The second messenger c-di-AMP inhibits the osmolyte uptake system OpuC in Staphylococcus aureus

Staphylococcus aureus is an important opportunistic human pathogen that is highly resistant to osmotic stresses. In order to survive an increase in osmolarity, bacteria immediately take up potassium and small organic compounds, also referred to as compatible solutes. The second messenger c-di-AMP binds to several receptor proteins, most of which are involved in ion and potassium uptake, that help bacteria cope with osmotic stress. In this study, we identified OpuCA, the ATPase component of an uptake system for the compatible solute carnitine, as a cdi-AMP target protein in S. aureus and found that a strain overproducing c-di-AMP showed reduced carnitine uptake. The CBS domains of OpuCA bound to c-di-AMP, and a crystal structure revealed a putative binding pocket for c-di-AMP in the cleft between the two CBS domains. Thus, c-di-AMP is involved in regulating both branches of osmoprotection (potassium uptake and compatible solute uptake), suggesting that c-di-AMP is a general osmotic stress regulato

Pathological macromolecular crystallographic data affected by twinning, partial-disorder and exhibiting multiple lattices for testing of data processing and refinement tools

Twinning is a crystal growth anomaly, which has posed a challenge in macromolecular crystallography (MX) since the earliest days. Many approaches have been used to treat twinned data in order to extract structural information. However, in most cases it is usually simpler to rescreen for new crystallization conditions that yield an untwinned crystal form or, if possible, collect data from non-twinned parts of the crystal. Here, we report 11 structures of engineered variants of the E. coli enzyme N-acetyl-neuraminic lyase which, despite twinning and incommensurate modulation, have been successfully indexed, solved and deposited. These structures span a resolution range of 1.45–2.30 Å, which is unusually high for datasets presenting such lattice disorders in MX and therefore these data provide an excellent test set for improving and challenging MX data processing programs

Fecal Calprotectin Excretion in Preterm Infants during the Neonatal Period

Fecal calprotectin has been proposed as a non-invasive marker of intestinal inflammation in inflammatory bowel disease in adults and children. Fecal calprotectin levels have been reported to be much higher in both healthy full-term and preterm infants than in children and adults.To determine the time course of fecal calprotectin (f-calprotectin) excretion in preterm infants from birth until hospital discharge and to identify factors influencing f-calprotectin levels in the first weeks of life, including bacterial establishment in the gut.F-calprotectin was determined using an ELISA assay in 147 samples obtained prospectively from 47 preterm infants (gestational age, and birth-weight interquartiles 27–29 weeks, and 880–1320 g, respectively) at birth, and at 2-week intervals until hospital discharge. (p = 0.047).During the first weeks of life, the high f-calprotectin values observed in preterm infants could be linked to the gut bacterial establishment

Clostridia in Premature Neonates' Gut: Incidence, Antibiotic Susceptibility, and Perinatal Determinants Influencing Colonization

Although premature neonates (PN) gut microbiota has been studied, data about gut clostridial colonization in PN are scarce. Few studies have reported clostridia colonization in PN whereas Bacteroides and bifidobacteria have been seldom isolated. Such aberrant gut microbiota has been suggested to be a risk factor for the development of intestinal infections. Besides, PN are often treated by broad spectrum antibiotics, but little is known about how antibiotics can influence clostridial colonization based on their susceptibility patterns. The aim of this study was to report the distribution of Clostridium species isolated in feces from PN and to determine their antimicrobial susceptibility patterns. Additionally, clostridial colonization perinatal determinants were analyzed.Of the 76 PN followed until hospital discharge in three French neonatal intensive care units (NICUs), 79% were colonized by clostridia. Clostridium sp. colonization, with a high diversity of species, increased throughout the hospitalization. Antibiotic courses had no effect on the clostridial colonization incidence although strains were found susceptible (except C. difficile) to anti-anaerobe molecules tested. However, levels of colonization were decreased by either antenatal or neonatal (during more than 10 days) antibiotic courses (p = 0.006 and p = 0.001, respectively). Besides, incidence of colonization was depending on the NICU (p = 0.048).This study shows that clostridia are part of the PN gut microbiota. It provides for the first time information on the status of clostridia antimicrobial susceptibility in PN showing that strains were susceptible to most antibiotic molecules. Thus, the high prevalence of this genus is not linked to a high degree of resistance to antimicrobial agents or to the use of antibiotics in NICUs. The main perinatal determinant influencing PN clostridia colonization appears to be the NICU environment

Faecal calprotectin concentrations in apparently healthy children aged 0-12 years in urban Kampala, Uganda: a community-based survey

<p>Abstract</p> <p>Background</p> <p>Calprotectin is a calcium and zinc binding protein, abundant in neutrophils and is extremely stable in faeces. Faecal calprotectin is used as a non-specific marker for gastrointestinal inflammation. It has a good diagnostic precision to distinguish between irritable bowel syndrome and inflammatory bowel disease. Studies have established normal concentrations in healthy children; all these studies have been performed in high-income countries. The objective of this study was to determine the concentration of faecal calprotectin in apparently healthy children aged 0-12 years in urban Kampala, Uganda.</p> <p>Method</p> <p>We tested 302 apparently healthy children aged, age 0-12 years (162 female, 140 male) in urban Kampala, Uganda. The children were recruited consecutively by door-to-door visits. Faecal calprotectin was analyzed using a quantitative enzyme-linked immunosorbent assay. Faeces were also tested for <it>Helicobacter pylori (H. pylori) </it>antigen, for growth of enteropathogens and microscopy was performed to assess protozoa and helminths. A short standardized interview with socio-demographic information and medical history was obtained to assess health status of the children.</p> <p>Results</p> <p>In the different age groups the median faecal calprotectin concentrations were 249 mg/kg in 0 < 1 year (n = 54), 75 mg/kg in 1 < 4 years (n = 89) and 28 mg/kg in 4 < 12 years (n = 159). There was no significant difference in faecal calprotectin concentrations and education of female caretaker, wealth index, gender, habits of using mosquito nets, being colonized with <it>H. pylori </it>or having other pathogens in the stool.</p> <p>Conclusion</p> <p>Concentrations of faecal calprotectin among healthy children, living in urban Ugandan, a low-income country, are comparable to those in healthy children living in high-income countries. In children older than 4 years, the faecal calprotectin concentration is low. In healthy infants faecal calprotectin is high. The suggested cut-off concentrations in the literature can be used in apparently healthy Ugandan children. This finding also shows that healthy children living under poor circumstances do not have a constant inflammation in the gut. We see an opportunity to use this relatively inexpensive test for further understanding and investigations of gut inflammation in children living in low-income countries.</p

Branched Chain Fatty Acids Reduce the Incidence of Necrotizing Enterocolitis and Alter Gastrointestinal Microbial Ecology in a Neonatal Rat Model

Branched chain fatty acids (BCFA) are found in the normal term human newborn's gut, deposited as major components of vernix caseosa ingested during late fetal life. We tested the hypothesis that premature infants' lack of exposure to gastrointestinal (GI) BCFA is associated with their microbiota and risk for necrotizing enterocolitis (NEC) using a neonatal rat model.Pups were collected one day before scheduled birth. The pups were exposed to asphyxia and cold stress to induce NEC. Pups were assigned to one of three experimental treatments. DF (dam-fed); Control, hand-fed rat milk substitute; BCFA, hand-fed rat milk substitute with 20%w/w BCFA. Total fat was equivalent (11%wt) for both the Control and BCFA groups. Cecal microbiota were characterized by 16S rRNA gene pyrosequencing, and intestinal injury, ileal cytokine and mucin gene expression, interleukin-10 (IL-10) peptide immunohistochemistry, and BCFA uptake in ileum phospholipids, serum and liver were assessed.NEC incidence was reduced by over 50% in the BCFA group compared to the Control group as assessed in ileal tissue; microbiota differed among all groups. BCFA-fed pups harbored greater levels of BCFA-associated Bacillus subtilis and Pseudomonas aeruginosa compared to Controls. Bacillus subtilis levels were five-fold greater in healthy pups compared to pups with NEC. BCFA were selectively incorporated into ileal phospholipids, serum and liver tissue. IL-10 expression increased three-fold in the BCFA group versus Controls and no other inflammatory or mucosal mRNA markers changed.At constant dietary fat level, BCFA reduce NEC incidence and alter microbiota composition. BCFA are also incorporated into pup ileum where they are associated with enhanced IL-10 and may exert other specific effects

Production, quality control, stability, and potency of cGMP-produced Plasmodium falciparum RH5.1 protein vaccine expressed in Drosophila S2 cells

Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is a leading asexual blood-stage vaccine candidate for malaria. In preparation for clinical trials, a full-length PfRH5 protein vaccine called “RH5.1” was produced as a soluble product under cGMP using the ExpreS2 platform (based on a Drosophila melanogaster S2 stable cell line system). Following development of a highproducing monoclonal S2 cell line, a master cell bank was produced prior to the cGMP campaign. Culture supernatants were processed using C-tag affinity chromatography followed by size exclusion chromatography and virus-reduction filtration. The overall process yielded >400 mg highly pure RH5.1 protein. QC testing showed the MCB and the RH5.1 product met all specified acceptance criteria including those for sterility, purity, and identity. The RH5.1 vaccine product was stored at −80 °C and is stable for over 18 months. Characterization of the protein following formulation in the adjuvant system AS01B showed that RH5.1 is stable in the timeframe needed for clinical vaccine administration, and that there was no discernible impact on the liposomal formulation of AS01B following addition of RH5.1. Subsequent immunization of mice confirmed the RH5.1/AS01B vaccine was immunogenic and could induce functional growth inhibitory antibodies against blood-stage P. falciparum in vitro. The RH5.1/AS01B was judged suitable for use in humans and has since progressed to phase I/IIa clinical trial. Our data support the future use of the Drosophila S2 cell and C-tag platform technologies to enable cGMP-compliant biomanufacture of other novel and “difficult-to-express” recombinant protein-based vaccines

Complex structure and biochemical characterization of the Staphylococcus aureus cyclic diadenylate monophosphate (c-di-AMP)-binding protein PstA, the founding member of a new signal transduction protein family

Signaling nucleotides are integral parts of signal transduction systems allowing bacteria to cope with and rapidly respond to changes in the environment. The Staphylococcus aureus PII-like signal transduction protein PstA was recently identified as a cyclic diadenylate monophosphate (c-di-AMP)-binding protein. Here, we present the crystal structures of the apo- and c-di-AMP-bound PstA protein, which is trimeric in solution as well as in the crystals. The structures combined with detailed bioinformatics analysis revealed that the protein belongs to a new family of proteins with a similar core fold but with distinct features to classical PII proteins, which usually function in nitrogen metabolism pathways in bacteria. The complex structure revealed three identical c-di-AMP-binding sites per trimer with each binding site at a monomer-monomer interface. Although distinctly different from other cyclic-di-nucleotide-binding sites, as the half-binding sites are not symmetrical, the complex structure also highlighted common features for c-di-AMP-binding sites. A comparison between the apo and complex structures revealed a series of conformational changes that result in the ordering of two anti-parallel β-strands that protrude from each monomer and allowed us to propose a mechanism on how the PstA protein functions as a signaling transduction protein

A GPU implementation of the ASP computation

General Purpose Graphical Processing Units (GPUs) are affordable multi-core platforms, providing access to large number of cores, but at the price of a complex architecture with non-trivial synchronization and communication costs. This paper presents the design and implementation of a conflict-driven ASP solver, that is capable of exploiting the parallelism offered by GPUs. The proposed system builds on the notion of ASP computation, that avoids the generation of unfounded sets, enhanced by conflict analysis and learning. The proposed system uses the CPU exclusively for input and output, in order to reduce the negative impact of the expensive data transfers between the CPU and the GPU. All the solving components, i.e., the management of nogoods, the search strategy, backjumping, the search heuristics, conflict analysis and learning, and unit propagation, are performed on the GPU, by exploiting Single Instruction Multiple Threads (SIMT) parallelism. The preliminary experimental results confirm the feasibility and scalability of the approach, and the potential to enhance performance of ASP solvers