aortic valve replacement surgery (with or without coronary artery bypass grafting) with a biological (n = 15 series) or mechanical (n = 10 series) prosthetic valve from 5 primary suppliers, and satisfying prespecified data quality criteria (n = 43 782 biological; n = 11 084 mechanical) between 1998 and 2013 were included. Valves were classified into series of related models. Outcome tracking was performed using multifaceted record linkage. The median follow-up was 4.1 years (maximum, 15.3 years). Cox proportional hazards regression with random effects (frailty models) were used to model valve effects on the outcomes, with and without adjustment for preoperative and intraoperative covariates. Main Outcomes and Measures: Time to all-cause mortality or aortic valve reintervention (surgical or transcatheter). There were 13 104 deaths and 723 reinterventions during follow-up. Results: Of 79 345 isolated aortic valve replacement procedures with or without coronary artery bypass grafting, 54 866 were analyzed. Biological valve implantation rates increased from 59% in 1998 and 1999 to 86% in 2012 and 2013. Two series of valves associated with significantly increased hazard of death or reintervention were identified (first series: frailty, 1.18; 95% prediction interval [PI], 1.06-1.32 and second series: frailty, 1.19; 95% PI, 1.09-1.31). These results were robust to covariate adjustment and sensitivity analyses. There were 3 prosthetic valves with a significant reduction in hazard (valve 1: frailty, 0.88; 95% PI, 0.80-0.96; valve 2: frailty, 0.88; 95% PI, 0.80-0.96; and valve 3: frailty, 0.88; 95% PI, 0.78-0.98). Conclusions and Relevance: Meaningful evidence from the analysis of routinely collected registry data can inform postmarket surveillance of medical devices. Although the findings are associated with a number of caveats, 2 specific biological aortic valve series identified in this study may warrant further investigation.

Metabolomic Profiling of the Effects of Melittin on Cisplatin Resistant and Cisplatin Sensitive Ovarian Cancer Cells Using Mass Spectrometry and Biolog Microarray Technology.

In the present study, liquid chromatography-mass spectrometry (LC-MS) was employed to characterise the metabolic profiles of two human ovarian cancer cell lines A2780 (cisplatin-sensitive) and A2780CR (cisplatin-resistant) in response to their exposure to melittin, a cytotoxic peptide from bee venom. In addition, the metabolomics data were supported by application of Biolog microarray technology to examine the utilisation of carbon sources by the two cell lines. Data extraction with MZmine 2.14 and database searching were applied to provide metabolite lists. Principal component analysis (PCA) gave clear separation between the cisplatin-sensitive and resistant strains and their respective controls. The cisplatin-resistant cells were slightly more sensitive to melittin than the sensitive cells with IC50 values of 4.5 and 6.8 µg/mL respectively, although the latter cell line exhibited the greatest metabolic perturbation upon treatment. The changes induced by melittin in the cisplatin-sensitive cells led mostly to reduced levels of amino acids in the proline/glutamine/arginine pathway, as well as to decreased levels of carnitines, polyamines, adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+). The effects on energy metabolism were supported by the data from the Biolog assays. The lipid compositions of the two cell lines were quite different with the A2780 cells having higher levels of several ether lipids than the A2780CR cells. Melittin also had some effect on the lipid composition of the cells. Overall, this study suggests that melittin might have some potential as an adjuvant therapy in cancer treatment.

Chemotactic Signaling by Single-Chain Chemoreceptors.

Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit swapping interactions that will need to be taken into account in experimental applications of single-chain chemoreceptors.

Identification of Candidate Adherent-Invasive E. coli Signature Transcripts by Genomic/Transcriptomic Analysis.

Adherent-invasive Escherichia coli (AIEC) strains are detected more frequently within mucosal lesions of patients with Crohn's disease (CD). The AIEC phenotype consists of adherence and invasion of intestinal epithelial cells and survival within macrophages of these bacteria in vitro. Our aim was to identify candidate transcripts that distinguish AIEC from non-invasive E. coli (NIEC) strains and might be useful for rapid and accurate identification of AIEC by culture-independent technology. We performed comparative RNA-Sequence (RNASeq) analysis using AIEC strain LF82 and NIEC strain HS during exponential and stationary growth. Differential expression analysis of coding sequences (CDS) homologous to both strains demonstrated 224 and 241 genes with increased and decreased expression, respectively, in LF82 relative to HS. Transition metal transport and siderophore metabolism related pathway genes were up-regulated, while glycogen metabolic and oxidation-reduction related pathway genes were down-regulated, in LF82. Chemotaxis related transcripts were up-regulated in LF82 during the exponential phase, but flagellum-dependent motility pathway genes were down-regulated in LF82 during the stationary phase. CDS that mapped only to the LF82 genome accounted for 747 genes. We applied an in silico subtractive genomics approach to identify CDS specific to AIEC by incorporating the genomes of 10 other previously phenotyped NIEC. From this analysis, 166 CDS mapped to the LF82 genome and lacked homology to any of the 11 human NIEC strains. We compared these CDS across 13 AIEC, but none were homologous in each. Four LF82 gene loci belonging to clustered regularly interspaced short palindromic repeats region (CRISPR)--CRISPR-associated (Cas) genes were identified in 4 to 6 AIEC and absent from all non-pathogenic bacteria. As previously reported, AIEC strains were enriched for pdu operon genes. One CDS, encoding an excisionase, was shared by 9 AIEC strains. Reverse transcription quantitative polymerase chain reaction assays for 6 genes were conducted on fecal and ileal RNA samples from 22 inflammatory bowel disease (IBD), and 32 patients without IBD (non-IBD). The expression of Cas loci was detected in a higher proportion of CD than non-IBD fecal and ileal RNA samples (p <0.05). These results support a comparative genomic/transcriptomic approach towards identifying candidate AIEC signature transcripts.

Systems-based analysis of the Sarcocystis neurona genome identifies pathways that contribute to a heteroxenous life cycle.

UNLABELLED: Sarcocystis neurona is a member of the coccidia, a clade of single-celled parasites of medical and veterinary importance including Eimeria, Sarcocystis, Neospora, and Toxoplasma. Unlike Eimeria, a single-host enteric pathogen, Sarcocystis, Neospora, and Toxoplasma are two-host parasites that infect and produce infectious tissue cysts in a wide range of intermediate hosts. As a genus, Sarcocystis is one of the most successful protozoan parasites; all vertebrates, including birds, reptiles, fish, and mammals are hosts to at least one Sarcocystis species. Here we sequenced Sarcocystis neurona, the causal agent of fatal equine protozoal myeloencephalitis. The S. neurona genome is 127 Mbp, more than twice the size of other sequenced coccidian genomes. Comparative analyses identified conservation of the invasion machinery among the coccidia. However, many dense-granule and rhoptry kinase genes, responsible for altering host effector pathways in Toxoplasma and Neospora, are absent from S. neurona. Further, S. neurona has a divergent repertoire of SRS proteins, previously implicated in tissue cyst formation in Toxoplasma. Systems-based analyses identified a series of metabolic innovations, including the ability to exploit alternative sources of energy. Finally, we present an S. neurona model detailing conserved molecular innovations that promote the transition from a purely enteric lifestyle (Eimeria) to a heteroxenous parasite capable of infecting a wide range of intermediate hosts. IMPORTANCE: Sarcocystis neurona is a member of the coccidia, a clade of single-celled apicomplexan parasites responsible for major economic and health care burdens worldwide. A cousin of Plasmodium, Cryptosporidium, Theileria, and Eimeria, Sarcocystis is one of the most successful parasite genera; it is capable of infecting all vertebrates (fish, reptiles, birds, and mammals-including humans). The past decade has witnessed an increasing number of human outbreaks of clinical significance associated with acute sarcocystosis. Among Sarcocystis species, S. neurona has a wide host range and causes fatal encephalitis in horses, marine mammals, and several other mammals. To provide insights into the transition from a purely enteric parasite (e.g., Eimeria) to one that forms tissue cysts (Toxoplasma), we present the first genome sequence of S. neurona. Comparisons with other coccidian genomes highlight the molecular innovations that drive its distinct life cycle strategies.

Signalling-dependent interactions between the kinase-coupling protein CheW and chemoreceptors in living cells.

Chemical signals sensed on the periplasmic side of bacterial cells by transmembrane chemoreceptors are transmitted to the flagellar motors via the histidine kinase CheA, which controls the phosphorylation level of the effector protein CheY. Chemoreceptor arrays comprise remarkably stable supramolecular structures in which thousands of chemoreceptors are networked through interactions between their cytoplasmic tips, CheA, and the small coupling protein CheW. To explore the conformational changes that occur within this protein assembly during signalling, we used in vivo cross-linking methods to detect close interactions between the coupling protein CheW and the serine receptor Tsr in intact Escherichia coli cells. We identified two signal-sensitive contacts between CheW and the cytoplasmic tip of Tsr. Our results suggest that ligand binding triggers changes in the receptor that alter its signalling contacts with CheW (and/or CheA).

Molecular speciation and mesoscale clustering in formaldehyde-methanol-water solutions in the presence of sodium carbonate.

Nanoporous organic gels can be synthesized from aqueous solutions of formaldehyde and resorcinol in the presence of basic electrolytes such as sodium carbonate. It is well known that formaldehyde is present in the form of methylene glycols or methoxy-glycols in aqueous and methanolic solutions, but influence of pH or electrolytes on speciation in these solutions has not been previously studied. Here we investigated effects of sodium carbonate on the speciation and colloidal scale clustering in formaldehyde-methanol-water solutions in the absence of resorcinol. We used (13)C NMR spectroscopy to quantitatively characterize molecular speciation in solutions and to estimate corresponding equilibrium constants for glycol dimerization and methoxylation. We found that species distribution is essentially independent of carbonate concentration for pH values between 3.4 (no carbonate) and 10.6. This was also consistent with ATR IR measurements of the same solutions. However, NMR spin-spin relaxation time measurements showed an unexpected behavior for glycols and especially for diglycol (but not for methanol), with relaxation times strongly decreasing with increasing carbonate concentration, indicating differences in local molecular environment of glycols. We further used dynamic light scattering to confirm the presence of mesoscale clustering in formaldehyde-methanol-water (for both H2O and D2O) solutions in the presence of sodium carbonate. We propose that the observed phenomena are due to glycol-rich cluster mesospecies in equilibrium bulk solution, together forming a thermodynamically stable mesostructured liquid phase.