Glycerol metabolism impacts biofilm phenotypes and virulence in Pseudomonas aeruginosa via the Entner-Doudoroff pathway.

Pseudomonas aeruginosa is a ubiquitous bacterium and a notorious opportunistic pathogen that forms biofilm structures in response to many environmental cues. Biofilm formation includes attachment to surfaces and the production of the exopolysaccharide Pel, which is present in both the PAO1 and PA14 laboratory strains of P. aeruginosa. Biofilms help protect bacterial cells from host defenses and antibiotics and abet infection. The carbon source used by the cells also influences biofilm, but these effects have not been deeply studied. We show here that glycerol, which can be liberated from host surfactants during infection, encourages surface attachment and magnifies colony morphology differences. We find that glycerol kinase is important but not essential for glycerol utilization and relatively unimportant for biofilm behaviors. Among downstream enzymes predicted to take part in glycerol utilization, Edd stood out as being important for glycerol utilization and for enhanced biofilm phenotypes in the presence of glycerol. Thus, gluconeogenesis and catabolism of anabolically produced glucose appear to impact not only the utilization of glycerol but also glycerol-stimulated biofilm phenotypes. Finally, waxworm moth larvae and nematode infection models reveal that interruption of the Entner-Doudoroff pathway, but not abrogation of glycerol phosphorylation, unexpectedly increases P. aeruginosa lethality in both acute and chronic infections, even while stimulating a stronger immune response by Caenorhabditis elegans.IMPORTANCEPseudomonas aeruginosa, the ubiquitous environmental bacterium and human pathogen, forms multicellular communities known as biofilms in response to various stimuli. We find that glycerol, a common carbon source that bacteria can use for energy and biosynthesis, encourages biofilm behaviors such as surface attachment and colony wrinkling by P. aeruginosa. Glycerol can be derived from surfactants that are present in the human lungs, a common infection site. Glycerol-stimulated biofilm phenotypes do not depend on phosphorylation of glycerol but are surprisingly impacted by a glucose breakdown pathway, suggesting that it is glycerol utilization, and not its mere presence or cellular import, that stimulates biofilm phenotypes. Moreover, the same mutations that block glycerol-stimulated biofilm phenotypes also impact P. aeruginosa virulence in both acute and chronic animal models. Notably, a glucose-breakdown mutant (Δedd) counteracts biofilm phenotypes but shows enhanced virulence and stimulates a stronger immune response in Caenorhabditis elegans.

PtsN in Pseudomonas aeruginosa Is Phosphorylated by Redundant Upstream Proteins and Impacts Virulence-Related Genes.

The bacterial nitrogen-related phosphotransfer (PTSNtr; here, Nitro-PTS) system bears homology to well-known PTS systems that facilitate saccharide import and phosphorylation. The Nitro-PTS comprises an enzyme I (EI), PtsP; an intermediate phosphate carrier, PtsO; and a terminal acceptor, PtsN, which is thought to exert regulatory effects that depend on its phosphostate. For instance, biofilm formation by Pseudomonas aeruginosa can be impacted by the Nitro-PTS, as deletion of either ptsP or ptsO suppresses Pel exopolysaccharide production and additional deletion of ptsN elevates Pel production. However, the phosphorylation state of PtsN in the presence and absence of its upstream phosphotransferases has not been directly assessed, and other targets of PtsN have not been well defined in P. aeruginosa. We show that PtsN phosphorylation via PtsP requires the GAF domain of PtsP and that PtsN is phosphorylated on histidine 68, as in Pseudomonas putida. We also find that FruB, the fructose EI, can substitute for PtsP in PtsN phosphorylation but only in the absence of PtsO, implicating PtsO as a specificity factor. Unphosphorylatable PtsN had a minimal effect on biofilm formation, suggesting that it is necessary but not sufficient for the reduction of Pel in a ptsP deletion. Finally, we use transcriptomics to show that the phosphostate and the presence of PtsN do not appear to alter the transcription of biofilm-related genes but do influence genes involved in type III secretion, potassium transport, and pyoverdine biosynthesis. Thus, the Nitro-PTS influences several P. aeruginosa behaviors, including the production of its signature virulence factors. IMPORTANCE The PtsN protein impacts the physiology of a number of bacterial species, and its control over downstream targets can be altered by its phosphorylation state. Neither its upstream phosphotransferases nor its downstream targets are well understood in Pseudomonas aeruginosa. Here, we examine PtsN phosphorylation and find that the immediate upstream phosphotransferase acts as a gatekeeper, allowing phosphorylation by only one of two potential upstream proteins. We use transcriptomics to discover that PtsN regulates the expression of gene families that are implicated in virulence. One emerging pattern is a repression hierarchy by different forms of PtsN: its phosphorylated state is more repressive than its unphosphorylated state, but the expression of its targets is even higher in its complete absence.

Redundancy in Citrate and cis-Aconitate Transport in Pseudomonas aeruginosa.

Tricarboxylates such as citrate are the preferred carbon sources for Pseudomonas aeruginosa, an opportunistic pathogen that causes chronic human infections. However, the membrane transport process for the tricarboxylic acid cycle intermediates citrate and cis-aconitate is poorly characterized. Transport is thought to be controlled by the TctDE two-component system, which mediates transcription of the putative major transporter OpdH. Here, we search for previously unidentified transporters of citrate and cis-aconitate using both protein homology and RNA sequencing approaches. We uncover new transporters and show that OpdH is not the major citrate importer; instead, citrate transport primarily relies on the tripartite TctCBA system, which is encoded in the opdH operon. Deletion of tctA causes a growth lag on citrate and loss of growth on cis-aconitate. Combinatorial deletion of newly discovered transporters can fully block citrate utilization. We then characterize transcriptional control of the opdH operon in tctDE mutants and show that loss of tctD blocks citrate utilization due to an inability to express opdH-tctCBA. However, tctE and tctDE mutants evolve heritable adaptations that restore growth on citrate as the sole carbon source. IMPORTANCE Pseudomonas aeruginosa is a bacterium that infects hospitalized patients and is often highly resistant to antibiotic treatment. It preferentially uses small organic acids called tricarboxylates rather than sugars as a source of carbon for growth. The transport of many of these molecules from outside the cell to the interior occurs through unknown channels. Here, we examined how the tricarboxylates citrate and cis-aconitate are transported in P. aeruginosa. We then sought to understand how production of proteins that permit citrate and cis-aconitate transport is regulated by a signaling system called TctDE. We identified new transporters for these molecules, clarified the function of a known transport system, and directly tied transporter expression to the presence of an intact TctDE system.

Enhanced Acceptance Specification of Asphalt Binder to Drive Sustainability in the Paving Industry.

Testing small amounts of extracted and recovered asphalt binder as used in construction allows for the acceptance of materials in accordance with traffic and climate requirements. This approach facilitates the sustainable use of resources and thus prepares the paving industry for the true circular economy. Oscillatory, creep, and failure tests in a rheometer are compared for the performance grading of 32 asphalt binders extracted and recovered from real-world contract samples. Films 8 mm in diameter and 0.5 mm thick were tested from 35 to -5 °C in dynamic shear, followed by shear creep at 0 and 5 °C, and finally in tertiary tensile creep at 15 °C. The enhanced protocol uses a very small amount of material in contrast to current methods, yet it provides comparable results. Phase angle measurements appear to be optimal for performance grading, but further field study is required to determine if additional binder properties such as stiffness and/or failure strain would be required for the control of cracking.

Abnormal lymphatic phenotype in a CRISPR mouse model of the human lymphedema-causing Connexin47 R260C point mutation.

Connexin proteins form gap junctions controlling exchange of ions and small molecules between cells and play an important role in movement of lymph within lymphatic vessels. Connexin47 (CX47) is highly expressed in lymphatic endothelial cells and CX47 missense mutations, i.e., R260C, cosegregate with primary lymphedema in humans. However, studies utilizing CX47 knockout mice have failed to demonstrate any lymphatic anomalies. To unravel the lymphatic consequences of expressing a mutant CX47 protein, we used CRISPR technology to create a mouse carrying a Cx47 missense mutation (Cx47R259C) equivalent to the human CX47R260C missense mutation associated with human primary lymphedema. Intradermal Evans Blue dye injection identified a 2-fold increase in regional lymph nodes in homozygous Cx47R259C mice compared to wildtype, particularly in the jugular region (4.8 ± 0.4 and 2.0 ± 0.0, respectively, p<0.01). Associated lymphatic channels were increased in Cx47R259C mice and mesenteric lymph reflux occurred in homozygous Cx47R259C mice but not in wildtype. Contractility of superficial cervical lymphatics, assessed by pressure myography, was reduced in homozygous Cx47R259C mice compared to wildtype. In conclusion, our data are the first to demonstrate a role for the Cx47 protein in lymphatic anatomy and function. This phenotype is similar to that found with other valve deficient mouse mutants, e.g., in Foxc2. Of significance, this study is the first to use CRISPR technology to develop a pre-clinical model of primary lymphedema and demonstrates the importance of distinguishing between lack of and presence of mutant protein when developing clinically relevant animal models for translation of pre-clinical findings.

Side Effect Patterns in a Crossover Trial of Statin, Placebo, and No Treatment.

BACKGROUND: Most people who begin statins abandon them, most commonly because of side effects. OBJECTIVES: The purpose of this study was to assess daily symptom scores on statin, placebo, and no treatment in participants who had abandoned statins. METHODS: Participants received 12 1-month medication bottles, 4 containing atorvastatin 20 mg, 4 placebo, and 4 empty. We measured daily symptom intensity for each using an app (scale 1-100). We also measured the "nocebo" ratio: the ratio of symptoms induced by taking statin that was also induced by taking placebo. RESULTS: A total of 60 participants were randomized and 49 completed the 12-month protocol. Mean symptom score was 8.0 (95% CI: 4.7-11.3) in no-tablet months. It was higher in statin months (16.3; 95% CI: 13.0-19.6; P < 0.001), but also in placebo months (15.4; 95% CI: 12.1-18.7; P < 0.001), with no difference between the 2 (P = 0.388). The corresponding nocebo ratio was 0.90. In the individual-patient daily data, neither symptom intensity on starting (OR: 1.02; 95% CI: 0.98-1.06; P = 0.28) nor extent of symptom relief on stopping (OR: 1.01; 95% CI: 0.98-1.05; P = 0.48) distinguished between statin and placebo. Stopping was no more frequent for statin than placebo (P = 0.173), and subsequent symptom relief was similar between statin and placebo. At 6 months after the trial, 30 of 60 (50%) participants were back taking statins. CONCLUSIONS: The majority of symptoms caused by statin tablets were nocebo. Clinicians should not interpret symptom intensity or timing of symptom onset or offset (on starting or stopping statin tablets) as indicating pharmacological causation, because the pattern is identical for placebo. (Self-Assessment Method for Statin Side-effects Or Nocebo [SAMSON]; NCT02668016).

Omega-3 index and blood pressure responses to eating foods naturally enriched with omega-3 polyunsaturated fatty acids: a randomized controlled trial.

Diets low in seafood omega-3 polyunsaturated fatty acids (PUFAs) are very prevalent. Such diets have recently been ranked as the sixth most important dietary risk factor-1.5 million deaths and 33 million disability-adjusted life-years worldwide are attributable to this deficiency. Wild oily fish stocks are insufficient to feed the world's population, and levels of eicosapentaenoic acid and docosahexaenoic acid (DHA) in farmed fish have more than halved in the last 20 years. Here we report on a double-blinded, controlled trial, where 161 healthy normotensive adults were randomly allocated to eat at least three portions/week of omega-3-PUFA enriched (or control) chicken-meat, and to eat at least three omega-3-PUFA enriched (or control) eggs/week, for 6 months. We show that regular consumption of omega-3-PUFA enriched chicken-meat and eggs significantly increased the primary outcome, the red cell omega-3 index (mean difference [98.75% confidence interval] from the group that ate both control foods, 1.7% [0.7, 2.6]). Numbers of subjects with a very high-risk omega-3 index (index < 4%) were more than halved amongst the group that ate both enriched foods. Furthermore, eating the enriched foods resulted in clinically relevant reductions in diastolic blood pressure (- 3.1 mmHg [- 5.8, - 0.3]). We conclude that chicken-meat and eggs, naturally enriched with algae-sourced omega-3-PUFAs, may serve as alternative dietary sources of these essential micronutrients. Unlike many lifestyle interventions, long-term population health benefits do not depend on willingness of individuals to make long-lasting difficult dietary changes, but on the availability of a range of commonly eaten, relatively inexpensive, omega-3-PUFA enriched foods.

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.

A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

Comparison of desferrioxamine and NODAGA for the gallium-68 labeling of exendin-4.

INTRODUCTION: Radiolabeled exendin-4 (Ex4) derivatives are used to target the glucagon-like peptide-1 receptor (GLP-1R) for the clinical diagnosis of insulinomas, a rare type of neuroendocrine tumor. Gallium-68 is an ideal diagnostic nuclide for this application and a study evaluating an exendin-4-NODAGA conjugate is currently underway. However, in complexion with the chelator DFO, its in vivo stability has been a matter of dispute. The aim of this work was to directly compare [68Ga]Ga-Ex4NOD with [68Ga]Ga-Ex4DFO in vitro and in vivo. METHODS: In our approach, we directly compared N'-[5-(acetyl-hydroxy-amino)pentyl]-N-[5-[3-(5-aminopentyl-hydroxy-carbamoyl)propanoylamino]pentyl]-N-hydroxy-butane diamide (desferriox-amine B, DFO) and 2-(4,7-bis (carboxymethyl)-1,4,7-triazonan-1-yl) pentanedioic acid (NODAGA) conjugated to exendin-4 in vitro and in vivo. We radiolabeled the peptides with gallium-68, followed by HPLC quality control. In vitro characterization was performed in CHL cells overexpressing the GLP-1R and in vivo studies were conducted with CD1 nu/nu mice carrying tumors derived from these cells. RESULTS: We found that both peptides could be radiolabeled with a molar activity of about 9.33 MBq/nmol without further purification. They internalized equally well into GLP-1R-expressing cells and their IC50 was similar with 15.6 ± 7.8 nM and 18.4 ± 3.0 nM for [natGa]Ga-Ex4NOD and [natGa]Ga-Ex4DFO, respectively. In vivo, [68Ga]Ga-Ex4NOD accumulated more in all tissue, while [68Ga]Ga-Ex4DFO exhibited a more favorable target-to-kidney ratio. CONCLUSION AND RELEVANCE: DFO is a suitable chelator for the radiolabeling of exendin-4 derivatives with gallium-68 for in vitro and preclinical in vivo studies. DFO performed better in vivo due to its significantly lower kidney accumulation (p < 0.0001). It was also found to be stable in vivo in mice, contrary to earlier reports. Based on our results, the DFO chelating system in combination with exendin-4 would be an interesting option for clinical imaging of insulinomas.

Carbohydrate and PepO control bimodality in competence development by Streptococcus mutans.

In Streptococcus mutans, the alternative sigma factor ComX controls entry into genetic competence. Competence stimulating peptide (CSP) induces bimodal expression of comX, with only a fraction of the population becoming transformable. Curiously, the bimodality of comX is affected by peptides in the growth medium and by carbohydrate source. CSP elicits bimodal expression of comX in media rich in small peptides, but CSP elicits no response in defined media lacking small peptides. In addition, growth on certain sugars increases the proportion of the population that activates comX in response to CSP. By investigating the connection between media and comX bimodality, we find evidence for two mechanisms that modulate transcriptional positive feedback in the ComRS system, where comX bimodality originates. We find that the endopeptidase PepO suppresses the ComRS feedback loop, most likely by degrading the XIP/ComS feedback signal. Deletion of pepO eliminates comX bimodality, leading to a unimodal comX response to CSP in both defined and complex media. We also find that CSP stimulates the ComRS feedback system by upregulating comR in a carbohydrate source-dependent fashion. Our data provide mechanistic insight into how S. mutans regulates bimodality and explain the puzzle of growth medium effects on competence induction by CSP.

Exendin-4 Derivatives with an Albumin-Binding Moiety Show Decreased Renal Retention and Improved GLP-1 Receptor Targeting.

The glucagon-like peptide-1 receptor (GLP-1R) is highly and specifically expressed on the pancreatic ß-cells. It plays an important role in glucose metabolism as well as in ß-cell-derived diseases like diabetes, insulinoma, or congenital and adult hyperinsulinemic hypoglycemia. Radiolabeled exendin-4, a ligand of GLP-1R, has routinely been used in clinics to image insulinomas. However, its major drawback is the high kidney accumulation. Here, we show that the addition of an albumin-binding moiety (ABM) to radiolabeled exendin-4 results in a significant reduction of kidney uptake while retaining its high affinity and specificity to GLP-1R. The four tested peptides were shown to have high affinity to the GLP-1 receptor (IC50 of 3.7 ± 0.6 to 15.1 ± 0.8 nM). The radiolabeled derivatives were taken up into cells efficiently, internalizing between 39 ± 2 and 56 ± 2% after 2 h. Thus, the derivatives with ABM outperformed the reference peptide with its IC50 of 22.5 ± 2.9 nM and internalization of 41 ± 4%. Stability in human blood plasma was slightly enhanced by the addition of the albumin binder. In biodistribution studies, the radioligands exhibited an improved target-to-kidney ratio in comparison to the reference peptide of up to seven-fold. This was confirmed qualitatively in single-photon-emission computed tomography (SPECT)/CT imaging. This study demonstrated in vitro and in vivo that the addition of an ABM to radiolabeled exendin-4 strongly decreased kidney accumulation while retaining affinity to GLP-1R. Thus, exendin-4 derivatives with an albumin-binding moiety could present a viable class of diagnostic tracers for the detection of insulinomas and other GLP-1R-positive tissue in clinical application.

Automatic optic disc detection in colour fundus images by means of multispectral analysis and information content.

The optic disc (OD) in retinal fundus images is widely used as a reference in computer-based systems for the measurement of the severity of retinal disease. A number of algorithms have been published in the past 5 years to locate and measure the OD in digital fundus images. Our proposed algorithm, automatically: (i) uses the three channels (RGB) of the digital colour image to locate the region of interest (ROI) where the OD lies, (ii) measures the Shannon information content per channel in the ROI, to decide which channel is most appropriate for searching for the OD centre using the circular Hough transform. A series of evaluations were undertaken to test our hypothesis that using the three channels gives a better performance than a single channel. Three different databases were used for evaluation purposes with a total of 2,371 colour images giving a misdetection error of 3% in the localisation of the centre of the OD. We find that the area determined by our algorithm which assumes that the OD is circular, is similar to that found by other algorithms that detected the shape of the OD. Five metrics were measured for comparison with other recent studies. Combining the two databases where expert delineation of the OD is available (1,240 images), the average results for our multispectral algorithm are: TPR = 0.879, FPR = 0.003, Accuracy = 0.994, Overlap = 80.6% and Dice index = 0.878.

Intracellular Signaling by the comRS System in Streptococcus mutans Genetic Competence.

Entry into genetic competence in streptococci is controlled by ComX, an alternative sigma factor for genes that enable the import of exogenous DNA. In Streptococcus mutans, the immediate activator of comX is the ComRS quorum system. ComS is the precursor of XIP, a seven-residue peptide that is imported into the cell and interacts with the cytosolic receptor ComR to form a transcriptional activator for both comX and comS Although intercellular quorum signaling by ComRS has been demonstrated, observations of bimodal expression of comX suggest that comRS may also function as an intracellular feedback loop, activating comX without export or detection of extracellular XIP. Here we used microfluidic and single-cell methods to test whether ComRS induction of comX requires extracellular XIP or ComS. We found that individual comS-overexpressing cells activate their own comX, independently of the rate at which their growth medium is replaced. However, in the absence of lysis they do not activate comS-deficient mutants growing in coculture. We also found that induction of comR and comS genes introduced into Escherichia coli cells leads to activation of a comX reporter. Therefore, ComRS control of comX does not require either the import or extracellular accumulation of ComS or XIP or specific processing of ComS to XIP. We also found that endogenously and exogenously produced ComS and XIP have inequivalent effects on comX activation. These data are fully consistent with identification of intracellular positive feedback in comS transcription as the origin of bimodal comX expression in S. mutansIMPORTANCE The ComRS system can function as a quorum sensing trigger for genetic competence in S. mutans The signal peptide XIP, which is derived from the precursor ComS, enters the cell and interacts with the Rgg-type cytosolic receptor ComR to activate comX, which encodes the alternative sigma factor for the late competence genes. Previous studies have demonstrated intercellular signaling via ComRS, although release of the ComS or XIP peptide to the extracellular medium appears to require lysis of the producing cells. Here we tested the complementary hypothesis that ComRS can drive comX through a purely intracellular mechanism that does not depend on extracellular accumulation or import of ComS or XIP. By combining single-cell, coculture, and microfluidic approaches, we demonstrated that endogenously produced ComS can enable ComRS to activate comX without requiring processing, export, or import. These data provide insight into intracellular mechanisms that generate noise and heterogeneity in S. mutans competence.

Online adaptive neural control of a robotic lower limb prosthesis.

OBJECTIVE: The purpose of this study was to develop and evaluate an adaptive intent recognition algorithm that continuously learns to incorporate a lower limb amputee's neural information (acquired via electromyography (EMG)) as they ambulate with a robotic leg prosthesis. APPROACH: We present a powered lower limb prosthesis that was configured to acquire the user's neural information and kinetic/kinematic information from embedded mechanical sensors, and identify and respond to the user's intent. We conducted an experiment with eight transfemoral amputees over multiple days. EMG and mechanical sensor data were collected while subjects using a powered knee/ankle prosthesis completed various ambulation activities such as walking on level ground, stairs, and ramps. Our adaptive intent recognition algorithm automatically transitioned the prosthesis into the different locomotion modes and continuously updated the user's model of neural data during ambulation. MAIN RESULTS: Our proposed algorithm accurately and consistently identified the user's intent over multiple days, despite changing neural signals. The algorithm incorporated 96.31% [0.91%] (mean, [standard error]) of neural information across multiple experimental sessions, and outperformed non-adaptive versions of our algorithm-with a 6.66% [3.16%] relative decrease in error rate. SIGNIFICANCE: This study demonstrates that our adaptive intent recognition algorithm enables incorporation of neural information over long periods of use, allowing assistive robotic devices to accurately respond to the user's intent with low error rates.

Intercellular Communication via the comX-Inducing Peptide (XIP) of Streptococcus mutans.

Gram-positive bacteria utilize exported peptides to coordinate genetic and physiological processes required for biofilm formation, stress responses, and ecological competitiveness. One example is activation of natural genetic competence by ComR and the com X -inducing peptide (XIP) in Streptococcus mutans Although the competence pathway can be activated by the addition of synthetic XIP in defined medium, the hypothesis that XIP is able to function as an intercellular signaling molecule has not been rigorously tested. Coculture model systems were developed that included a "sender" strain that overexpressed the XIP precursor (ComS) and a "responder" strain harboring a green fluorescent protein (GFP) reporter fused to a ComR-activated gene (comX) promoter. The ability of the sender strain to provide a signal to activate GFP expression was monitored at the individual cell and population levels using (i) planktonic culture systems, (ii) cells suspended in an agarose matrix, or (iii) cells growing in biofilms. XIP was shown to be freely diffusible, and XIP signaling between the S. mutans sender and responder strains did not require cell-to-cell contact. The presence of a sucrose-derived exopolysaccharide matrix diminished the efficiency of XIP signaling in biofilms, possibly by affecting the spatial distribution of XIP senders and potential responders. Intercellular signaling was greatly impaired in a strain lacking the primary autolysin, AtlA, and was substantially greater when the sender strain underwent lysis. Collectively, these data provide evidence that S. mutans XIP can indeed function as a peptide signal between cells and highlight the importance of studying signaling with an endogenously produced peptide(s) in populations in various environments and physiologic states.IMPORTANCE The comX-inducing peptide (XIP) of Streptococcus mutans is a key regulatory element in the activation of genetic competence, which allows cells to take up extracellular DNA. XIP has been found in cell culture fluids, and the addition of synthetic XIP to physiologically receptive cells can robustly induce competence gene expression. However, there is a lack of consensus as to whether XIP can function as an intercellular communication signal. Here, we show that XIP indeed signals between cells in S. mutans, but that cell lysis may be a critical factor, as opposed to a dedicated secretion/processing system, in allowing for release of XIP into the environment. The results have important implications in the context of the ecology, virulence, and evolution of a ubiquitous human pathogen and related organisms.

Evaluation of C-reactive protein before and on-treatment as a predictor of benefit of atorvastatin: a cohort analysis from the Anglo-Scandinavian Cardiac Outcomes Trial lipid-lowering arm.

OBJECTIVES: The aim of this study was to determine whether baseline and on-statin C-reactive protein (CRP) are independent predictors of cardiovascular (CV) outcome beyond low-density lipoprotein cholesterol (LDL-C). BACKGROUND: Use of CRP as a predictor of statin treatment remains controversial. METHODS: We investigated the relationship of baseline and on-treatment CRP with subsequent CV events in Cox models using a subset of white subjects with no history of CV disease from the UK ASCOT (Anglo-Scandinavian Cardiac Outcomes Trial). RESULTS: During 5.5 years of follow-up, a total of 488 subjects experienced a CV event. CV risk increased with baseline CRP (hazard ratio [HR] per 1 SD: 1.21; 95% confidence interval [CI]: 1.09 to 1.33) in an adjusted model. In ASCOT Lipid-Lowering Arm, the relative statin effect in preventing CV events did not differ according to tertiles of baseline CRP (p = 0.69). After 6 months of atorvastatin therapy, the median LDL-C and CRP were reduced by 38.7% and 25.8%, respectively. Those who achieved LDL-C below the median had a reduced CV risk (HR: 0.58; 95% CI: 0.34 to 0.97) compared with those who did not. In contrast, those who achieved a CRP level below the median did not have a reduced risk of CV events (HR: 0.95; 95% CI: 0.59 to 1.55). Among those who achieved LDL-C below the median, there was no difference in CV risk whether they also achieved a CRP level below (HR: 0.55; 95% CI: 0.30 to 1.02) or above the median (HR: 0.56; 95% CI: 0.30 to 1.03). CONCLUSIONS: In these primary prevention patients, although baseline CRP independently predicted CV event risk, the achieved CRP level on while statin therapy did not predict CV events, either alone or in combination with LDL-C.

[Factors associated with non-compliance to surgical antimicrobial prophylaxis guidelines during a prospective audit]. / Facteurs prédictifs de non-conformité d'antibioprophylaxie chirurgicale au cours d'un audit clinique prospectif.

OBJECTIVES: To assess physician compliance with guidelines for surgical antimicrobial prophylaxis practices and to identify factors associated with non-compliance. PATIENTS AND METHOD: In a prospective study of patients admitted for clean or clean contaminated surgery from February 1 to April 28, 2008, we assessed appropriateness of surgical antimicrobial prophylaxis indication (recommended and prescribed or not recommended and not prescribed) and administration (antimicrobial choice, dose, timing and duration). Overall compliance was achieved if all criteria of indication and administration were met. The overall non-compliance rate was estimated and was used as a dependent variable in a multivariate logistic regression analysis to identify factors associated with non-compliance. RESULTS: In a total of 481 practices evaluated, appropriateness of indication and administration of surgical prophylaxis were respectively 83% and 56%. The overall compliance rate to guidelines for surgical antimicrobial prophylaxis was 37%. In a multivariate regression analysis, prescription of antimicrobial prophylaxis by a surgeon (RR: 3.4; CI 95%: 1.6-7.5), clean-contaminated surgery (RR: 2.2; CI 95%: 1.4-3.7), traumatology surgery (1.87; 1.1-3.3), digestive surgery (3.7; 1.8-7.5) and head or neck-related surgery (11.4; 2.3-56.3) were independent factors associated with non-compliance to guidelines for surgical antimicrobial prophylaxis. CONCLUSION: This study confirmed previous findings on the impact of the type of surgery and the wound class contamination on the overall compliance rate. Furthermore, non-compliance was related to surgeon prescriptions. The compliance should be improved by specific measures after professional feedback.

Lymphatic communication: connexin junction, what's your function?

This article reviews recent findings on expression and function of connexin proteins--the structural subunits of gap junction intercellular channels in the lymphatic vasculature--both during development and in the mature lymphatic vessel. Highlighted in particular are recent mouse connexin knockout studies which show that connexins are crucial for normal lymphatic development. We discuss, in general terms, both channel-dependent as well as channel-independent functions of connexins and raise some of the many unanswered questions about the mechanism(s) of action and physiological roles of connexins in the lymphatic vasculature.