Mutational analysis of fructose-1,6-bisphosphate aldolase of Neisseria meningitidis serogroup B

Fructose-1,6-bisphosphate aldolase (FBA) is a classical cytoplasmic glycolytic enzyme which, despite lacking a predicted signal peptide, has been demonstrated to be expressed and transported to the surface of numerous Gram-positive bacteria and shown to interact with host molecules and perform non-glycolytic biological functions. Genome-based studies have also demonstrated that the glycolytic pathway appears to be non-functional in the meningococcus due to absence of phosphofructokinase, one of the important enzymes in this pathway. This study aimed to investigate whether the FBA, a so-called housekeeping enzyme, is required for maximal in vitro growth of N. meningitidis. An FBA knock-out mutant was created in N. meningitidis using an inverse polymerase chain reaction (PCR) and allelic exchange methodology. Phenotypic analysis of FBA-deficient mutant strains such as growth profiling experiments showed that the FBA-deficient mutant grew at the same rate (in broth culture and on solid media) as the wild-type strain, suggesting that FBA is not required for optimal growth of N. meningitidis under the in vitro conditions tested. No differences in either colony or bacterial cell morphology (using light microscopy) were observed. In summary, despite being a central enzyme in the glycolytic cycle, FBA is not required for in vitro growth of N. meningitidis.Key words: Neisseria meningitidis, aldolase, mutagenesis, growth kinetics, glycolytic cycle

A Project Based Approach to Statistics and Data Science

In an increasingly data-driven world, facility with statistics is more important than ever for our students. At institutions without a statistician, it often falls to the mathematics faculty to teach statistics courses. This paper presents a model that a mathematician asked to teach statistics can follow. This model entails connecting with faculty from numerous departments on campus to develop a list of topics, building a repository of real-world datasets from these faculty, and creating projects where students interface with these datasets to write lab reports aimed at consumers of statistics in other disciplines. The end result is students who are well prepared for interdisciplinary research, who are accustomed to coping with the idiosyncrasies of real data, and who have sharpened their technical writing and speaking skills

The role of glyceraldehyde 3-phosphate dehydrogenase (GapA-1) in Neisseria meningitidis adherence to human cells

BackgroundGlyceraldehyde 3-phosphate dehydrogenases (GAPDHs) are cytoplasmic glycolytic enzymes, which although lacking identifiable secretion signals, have also been found localized to the surface of several bacteria (and some eukaryotic organisms); where in some cases they have been shown to contribute to the colonization and invasion of host tissues. Neisseria meningitidis is an obligate human nasopharyngeal commensal which can cause life-threatening infections including septicaemia and meningitis. N. meningitidis has two genes, gapA-1 and gapA-2, encoding GAPDH enzymes. GapA-1 has previously been shown to be up-regulated on bacterial contact with host epithelial cells and is accessible to antibodies on the surface of capsule-permeabilized meningococcal cells. The aims of this study were: 1) to determine whether GapA-1 was expressed across different strains of N. meningitidis; 2) to determine whether GapA-1 surface accessibility to antibodies was dependant on the presence of capsule; 3) to determine whether GapA-1 can influence the interaction of meningococci and host cells, particularly in the key stages of adhesion and invasion.ResultsIn this study, expression of GapA-1 was shown to be well conserved across diverse isolates of Neisseria species. Flow cytometry confirmed that GapA-1 could be detected on the cell surface, but only in a siaD-knockout (capsule-deficient) background, suggesting that GapA-1 is inaccessible to antibody in in vitro-grown encapsulated meningococci. The role of GapA-1 in meningococcal pathogenesis was addressed by mutational analysis and functional complementation. Loss of GapA-1 did not affect the growth of the bacterium in vitro. However, a GapA-1 deficient mutant showed a significant reduction in adhesion to human epithelial and endothelial cells compared to the wild-type and complemented mutant. A similar reduction in adhesion levels was also apparent between a siaD-deficient meningococcal strain and an isogenic siaD gapA-1 double mutant.ConclusionsOur data demonstrates that meningococcal GapA-1 is a constitutively-expressed, highly-conserved surface-exposed protein which is antibody-accessible only in the absence of capsule. Mutation of GapA-1 does not affect the in vitro growth rate of N. meningitidis, but significantly affects the ability of the organism to adhere to human epithelial and endothelial cells in a capsule-independent process suggesting a role in the pathogenesis of meningococcal infection

Fructose-1,6-bisphosphate aldolase of Neisseria meningitidis binds human plasminogen via its C-terminal lysine residue

Neisseria meningitidis is a leading cause of fatal sepsis and meningitis worldwide. As for commensal species of human neisseriae, N. meningitidis inhabits the human nasopharynx and asymptomatic colonization is ubiquitous. Only rarely does the organism invade and survive in the bloodstream leading to disease. Moonlighting proteins perform two or more autonomous, often dissimilar, functions using a single polypeptide chain. They have been increasingly reported on the surface of both prokaryotic and eukaryotic organisms and shown to interact with a variety of host ligands. In some organisms moonlighting proteins perform virulence-related functions, and they may play a role in the pathogenesis of N. meningitidis. Fructose-1,6- bisphosphate aldolase (FBA) was previously shown to be surface-exposed in meningococci and involved in adhesion to host cells. In this study, FBA was shown to be present on the surface of both pathogenic and commensal neisseriae, and surface localization and anchoring was demonstrated to be independent of aldolase activity. Importantly, meningococcal FBA was found to bind to human glu- plasminogen in a dose-dependent manner. Site-directed mutagenesis demonstrated that the C-terminal lysine residue of FBA was required for this interaction, whereas # subterminal lysine residues were not involved

Climate change promotes parasitism in a coral symbiosis.

Coastal oceans are increasingly eutrophic, warm and acidic through the addition of anthropogenic nitrogen and carbon, respectively. Among the most sensitive taxa to these changes are scleractinian corals, which engineer the most biodiverse ecosystems on Earth. Corals' sensitivity is a consequence of their evolutionary investment in symbiosis with the dinoflagellate alga, Symbiodinium. Together, the coral holobiont has dominated oligotrophic tropical marine habitats. However, warming destabilizes this association and reduces coral fitness. It has been theorized that, when reefs become warm and eutrophic, mutualistic Symbiodinium sequester more resources for their own growth, thus parasitizing their hosts of nutrition. Here, we tested the hypothesis that sub-bleaching temperature and excess nitrogen promotes symbiont parasitism by measuring respiration (costs) and the assimilation and translocation of both carbon (energy) and nitrogen (growth; both benefits) within Orbicella faveolata hosting one of two Symbiodinium phylotypes using a dual stable isotope tracer incubation at ambient (26 °C) and sub-bleaching (31 °C) temperatures under elevated nitrate. Warming to 31 °C reduced holobiont net primary productivity (NPP) by 60% due to increased respiration which decreased host %carbon by 15% with no apparent cost to the symbiont. Concurrently, Symbiodinium carbon and nitrogen assimilation increased by 14 and 32%, respectively while increasing their mitotic index by 15%, whereas hosts did not gain a proportional increase in translocated photosynthates. We conclude that the disparity in benefits and costs to both partners is evidence of symbiont parasitism in the coral symbiosis and has major implications for the resilience of coral reefs under threat of global change

Fructose-1,6-bisphosphate aldolase of Neisseria meningitidis binds human plasminogen via its C-terminal lysine residue

Neisseria meningitidis is a leading cause of fatal sepsis and meningitis worldwide. As for commensal species of human neisseriae, N. meningitidis inhabits the human nasopharynx and asymptomatic colonization is ubiquitous. Only rarely does the organism invade and survive in the bloodstream leading to disease. Moonlighting proteins perform two or more autonomous, often dissimilar, functions using a single polypeptide chain. They have been increasingly reported on the surface of both prokaryotic and eukaryotic organisms and shown to interact with a variety of host ligands. In some organisms moonlighting proteins perform virulence-related functions, and they may play a role in the pathogenesis of N. meningitidis. Fructose-1,6- bisphosphate aldolase (FBA) was previously shown to be surface-exposed in meningococci and involved in adhesion to host cells. In this study, FBA was shown to be present on the surface of both pathogenic and commensal neisseriae, and surface localization and anchoring was demonstrated to be independent of aldolase activity. Importantly, meningococcal FBA was found to bind to human glu- plasminogen in a dose-dependent manner. Site-directed mutagenesis demonstrated that the C-terminal lysine residue of FBA was required for this interaction, whereas # subterminal lysine residues were not involved

National business regulations and city entrepreneurship in Europe: a multilevel nested analysis

This article provides and tests a theoretical framework with a multilevel (country–city) nested model to analyze the relationship between national business regulations (NBRs) and city level entrepreneurship. While public interest theory predicts a positive relationship between NBR and city level entrepreneurship, public choice theory predicts the opposite, a negative relationship. Based on multilevel analysis for a matched country–city panel of 228 cities across 20 European countries for the years 2004 to 2009, the empirical evidence from panel data estimation explains how changes in NBRs influence changes in city level entrepreneurial activity over time

Draft genome sequences of the type strains of Shigella flexneri held at Public Health England: comparison of classical phenotypic and novel molecular assays with whole genome sequence.

BACKGROUND: Public Health England (PHE) holds a collection of Shigella flexneri Type strains isolated between 1949 and 1972 representing 15 established serotypes and one provisional type, E1037. In this study, the genomes of all 16 PHE Type strains were sequenced using the Illumina HiSeq platform. The relationship between core genome phylogeny and serotype was examined. RESULTS: The most common target gene for the detection of Shigella species in clinical PCR assays, ipaH, was detected in all genomes. The type-specific target genes were correctly identified in each genome sequence. In contrast to the S. flexneri in serotype 5 strain described by Sun et al. (2012), the two PHE serotype 5 Type strains possessed an additional oac gene and were differentiated by the presence (serotype 5b) or absence (serotype 5a) of gtrX. The somatic antigen structure and phylogenetic relationship were broadly congruent for strains expressing serotype specific antigens III, IV and V, but not for those expressing I and II. The whole genome phylogenies of the 15 isolates sequenced showed that the serotype 6 Type Strain was phylogenetically distinct from the other S. flexneri serotypes sequenced. The provisional serotype E1037 fell within the serotype 4 clade, being most closely related to the Serotype 4a Type Strain. CONCLUSIONS: The S. flexneri genome sequences were used to evaluate phylogenetic relationships between Type strains and validate genotypic and phenotypic assays. The analysis confirmed that the PHE S. flexneri Type strains are phenotypically and genotypically distinct. Novel variants will continue to be added to this archive

Multi-agent simulation of hydraulic transient equations in pressurized systems

Computational modeling pervades virtually every industrial process. By using numerical representations of the behavior of elements that constitute a system it is possible to obtain efficient and safe designs. Moreover, system operation can be better defined by using such models, thus enabling greater reliability and control. In this paper the use of agents to solve the equations describing fast transients in water networks is investigated. As the simulation of hydraulic transients in pressurized systems is a naturally distributed problem, the authors argue that a multi-agent based system is very suitable for the solution of this complex engineering phenomenon. A hybrid solution is built by deploying agents to work with sets of equations describing hydraulic transient behavior in pipeline systems. The details necessary to assemble a complete and lubricated machine to model the complex phenomenon of hydraulic transients in pressurized systems are described. This research develops a platform that constitutes an efficient and versatile tool of great interest for water supply managers when analyzing water hammer effects in their networks.Izquierdo Sebastián, J.; Montalvo Arango, I.; Pérez García, R.; Ayala Cabrera, D. (2015). Multi-agent simulation of hydraulic transient equations in pressurized systems. Journal of Computing in Civil Engineering. 04015071:1-14. doi:10.1061/(ASCE)CP.1943-5487.0000549S1140401507