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Modern digital technologies enable the efficient collection and processing of student perceptions of teaching quality. However, students’ ratings could be confounded by student, teacher, and classroom characteristics. We investigated students’ ratings of 26 teachers who used the digital tool Impact! in their mathematics lessons with 14- and 15-year-old students (n = 717). A Bayesian item response theory (IRT)-model approach was used to model potential associations. High-performing students on average rated their teacher higher than low- and middle-performing students. More likeable and more experienced teachers received higher ratings from their students, and the higher the class’s average math grade, the higher the students rated their teachers. Other variables investigated in this study (e.g., student and teacher gender, class size) were not associated with student perceptions of teaching quality. Both related and unrelated factors are discussed. Some implications of the findings for practice, limitations of the study, and suggestions for further research are presented.

Mycoplasma hyopneumoniae is cultured on large-scale to produce antigen for inactivated whole-cell vaccines against respiratory disease in pigs. However, the fastidious nutrient requirements of this minimal bacterium and the low growth rate make it challenging to reach sufficient biomass yield for antigen production. In this study, we sequenced the genome of M. hyopneumoniae strain 11 and constructed a high quality constraint-based genome-scale metabolic model of 284 chemical reactions and 298 metabolites. We validated the model with time-series data of duplicate fermentation cultures to aim for an integrated model describing the dynamic profiles measured in fermentations. The model predicted that 84% of cellular energy in a standard M. hyopneumoniae cultivation was used for non-growth associated maintenance and only 16% of cellular energy was used for growth and growth associated maintenance. Following a cycle of model-driven experimentation in dedicated fermentation experiments, we were able to increase the fraction of cellular energy used for growth through pyruvate addition to the medium. This increase in turn led to an increase in growth rate and a 2.3 times increase in the total biomass concentration reached after 3-4 days of fermentation, enhancing the productivity of the overall process. The model presented provides a solid basis to understand and further improve M. hyopneumoniae fermentation processes. This article is protected by copyright. All rights reserved

Background and aims: Wastewater-based epidemiology is an additional indicator of drug use that is gaining reliability to complement the current established panel of indicators. The aims of this study were to: (i) assess spatial and temporal trends of population-normalized mass loads of benzoylecgonine, amphetamine, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) in raw wastewater over 7 years (2011–17); (ii) address overall drug use by estimating the average number of combined doses consumed per day in each city; and (iii) compare these with existing prevalence and seizure data. Design: Analysis of daily raw wastewater composite samples collected over 1 week per year from 2011 to 2017. Setting and Participants: Catchment areas of 143 wastewater treatment plants in 120 cities in 37 countries. Measurements: Parent substances (amphetamine, methamphetamine and MDMA) and the metabolites of cocaine (benzoylecgonine) and of Δ9-tetrahydrocannabinol (11-nor-9-carboxy-Δ9-tetrahydrocannabinol) were measured in wastewater using liquid chromatography–tandem mass spectrometry. Daily mass loads (mg/day) were normalized to catchment population (mg/1000 people/day) and converted to the number of combined doses consumed per day. Spatial differences were assessed world-wide, and temporal trends were discerned at European level by comparing 2011–13 drug loads versus 2014–17 loads. Findings: Benzoylecgonine was the stimulant metabolite detected at higher loads in southern and western Europe, and amphetamine, MDMA and methamphetamine in East and North–Central Europe. In other continents, methamphetamine showed the highest levels in the United States and Australia and benzoylecgonine in South America. During the reporting period, benzoylecgonine loads increased in general across Europe, amphetamine and methamphetamine levels fluctuated and MDMA underwent an intermittent upsurge. Conclusions: The analysis of wastewater to quantify drug loads provides near real-time drug use estimates that globally correspond to prevalence and seizure data. © 2019 The Authors. Addiction published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction

Design of a reliable process for bacterial antigen production requires understanding of and control over critical process parameters. Current methods for process design use extensive screening experiments for determining ranges of critical process parameters yet fail to give clear insights into how they influence antigen potency. To address this gap, we propose to apply constraint-based, genome-scale metabolic models to reduce the need of experimental screening for strain selection and to optimize strains based on model driven iterative Design–Build–Test–Learn (DBTL) cycles. Application of these systematic methods has not only increased the understanding of how metabolic network properties influence antigen potency, but also allows identification of novel critical process parameters that need to be controlled to achieve high process reliability.

Background Elevated expression of type I IFN response genes (IRGs) was previously described to be associated with a poor response to rituximab in rheumatoid arthritis (RA). In the present study, we aimed to validate this association and assessed the predictive performance upon combination of the predictive IRG gene set with clinical parameters. Materials and methods In two independent cohorts of 93 (Cohort I) and 133 (Cohort II) rituximab-starting RA patients, baseline peripheral blood expression of eight selected IRGs was determined, and averaged into an IFN score. Individual predictive performance of the IFN score and clinical parameters was assessed by logistic regression. A multivariate prediction model was developed using a forward stepwise selection procedure. Patients with a decrease in disease activity score (ΔDAS28)≥1.8 after 6 months of therapy were considered responders. Results A higher IFN score was observed in RTX non-responders compared to RTX responders from both Cohort I and II, but this difference was most pronounced in patients who did not use prednisone (PREDN-), as described before. Univariate analysis showed that baseline DAS28, IFN score and DMARD use were associated with non-response to rituximab, whereas positivity for IgM-RF and ACPA was associated with a good response to RTX. The multivariate model consisted of DAS28, IFN score and DMARD use, which showed an area under the curve (AUC) of 0.82. Validation of this multivariate model in Cohort II revealed a comparable AUC in PREDN- patients (0.78), but the AUC in PREDN+ patients was significantly lower (0.63), which seemed due to effect modification of the IFN score by prednisone. Multivariate analysis of the PREDN+ subgroup of Cohort II revealed a model containing DAS28 and positivity for IgM-RF and ACPA, which showed an AUC of 0.75. Conclusions The combination of predictive parameters, including IFN score, provided a promising model for the prediction of non-response to rituximab, with possibilities for optimisation via definition of the exact interfering effect of prednisone on IFN score.

Surface proteins are important for the fitness and virulence of the Gram-positive pathogen Streptococcus pneumoniae. They are crucial for interaction of the pathogen with its human host during infection. Therefore, the analysis of the pneumococcal surface proteome is an important task that requires powerful tools. In this study, two different methods, an optimized biotinylation approach and shaving with trypsin beads, were applied to study the pneumococcal surface proteome and to identify surface-exposed protein domains, respectively. The identification of nearly 95% of the predicted lipoproteins and 75% of the predicted sortase substrates reflects the high coverage of the two classical surface protein classes accomplished in this study. Furthermore, the biotinylation approach was applied to study the impact of an impaired lipoprotein maturation pathway on the cell envelope proteome and exoproteome. Loss of the lipoprotein diacylglyceryl transferase Lgt leads to striking changes in the lipoprotein distribution. Many lipoproteins disappear from the surface proteome and accumulate in the exoproteome. Further insights into lipoprotein processing in pneumococci are provided by immunoblot analyses of bacterial lysates and corresponding supernatant fractions. Taken together, the first comprehensive overview of the pneumococcal surface and exoproteome is presented, and a model for lipoprotein processing in S. pneumoniae is proposed.

Streptococcus pneumoniae (the pneumococcus) is an opportunistic human pathogen, which causes serious invasive disease such as pneumonia, bacteraemia and meningitis. The interaction of the bacteria with host receptors precedes the development of invasive disease. One host receptor implicated in pneumococcal adhesion to, invasion of and ultimately translocation of cell layers is the platelet-activating factor receptor (PAFR). PAFR is a G-protein coupled receptor which binds PAF, a potent phospholipid activator involved in many leucocyte functions, platelet aggregation and inflammation. PAFR has been proposed to bind S. pneumoniae and as such facilitate adhesion to, uptake by and transcytosis of endothelial cells leading to invasive disease. However, there is a shortage of biochemical data supporting direct interaction between PAFR and the bacteria, in addition to conflicting data on its role in development of invasive pneumococcal disease (IPD). In this review, we will discuss current literature on PAFR and S. pneumoniae and other pathogens,including data concerning human PAFR genetic variation related to IPD clinical aspects, to shed light on the importance of PAFR in IPD. Clarification of the role of this receptor in IPD development has the potential to enable the development of novel therapeutic strategies for treating pneumococcal disease by interfering with the PAFR.

Streptococcus pneumoniae is a Gram-positive bacterium which is a member of the normal human nasopharyngeal flora but can also cause serious disease such as pneumonia, bacteremia, and meningitis. Throughout its life cycle, S. pneumoniae is exposed to significant oxidative stress derived from endogenously produced hydrogen peroxide (H 2 O 2 ) and from the host through the oxidative burst. How S. pneumoniae , an aerotolerant anaerobic bacterium that lacks catalase, protects itself against hydrogen peroxide stress is still unclear. Bioinformatic analysis of its genome identified a hypothetical open reading frame belonging to the thiol-specific antioxidant (TlpA/TSA) family, located in an operon consisting of three open reading frames. For all four strains tested, deletion of the gene resulted in an approximately 10-fold reduction in survival when strains were exposed to external peroxide stress. However, no role for this gene in survival of internal superoxide stress was observed. Mutagenesis and complementation analysis demonstrated that all three genes are necessary and sufficient for protection against oxidative stress. Interestingly, in a competitive index mouse pneumonia model, deletion of the operon had no impact shortly after infection but was detrimental during the later stages of disease. Thus, we have identified a gene complex involved in the protection of S. pneumoniae against external oxidative stress, which plays an important role during invasive disease.

Streptococcus pneumoniae is a significant human pathogen which causes respiratory and serious invasive diseases. Mg 2+ is essential for life, and its concentration varies throughout the human body. Magnesium uptake plays an important role in the virulence of many bacterial pathogens. To study the Mg 2+ uptake of S. pneumoniae strain D39, a mutant was generated in SPD1383, a P-type ATPase with homology to the Salmonella Mg 2+ transporter MgtA, which has also been shown to be a Ca 2+ exporter in strain TIGR4. Under low-Ca 2+ conditions, mutation led to a growth defect in complex medium and the gene was nearly essential for growth under low-Mg 2+ conditions. Addition of Mg 2+ restored the normal growth of the mutant in all cases, but the addition of other divalent cations had no effect. Addition of Ca 2+ , Mn 2+ , and Zn 2+ in the presence of high Mg 2+ concentrations inhibited restoration of growth. The mutant was unable to proliferate in blood, which was also alleviated by the addition of Mg 2+ . The protein was located in the membrane and produced in various S. pneumoniae strains and pathogenic streptococcal species. Surprisingly, mutation of the gene led to an elevated toxicity for endothelial cells. This was caused by an increased amount of pneumolysin in the medium, mediated by elevated lysis of the mutant. Thus, in this study, we uncovered a role for SPD1383 in Mg 2+ uptake and hypothesize that the protein is a Mg 2+/ Ca 2+ antiporter. Furthermore, a disturbance in Mg 2+ homeostasis seems to promote lysis of S. pneumoniae .

The respiratory tract pathogen Streptococcus pneumoniae needs to adapt to the different levels of carbon dioxide (CO 2 ) it encounters during transmission, colonization, and infection. Since CO 2 is important for various cellular processes, factors that allow optimal CO 2 sequestering are likely to be important for pneumococcal growth and survival. In this study, we showed that the putative pneumococcal carbonic anhydrase (PCA) is essential for in vitro growth of S. pneumoniae under the CO 2 -poor conditions found in environmental ambient air. Enzymatic analysis showed that PCA catalyzes the reversible hydration of CO 2 to bicarbonate (HCO 3 − ), an essential step to prevent the cellular release of CO 2 . The addition of unsaturated fatty acids (UFAs) reversed the CO 2 -dependent in vitro growth inhibition of S. pneumoniae strains lacking the pca gene (Δ pca ), indicating that PCA-mediated CO 2 fixation is at least associated with HCO 3 − -dependent de novo biosynthesis of UFAs. Besides being necessary for growth in environmental ambient conditions, PCA-mediated CO 2 fixation pathways appear to be required for intracellular survival in host cells. This effect was especially pronounced during invasion of human brain microvascular endothelial cells (HBMEC) and uptake by murine J774 macrophage cells but not during interaction of S. pneumoniae with Detroit 562 pharyngeal epithelial cells. Finally, the highly conserved pca gene was found to be invariably present in both CO 2 -independent and naturally circulating CO 2 -dependent strains, suggesting a conserved essential role for PCA and PCA-mediated CO 2 fixation pathways for pneumococcal growth and survival.

Objective:Nonadherence to prescribed medication is associated with increased morbidity and mortality as well as the increased use of health services. The main objective of our study was to assess the incidence of prescription-filling and medication adherence in patients discharged from the emergency department (ED).Methods:This was a prospective, observational study carried out at a Canadian tertiary care ED with an annual census of 69 000. We enrolled a convenience sample of patients being discharged with a prescription. We queried a provincial prescription-dispensing database 2 weeks later to determine whether prescriptions had been filled. We used a standardized follow-up interview to assess adherence and whether or not the patient experienced an adverse drug-related event (ADRE) or an unplanned revisit to an ED or clinic.Results:Of the 301 patients who agreed to participate, follow-up was successful for 258 (85.7%). Fifty-one patients (19.8%, 95% confidence interval [CI] 15.4%–25.1%) failed to fill their discharge prescriptions and 104 (40.3%, 95% CI 34.5%–46.4%) did not adhere to 1 or more medications. Antibiotics were associated with a lower odds ratio (OR) of nonadherence (OR 0.21, 95% CI 0.08–0.52). There was a trend toward increasing nonadherence in patients who reported an ADRE (OR 1.84, 95% CI 0.98–3.48) or had 2 or more medications coprescribed (OR 1.71, 95% CI 0.95–3.09). There was also a trend toward a higher risk of a revisit to an ED or clinic in nonadherent patients (OR 1.75, 95% CI 0.94–3.25).Conclusion:Approximately 4 in 10 patients discharged from the ED did not adhere to his or her prescribed medication. Our results suggest that patients who are prescribed antibiotics are more likely to be adherent, and that further evaluation of the associations between nonadherence, ADREs, the coprescription of 2 or more medications and the use of health services is warranted.

Homeostasis of Zn 2+ and Mn 2+ is important for the physiology and virulence of the human pathogen Streptococcus pneumoniae . Here, transcriptome analysis was used to determine the response of S. pneumoniae D39 to a high concentration of Zn 2+ . Interestingly, virulence genes encoding the choline binding protein PcpA, the extracellular serine protease PrtA, and the Mn 2+ uptake system PsaBC(A) were strongly upregulated in the presence of Zn 2+ . Using random mutagenesis, a previously described Mn 2+ -responsive transcriptional repressor, PsaR, was found to mediate the observed Zn 2+ -dependent derepression. In addition, PsaR is also responsible for the Mn 2+ -dependent repression of these genes. Subsequently, we investigated how these opposite effects are mediated by the same regulator. In vitro binding of purified PsaR to the prtA , pcpA , and psaB promoters was stimulated by Mn 2+ , whereas Zn 2+ destroyed the interaction of PsaR with its target promoters. Mutational analysis of the pcpA promoter demonstrated the presence of a PsaR operator that mediates the transcriptional effects. In conclusion, PsaR is responsible for the counteracting effects of Mn 2+ and Zn 2+ on the expression of several virulence genes in S. pneumoniae , suggesting that the ratio of these metal ions exerts an important influence on pneumococcal pathogenesis.

In past years, several useful genetic tools have been developed to study the molecular biology of Streptococcus pneumoniae. In order to extend the existing spectrum of tools, advantage was taken of the toolbox originally developed for the closely related bacterium Lactococcus lactis, which was adapted for the manipulation of S. pneumoniae. The modified tools are as follows. (i) An improved nisin-inducible (over)expression system (NICE). The nisRK genes, encoding a two-component system essential for transcriptional activation in response to nisin, were integrated into the bgaA locus of S. pneumoniae D39. In this strain, D39nisRK, addition of nisin resulted in the overexpression of several genes placed under the control of the nisin-inducible promoter, while no detectable expression was observed in the absence of nisin. (ii) A lacZ reporter system. Using strain D39nisRK, which lacks endogenous β-galactosidase activity, the usefulness of the lacZ reporter vector pORI13 for the generation of chromosomal transcriptional fusions was demonstrated. In addition, the repA gene, necessary for the replication of pORI13, was introduced into the bgaA locus, thereby generating a background for plasmid-based promoter expression studies. (iii) A simplified chemically defined medium, which supports growth of all sequenced S. pneumoniae strains to a level comparable to that in complex medium. (iv) A system for the introduction of unmarked deletions and mutations into the chromosome, which is independent of the genotype of the target strain. Most of these systems were successfully applied in strains R6 and TIGR4 as well. In addition, the tools offer several improvements and advantages compared to existing ones. Thus, the molecular toolbox for S. pneumoniae has been successfully extended.

Spi/Ssa is a unique type three secretion system that functions exclusively when Salmonella enterica is inside eukaryotic cells. Expression of the Spi/Ssa system and its secreted effectors is dependent on SsrB/SpiR, a two-component regulatory system encoded in the SPI-2 pathogenicity island that also harbours the spi/ssa genes. Here we determine that the PhoP/PhoQ two-component system controls the intramacrophage expression of spi/ssa genes by regulating the SsrB/SpiR system. We establish that PhoP regulates transcription of the response regulator SsrB and demonstrate binding of the PhoP protein to the ssrB promoter both in vivo using chromatin immunoprecipitation in Salmonella-infected macrophages, and in vitro using purified PhoP protein. We show that PhoP controls the SpiR sensor post-transcriptionally and identify a region in the 5' untranslated region of the spiR message that is required for this effect. The demonstration that the PhoP/PhoQ system is directly involved in the regulation of the SPI-2 pathogenicity island highlights PhoP/PhoQ's central role in Salmonella virulence. We suggest that different regulatory systems convey distinct signals over time to produce the SsrB/SpiR system, which then modulates expression of the Spi/Ssa apparatus and secreted effectors.

We present a boy with blepharophimosis, ptosis, epicanthus inversus, microcephaly, mild mental retardation, and growth delay. Chromosomal analysis revealed a male karyotype with an interstitial deletion in the long arm of chromosome 3. DNA-analysis showed that the deletion is of maternal origin and encompasses the region between markers D3S1535 and D3S1593. The deletion contains not only the FOXL2 gene, but also the gene encoding ataxia-telangiectasia and Rad3-related protein (ATR). Mutations in FOXL2 have been shown to cause blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). ATR has been identified as a candidate gene for Seckel syndrome, an autosomal recessive syndrome that comprises growth retardation, microcephaly, and mental retardation. We hypothesize that our patient has a contiguous gene syndrome and that the non-BPES-associated abnormalities (microcephaly, mild mental retardation, and growth delay) are the result of the deletion of the maternal ATR gene. However, it has not yet been excluded that haploinsufficiency of some other gene in this region plays a role.

The only known niche of the human pathogen Helicobacter pylori is the gastric mucosa, where large fluctuations of pH occur, indicating that the bacterial response and resistance to acid are important for successful colonization. One of the few regulatory proteins in the H. pylori genome is a homologue of the ferric uptake regulator (Fur). In most bacteria, the main function of Fur is the regulation of iron homeostasis. However, in Salmonella enterica serovar Typhimurium, Fur also plays an important role in acid resistance. In this study, we determined the role of the H. pylori Fur homologue in acid resistance. Isogenic fur mutants were generated in three H. pylori strains (1061, 26695, and NCTC 11638). At pH 7 there was no difference between the growth rates of mutants and the parent strains. Under acidic conditions, growth of the fur mutants was severely impaired. No differences were observed between the survival of the fur mutant and parent strain 1061 after acid shock. Addition of extra iron or removal of iron from the growth medium did not improve the growth of the fur mutant at acidic pH. This indicates that the phenotype of the fur mutant at low pH was not due to increased iron sensitivity. Transcription of fur was repressed in response to low pH. From this we conclude that Fur is involved in the growth at acidic pH of H. pylori ; as such, it is the first regulatory protein implicated in the acid resistance of this important human pathogen.

Background. Availability of the essential nutrient iron is thought to vary greatly in the gastric mucosa, and thus the human gastric pathogen Helicobacter pylori requires regulatory responses to these environmental changes. Bacterial iron-responsive regulation is often mediated by Ferric Uptake Regulator (Fur) homologs, and in this study we have determined the role of H. pylori Fur in regulation of H. pylori iron uptake. Methods. Wild-type H. pylori and fur mutant derivatives were compared after growth in iron-restricted and iron-replete conditions. Iron-uptake was measured using 55Fe-labeled iron, whereas gene expression was monitored at the transcriptional level using Northern hybridization and lacZ reporter gene fusions. Results. Iron-uptake and total cellular iron content were approximately five-fold increased in the fur mutant compared with the wild-type strain, which indicated that in the fur mutant iron-uptake is not repressed by excess iron. A comprehensive screening of all H. pylori genes encoding putative iron-uptake proteins indicated that some of these H. pylori genes are constitutively expressed, while others are iron- and Fur-regulated. Conclusions. Iron uptake in H. pylori is in part differently regulated compared with other bacteria, since in H. pylori some iron-uptake systems are constitutively expressed. However, other iron uptake systems of H. pylori display the iron- and Fur-mediated repression that is common in bacteria. Taken together, this Fur-mediated modulation of iron-uptake capacity may be a specific adaptation to the conditions in the human stomach, where iron starvation and iron overload can be encountered in relatively short time intervals.

Streptococcus pneumoniae (pneumococcus) is a Gram-positive bacterium that causes serious invasive diseases, such as pneumonia, bacteremia, and meningitis, with high morbidity and mortality throughout the world. Before causing invasive disease, S. pneumoniae encounters cellular barriers, which are often composed of endothelial cells, like the alveolar-capillary barrier and the blood-brain barrier. S. pneumoniae adheres to endothelial cells and may invade them, which requires an efficient host response to the intracellular bacteria. The precise intracellular fate of S. pneumoniae during infection still remains a subject of debate. The proteasome-ubiquitin system is largely responsible for the degradation of misfolded, damaged, or no-longer-useful proteins. Recently, the role of the proteasome-ubiquitin system in the clearing of invading bacteria and viruses has been more closely studied. In this study, we show that inhibition of the proteasome-ubiquitin system leads to a marked increase in S. pneumoniae survival inside host cells. Immunofluorescence analysis showed that intracellular pneumococci colocalized with proteasome and ubiquitin in human endothelial cells in vitro. Confocal imaging analysis demonstrated that in the brains of mice intravenously infected with S. pneumoniae, the bacteria were inside endothelial cells, where they colocalized with proteasome and ubiquitin signals. In conclusion, our data indicate that a fully functional proteasome-ubiquitin system in endothelial cells is crucial for efficient killing of intracellular S. pneumoniae., IMPORTANCE Bacterial meningitis is a serious invasive disease with high morbidity and mortality. How bacteria traverse the blood-brain barrier in vivo and what mechanisms are employed by the host to prevent invasion are still unclear. Our data show that inhibition of the proteasome-ubiquitin system in vitro leads to a significant increase in S. pneumoniae survival inside brain endothelial cells. Confocal imaging analysis of brain tissue from mice intravenously infected with pneumococci demonstrated that the bacteria are inside brain microvascular endothelial cells, where they associate with the proteasome and ubiquitin. This is, as far as we know, the first report that demonstrates that Streptococcus pneumoniae invades endothelial cells of the blood-brain barrier in vivo. The host requires the proteasome-ubiquitin system for an efficient decimation of intracellular S. pneumoniae.

The Gram-positive bacterium Streptococcus pneumoniae is the main causative agent of bacterial meningitis. S. pneumoniae is thought to invade the central nervous system via the bloodstream by crossing the vascular endothelium of the blood-brain barrier. The exact mechanism by which pneumococci cross endothelial cell barriers before meningitis develops is unknown. Here, we investigated the role of PECAM-1/CD31, one of the major endothelial cell adhesion molecules, in S. pneumoniae adhesion to vascular endothelium of the blood-brain barrier. Mice were intravenously infected with pneumococci and sacrificed at various time points to represent stages preceding meningitis. Immunofluorescent analysis of brain tissue of infected mice showed that pneumococci colocalized with PECAM-1. In human brain microvascular endothelial cells (HBMEC) incubated with S. pneumoniae , we observed a clear colocalization between PECAM-1 and pneumococci. Blocking of PECAM-1 reduced the adhesion of S. pneumoniae to endothelial cells in vitro , implying that PECAM-1 is involved in pneumococcal adhesion to the cells. Furthermore, using endothelial cell protein lysates, we demonstrated that S. pneumoniae physically binds to PECAM-1. Moreover, both in vitro and in vivo PECAM-1 colocalizes with the S. pneumoniae adhesion receptor pIgR. Lastly, immunoprecipitation experiments revealed that PECAM-1 can physically interact with pIgR. In summary, we show for the first time that blood-borne S. pneumoniae colocalizes with PECAM-1 expressed by brain microvascular endothelium and that, in addition, they colocalize with pIgR. We hypothesize that this interaction plays a role in pneumococcal binding to the blood-brain barrier vasculature prior to invasion into the brain.

Background. Helicobacter pylori persists in the human stomach for decades. This requires an efficient adaptation of H. pylori to the gastric niche and involves the regulation of bacterial genes in response to environmental stress. Efficient molecular tools to identify regulated H. pylori genes are scarce, therefore we developed a genomic lacZ reporter gene fusion system in H. pylori to screen for stress-regulated genes. Materials and methods. The integration vector pBW was constructed and used to generate random genomic lacZ fusions in H. pylori. Two-hundred-and-fifty H. pylori transformants were selected from this library, replica-plated and screened for differential lacZ expression after exposure to two environmental stress conditions: increased temperature (42°C), and iron-limitation. Results. From a library of H. pylori transformants with random genomic transcriptional lacZ fusions, two stress-regulated H. pylori loci were identified. The transcription of a gene of unknown function (designated hsp12) was increased by incubation at 42°C. The transcription of a locus, consisting of the three fumarate reductase subunit genes (frdCAB) and the HP0190 gene from H. pylori strain 26695, was decreased under iron-limitation. Conclusions. This is the first time that a genomic transcriptional lacZ reporter gene H. pylori library has been used as a tool for the fast and efficient identification of environmental stress-regulated H. pylori genes.

To persist in the hostile acidic environment of the stomach, Helicobacter pylori must survive acid shock and grow at acidic pH. Of a library of 1250 random mutants screened for isolates unable to grow at low pH, 10 mutants were detected that were unable to grow at pH 4.8. However, all 10 mutants were resistant to acid shock. Four mutants had an insertion in genes of unknown function. One mutant was affected in lepA, an orthologue of a membrane GTPase. Three mutants were disrupted in loci involved in the transport of H(+) ions or other cations (FRaseI, czcA, and aldo-keto reductase). Two mutants were affected in loci that contribute to acid resistance in other microorganisms (uvrA and atpF'). Thus, at least 10 loci not related to urease are essential for the growth of H. pylori under acidic conditions and should be critical for lifelong infection by this pathogen.

Background. Self-regulation theory explains how patients' illness perceptions influence self-management behaviour (e. g. via adherence to treatment). Following these assumptions, we explored whether illness perceptions of ESRD-patients are related to mortality rates.Methods. Illness perceptions of 182 patients participating in the NECOSAD-2 study in the period between December 2004 and June 2005 were assessed. Cox proportional hazard models were used to estimate whether subsequent all-cause mortality could be attributed to illness perception dimensions.Results. One-third of the participants had died at the end of the follow-up. Mortality rates were higher among patients who believed that their treatment was less effective in controlling their disease (perceived treatment control; RR = 0.71, P = 0.028). This effect remained stable after adjusting for sociodemographic and clinical variables (RR = 0.65, P = 0.015).Conclusions. If we consider risk factors for mortality, we tend to rely on clinical parameters rather than on patients' representations of their illness. Nevertheless, results from the current exploration may suggest that addressing patients' personal beliefs regarding the effectiveness of treatment can provide a powerful tool for predicting and perhaps even enhancing survival.

The development of genetic competence in Bacillus subtilisis regulated by a complex signal transduction cascade, which leads to the synthesis of the competence transcription factor (CTF). Previous studies suggested that CTF is encoded by comK. ComK is required for the transcription of comK itself, as well as of the late competence genes encoding the DNA uptake machinery and of genes required for homologous recombination. Here, we used purified ComK to study its role in transcription and to determine the DNA recognition sequence for ComK. In vitro transcription from the comGpromoter, which depends on ComK in vivo, was observed on the addition of purified ComK together with Bacillus subtilis RNA polymerase, proving that ComK is CTF. To determine the DNA sequences involved in ComK recognition, footprinting analysis was performed with promoter fragments of the CTF-dependent genes: comC, comE, comF, comG, comK, and addAB. The ComK binding sites determined by DNase I protection experiments were unusually long, with average lengths of ∼65 bp, and displayed only weak sequence similarities. Hydroxy-radical footprinting, performed with the addABpromoter, revealed a unique arrangement of four short A/T-rich sequences. Gel retardation experiments indicated that four molecules of ComK bound the addAB promoter and the dyad symmetrical arrangement of the four A/T-rich sequences implied that ComK functions as a tetramer composed of two dimers each recognizing the motif AAAAN5TTTT. Comparable A/T-rich sequences were identified in all six DNase I footprints and could be used to predict ComK targets in the B. subtilis genome. On the basis of the variability in distance between the ComK–dimer binding sites, ComK-regulated promoters could be divided into three classes, demonstrating a remarkable flexibility in the binding of ComK.The pattern of hydroxy-radical protections suggested that ComK binds at one face of the DNA helix through the minor groove. This inference was strengthened by the observation that minor groove binding drugs inhibited the binding of ComK.

Aims: To perform wastewater analyses to assess spatial differences and temporal changes of illicit drug use in a large European population. Design: Analyses of raw wastewater over a 1-week period in 2012 and 2013. Setting and Participants: Catchment areas of wastewater treatment plants (WWTPs) across Europe, as follows: 2012: 25 WWTPs in 11 countries (23 cities, total population 11.50 million); 2013: 47 WWTPs in 21 countries (42 cities, total population 24.74 million). Measurements: Excretion products of five illicit drugs (cocaine, amphetamine, ecstasy, methamphetamine, cannabis) were quantified in wastewater samples using methods based on liquid chromatography coupled to mass spectrometry. Findings: Spatial differences were assessed and confirmed to vary greatly across European metropolitan areas. In general, results were in agreement with traditional surveillance data, where available. While temporal changes were substantial in individual cities and years (P ranging from insignificant to

Streptococcus pneumoniae (the pneumococcus) is an opportunistic human pathogen, which causes serious invasive disease such as pneumonia, bacteraemia and meningitis. The interaction of the bacteria with host receptors precedes the development of invasive disease. One host receptor implicated in pneumococcal adhesion to, invasion of and ultimately translocation of cell layers is the platelet-activating factor receptor (PAFR). PAFR is a G-protein coupled receptor which binds PAF, a potent phospholipid activator involved in many leucocyte functions, platelet aggregation and inflammation. PAFR has been proposed to bind S. pneumoniae and as such facilitate adhesion to, uptake by and transcytosis of endothelial cells leading to invasive disease. However, there is a shortage of biochemical data supporting direct interaction between PAFR and the bacteria, in addition to conflicting data on its role in development of invasive pneumococcal disease (IPD). In this review, we will discuss current literature on PAFR and S. pneumoniae and other pathogens,including data concerning human PAFR genetic variation related to IPD clinical aspects, to shed light on the importance of PAFR in IPD. Clarification of the role of this receptor in IPD development has the potential to enable the development of novel therapeutic strategies for treating pneumococcal disease by interfering with the PAFR.

Streptococcus pneumoniae can cause serious invasive pneumococcal 1 coccal disease (IPD) such as pneumonia, sepsis and meningitis and is the cause of major morbidity and mortality worldwide.…

We report four children (three sibs and one sporadic case) with congenital sutural cataract (opacity of the sutures of the crystalline lens), retinitis pigmentosa (leading to diminished visual acuity), microcephaly, and moderate to severe psychomotor retardation. The three sibs (two F and one M) were born to healthy, consanguineous Moroccan parents; the sporadic case is an 11-year-old Dutch girl who presented at the age of nine months with a small head circumference (third percentile) and sutural cataract. Psychomotor development was retarded in all cases, retinitis pigmentosa became evident during middle or late childhood. Congenital cataract has been described in association with a large number of various congenital abnormalities, such as renal, nervous system, skeletal, dermal and ocular (including retinal) defects. A computer-assisted literature search has not revealed similar cases to those presented here. The cases described here appear to have a previously undescribed combination of ophthalmological and cerebral abnormalities. The inheritance of the condition appears to be autosomal recessive as a brother and two sisters (offspring of normal consanguineous parents) are affected. The differential diagnosis is discussed.

A multidisciplinary working group has developed a practice guideline containing various recommendations on the responsible and efficient use of biologicals. These biologicals include both soluble immune-receptor proteins and monoclonal antibodies that are aimed at immune mediators, receptors or cells. They are produced by biotechnology. Biologicals are used to treat patients with immune-mediated inflammatory disorders (IMIDs) such as rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriatic arthritis, juvenile idiopathic arthritis (JIA), psoriasis, ulcerative colitis, Crohn's disease, uveitis and sarcoidosis. This article summarises the most important recommendations contained in the practice guideline. The practice guideline is intended for members of the medical profession in addition to patients, who are considering, or are already, using biologicals.

Where health care is concerned, the introduction of biologicals has greatly improved the treatment of chronic inflammatory disorders. This class of drugs is not only noticeably effective, but is also well tolerated. The cost effectiveness of these drugs means that there are limits to their use. This is the other face of success. The Dutch Ministry of Health, Welfare and Sport recently announced measures intended to limit the growth of costs while at the same time requiring that savings should be made on the current budget. Recently the Canadian Agency for Drugs and Technologies in Health (CADTH) produced a report on the effects and costs of biologicals for adults with rheumatoid arthritis (www.cadth.ca). Here we discuss their findings. Organizations for medical specialists should be societal aware and be able to deal with these drugs in a cost-conscious manner, including the development of dynamic guidelines and quality visitations.

It is becoming clearer that early treatment of rheumatoid arthritis (RA) is useful. Recently, Dutch investigators observed a difference between RA referred early (within 12 weeks) and RA referred later (after 12 weeks). Patients who were referred earlier had a better prognosis, less damage on radiology, and a higher chance of going into remission. We discuss that these data support the importance of the early recognition of RA. Educating the public and establishing early arthritis clinics could help to prevent delay for both patient and doctor.

We studied the effects of a single dose (100 mg orally) and repeated administration (100 mg o.d. for 7 days) of FK453, a novel adenosine-1 receptor antagonist, on renal sodium handling and blood pressure in eight patients with essential hypertension. Within 60 minutes after administration of FK453, sodium excretion increased threefold. This occurred in the absence of a change in renal hemodynamics, assessed from inulin and para-aminohippurate clearance, and was accompanied by increased fractional excretion of lithium, phosphate, and uric acid and by increased excretion of calcium and magnesium. Maximal free water clearance data showed an increase in maximal urine flow and distal delivery term and a decrease in the diluting segment reabsorption term. FK453 also decreased blood pressure and increased heart rate, but this did not occur until about 3 hours after ingestion, that is, when the natriuresis was already over. The natriuretic effect of FK453 was short-lasting, and continued use of FK453 was in fact accompanied by some net sodium retention. Blood pressure on the seventh day before FK453 treatment was not different from blood pressure before administration of the first dose of FK453. Again an acute natriuretic response followed, although less than after the first dose. Changes in intrarenal sodium handling parameters, blood pressure, and heart rate were similar to those seen after the first dose. The natriuretic and hypotensive effects of FK453 indicate that adenosine-1 receptor activity plays a role in the regulation of blood pressure and renal sodium handling in patients with essential hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

The workup of the emergency patient with a raised anion gap metabolic acidosis includes assessment of the components of “MUDPILES” (methanol; uremia; diabetic ketoacidosis; paraldehyde; isoniazid, iron or inborn errors of metabolism; lactic acid; ethylene glycol; salicylates). This approach is usually sufficient for the majority of cases in the emergency department; however, there are many other etiologies not addressed in this mnemonic. Organic acids including 5-oxoproline (pyroglutamic acid) are rare but important causes of anion gap metabolic acidosis. We present the case of a patient with profound metabolic acidosis with raised anion gap, due to pyroglutamic acid in the setting of malnutrition and chronic ingestion of acetaminophen.

Background Autoantigen-specific immunotherapy by mucosal tolerance induction via the intranasal route is an attractive therapeutic option for the treatment of autoimmune diseases, including rheumatoid arthritis (RA). Human cartilage glycoprotein-39 (HC gp-39) has been identified as a potential key autoantigen in RA. Based on animal studies, intranasal administration of the autoantigen is hypothesised to induce immunological tolerance in patients with RA and to ameliorate disease activity. In a phase I/IIA clinical trial in patients with RA, intranasal application of HC gp-39 was safe and well tolerated. Objective To investigate the efficacy of intranasally administered fully human, recombinant HC gp-39 (Org 39141) by a large clinical study. Methods In a 13-week multicentre, double-blind, randomised, placebo-controlled, parallel-group, dose-finding, proof-of-concept trial, patients with RA (disease-modifying antirheumatic drug (DMARD) naive or after washout of DMARD treatment) were randomised to receive either intranasal applications of placebo or HC gp-39 in doses of 30, 150, 300 or 600 µg, once a week. The primary efficacy variable was the 28 joint count Disease Activity Score (DAS28). Results During the treatment period the DAS28 decreased similarly for all treatment groups—including placebo—indicating lack of efficacy of intranasal HC gp-39 treatment in the current setting. Safety variables were similar for all study groups. Conclusion It was concluded that with the treatment protocol used (dose levels and frequency of dosing), intranasal treatment with Org 39141 was safe but did not result in more clinical improvement than in placebo-treated patients.

Polymyalgia rheumatica (PMR) is a chronic inflammatory rheumatic disorder, which predominantly occurs in patients aged 50 years and older. It can be difficult to differentiate PMR from other diseases, for general practitioners as well as for rheumatologists. Arteritis temporalis should not be overlooked in these patients, since missing this diagnosis may lead to irreversible visual loss. The majority of patients in whom PMR is suspected respond well to treatment with prednisone 15 mg per day. Methotrexate is an attractive alternative in patients who do not respond to prednisone or who experience side-effects due to prednisone.

The authors describe a female patient with unilateral malformations of skin, cerebrum and eye. The symptoms consisted of local skin hypoplasia, skin appendages and lipomatous tissue; cysts, hypoplasia and lipomatosis of the brain; and ocular malformations. In the newborn period the symptoms led to the diagnosis of oculocerebrocutaneous (OCC) syndrome. In the first year of life the clinical course deteriorated and the psychomotor development was progressively retarded. Evaluation at the age of 15 months prompted the authors to change the diagnosis to encephalocraniocutaneous lipomatosis (ECCL). The differential diagnosis of ECCL and OCC syndromes is discussed and a possible common pathogenetic pathway of these two rare disorders is proposed.

Influenza vaccination is important in order to limit flu-related morbidity and mortality. This especially applies to immunocompromised patients, such as HIV-infected individuals and patients on immunosuppressive treatment, who have an increased risk for developing complications from influenza. Influenza vaccine can be safely administered to all immunocompromised patients, but the effectiveness of the vaccine may be reduced. Uncertainty still exists concerning the value of an increased vaccine dose or booster vaccination: if positive effects exist, they are probably only of marginal clinical importance. Despite clearly reduced effectiveness, vaccination of immunocompromised patients is still valuable in view of the high absolute risk of infection and complications.

Some accidents due to methotrexate (MTX) have been reported to the Dutch Health Authorities. However, for modern, intensive treatment of patients with rheumatoid arthritis and various other chronic autoimmune diseases, we cannot do without the drug, because of its broad applications and unique properties. Fortunately, it is a safe drug, if used by doctors experienced in using it and who follow MTX guidelines.

Thanks to progress in genetic research, various genetic factors involved in the pathogenesis of rheumatoid arthritis (RA) have been discovered.There is a difference in genetic risk factors for RA in patients with or without antibodies against cyclic citrullinated peptides. This has led to the idea that RA is not a single disease, but a heterogeneous syndrome. Currently known genetic risk factors are not suitable for prediction of the risk of individual patients developing RA, nor for early diagnosis, nor for determining the therapeutic strategy.Research into functional defects of gene products of genetic risk variants will increase the understanding of the pathogenesis of RA. This could lead to insights for the development of new specific medication.In the future, research into genetic factors correlated with the severity of RA disease progression or with the effect of antirheumatic medication is expected to yield interesting and hopefully clinically relevant results.

In a recent equivalence clinical trial, prednisolone was reported to be equally effective as naproxen in the treatment of acute gout arthritis. The authors stated that the study provides a strong argument to consider prednisolone as first-line therapy for acute gout. For this double-blind placebo-controlled trial patients with the clinical diagnosis of acute gout (n = 381) were referred from primary care centers, but only those patients with crystal-proven gout and absence of contraindications for the trial medication were included (n = 120). They were randomly assigned to oral treatment for 5 days with either once-daily prednisolone 35 mg or twice-daily naproxen 500 mg and respective placebos. Points of discussion are whether there was indeed equivalence in effect, whether equivalence in adverse effects of the two treatments can be determined from the trial or from literature, and whether the results may be generalised. Although in selected acute gout patients prednisolone may be a good choice, available data still are too scarce to conclude that oral prednisolone is the first-line therapy for gout arthritis.

We found previously that calcium entry blockade with nitrendipine enhanced the natriuretic effect of high-dose atrial natriuretic peptide (ANP). It is unknown whether nitrendipine also influences the effect of physiological changes in ANP. We therefore studied the effect of nitrendipine on cumulative sodium balance during changes in sodium intake as well as on natriuresis after low-dosage ANP infusion during low and high sodium (LS and HS, respectively) intake. In eight healthy volunteers, sodium balance was recorded after the switch from LS (20 mmol/day) to HS (300 mmol/day) diet. Cumulative sodium balance was equal with (441 +/- 45 mmol) or without (458 +/- 45 mmol) nitrendipine treatment. The body weight curves were also fully congruent. ANP (0.005 micrograms/kg/min for 3 h) was administered during maximal water diuresis on both sodium intake levels with and without nitrendipine. Infusion of ANP increased sodium excretion (mumol/min) from 30 +/- 7 to 81 +/- 12 (LS) and from 316 +/- 27 to 469 +/- 46 (HS). During nitrendipine, similar increments (from 36 +/- 7 to 98 +/- 24 mumol/min, HS) were found. ANP had no effect on inulin clearance and fractional excretion of lithium, but consistently depressed diluting segment reabsorption. This pattern was also similar during nitrendipine. Apparently, under the conditions of this study in normal subjects, nitrendipine has no effect on sodium balance. ANP, in physiological concentrations, increases natriuresis mainly by depressing sodium reabsorption in the distal nephron, an effect not enhanced by nitrendipine.

Evidence is accumulating that insulin is a hypertensive factor in humans. The involved mechanism may be its sodium-retaining effect. We examined whether insulin causes sodium retention through a direct action on the kidney, as is generally assumed, or indirectly through hypokalemia. Insulin was infused (euglycemic clamp technique) with and without potassium infusion to prevent hypokalemia in six healthy subjects. Without potassium infusion, insulin caused a marked decrease in plasma potassium (-0.75 mmol/L), and decreased urinary sodium and potassium excretions by, approximately 38% and 65%, respectively. Simultaneous potassium infusion largely prevented the decrease in plasma potassium, as well as the decrease in urinary sodium and potassium excretions. These data suggest that the acute antinatriuretic effect of insulin may be largely mediated in an indirect way, ie, through hypokalemia.