Characterization and Redesign of the AFIT Multielectrode Array

Since 1978, faculty and graduate students from the Air Force Institute of Technology (AFIT) have been working on an implantable circuit array that can record visual signals from the brain or stimulate the brain. In the current design the circuit is a 16 x 16 array of pads, each pad being 160 x 160 micrometers with 250 micrometers spacing with an \u27L\u27 shaped reference pad used as a ground reference. The array is multiplexed so that only one I-O line is required to access all 256 pads. This research was twofold: first to analyze the existing circuit and identify sources for noise and then to redesign the circuit with the intent of reducing noise and power consumption. As a result of the research, a new 16 x 17 array has been developed. The new design incorporates new demultiplexing and synchronizing circuitry. The new circuitry exhibits lower noise and consumes much less power. The \u27L\u27 shaped reference pad has been removed-instead each pad and the pad to its right are sampled concurrent1y-one as the sample and one as a reference

Evolutionary pathway to increased virulence and epidemic group A Streptococcus disease derived from 3,615 genome sequences.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access.We sequenced the genomes of 3,615 strains of serotype Emm protein 1 (M1) group A Streptococcus to unravel the nature and timing of molecular events contributing to the emergence, dissemination, and genetic diversification of an unusually virulent clone that now causes epidemic human infections worldwide. We discovered that the contemporary epidemic clone emerged in stepwise fashion from a precursor cell that first contained the phage encoding an extracellular DNase virulence factor (streptococcal DNase D2, SdaD2) and subsequently acquired the phage encoding the SpeA1 variant of the streptococcal pyrogenic exotoxin A superantigen. The SpeA2 toxin variant evolved from SpeA1 by a single-nucleotide change in the M1 progenitor strain before acquisition by horizontal gene transfer of a large chromosomal region encoding secreted toxins NAD(+)-glycohydrolase and streptolysin O. Acquisition of this 36-kb region in the early 1980s into just one cell containing the phage-encoded sdaD2 and speA2 genes was the final major molecular event preceding the emergence and rapid intercontinental spread of the contemporary epidemic clone. Thus, we resolve a decades-old controversy about the type and sequence of genomic alterations that produced this explosive epidemic. Analysis of comprehensive, population-based contemporary invasive strains from seven countries identified strong patterns of temporal population structure. Compared with a preepidemic reference strain, the contemporary clone is significantly more virulent in nonhuman primate models of pharyngitis and necrotizing fasciitis. A key finding is that the molecular evolutionary events transpiring in just one bacterial cell ultimately have produced millions of human infections worldwide.Knut and Alice Wallenberg Foundation Swedish Research Council Houston Methodist Hospital Fondren Foundatio

Mechanism of Protection Induced by Group A Streptococcus Vaccine Candidate J8-DT: Contribution of B and T-Cells Towards Protection

Vaccination with J8-DT, a leading GAS vaccine candidate, results in protective immunity in mice. Analysis of immunologic correlates of protection indicated a role of J8-specific antibodies that were induced post-immunization. In the present study, several independent experimental approaches were employed to investigate the protective immunological mechanisms involved in J8-DT-mediated immunity. These approaches included the passive transfer of mouse or rabbit immune serum/antibodies in addition to selective depletion of T-cell subsets prior to bacterial challenge. Passive transfer of J8-DT antiserum/antibodies from mice and rabbits conferred significant resistance against challenge to mice. To exclude the possibility of involvement of other host immune factors, the studies were repeated in SCID mice, which highlighted the need for an ongoing immune response for long-lived protection. Depletion of CD4+ and CD8+ T-cell subsets confirmed that an active de novo immune response, involving CD4+ T-helper cells, is required for continued synthesis of antibodies resulting in protection against GAS infection. Taken together these results indicate an involvement of CD4+ T-cells in J8-DT-mediated protection possibly via an ability to maintain antibody levels. These results have considerable relevance to the development of a broad spectrum passive immunotherapy for GAS disease

Analysis of a Streptococcus pyogenes puerperal sepsis cluster using whole-genome sequencing

Between June and November 2010, a concerning rise in the number of cases of puerperal sepsis, a postpartum pelvic bacterial infection contracted by women after childbirth, was observed in the New South Wales, Australia, hospital system. Group A streptococcus (GAS; Streptococcus pyogenes) isolates PS001 to PS011 were recovered from nine patients. Pulsed-field gel electrophoresis and emm sequence typing revealed that GAS of emm1.40, emm75.0, emm77.0, emm89.0, and emm89.9 were each recovered from a single patient, ruling out a single source of infection. However, emm28.8 GAS were recovered from four different patients. To investigate the relatedness of these emm28 isolates, whole-genome sequencing was undertaken and the genome sequences were compared to the genome sequence of the emm28.4 reference strain, MGAS6180. A total of 186 single nucleotide polymorphisms were identified, for which the phylogenetic reconstruction indicated an outbreak of a polyclonal nature. While two isolates collected from different hospitals were not closely related, isolates from two puerperal sepsis patients from the same hospital were indistinguishable, suggesting patient-to-patient transmission or infection from a common source. The results of this study indicate that traditional typing protocols, such as pulsed-field gel electrophoresis, may not be sensitive enough to allow fine epidemiological discrimination of closely related bacterial isolates. Whole-genome sequencing presents a valid alternative that allows accurate fine-scale epidemiological investigation of bacterial infectious disease

Bacterial Phenotype Variants in Group B Streptococcal Toxic Shock Syndrome1

Variants with markedly different expression of virulence factors can arise in invasive infection in humans

Inhibition of Toxic Shock by Human Monoclonal Antibodies against Staphylococcal Enterotoxin B

BACKGROUND: Staphylococcus aureus is implicated in many opportunistic bacterial infections around the world. Rising antibiotic resistance and few alternative methods of treatment are just two looming problems associated with clinical management of S. aureus. Among numerous virulence factors produced by S. aureus, staphylococcal enterotoxin (SE) B is a secreted protein that binds T-cell receptor and major histocompatibility complex class II, potentially causing toxic shock mediated by pathological activation of T cells. Recombinant monoclonal antibodies that target SEB and block receptor interactions can be of therapeutic value. METHODOLOGY/PRINCIPAL FINDINGS: The inhibitory and biophysical properties of ten human monoclonal antibodies, isolated from a recombinant library by panning against SEB vaccine (STEBVax), were examined as bivalent Fabs and native full-length IgG (Mab). The best performing Fabs had binding affinities equal to polyclonal IgG, low nanomolar IC(50)s against SEB in cell culture assays, and protected mice from SEB-induced toxic shock. The orthologous staphylococcal proteins, SEC1 and SEC2, as well as streptococcal pyrogenic exotoxin C were recognized by several Fabs. Four Fabs against SEB, with the lowest IC(50)s, were converted into native full-length Mabs. Although SEB-binding kinetics were identical between each Fab and respective Mab, a 250-fold greater inhibition of SEB-induced T-cell activation was observed with two Mabs. CONCLUSIONS/SIGNIFICANCE: Results suggest that these human monoclonal antibodies possess high affinity, target specificity, and toxin neutralization qualities essential for any therapeutic agent

Streptococcal necrotising fasciitis from diverse strains of Streptococcus pyogenes in tropical northern Australia: case series and comparison with the literature

BACKGROUND: Since the mid-1980's there has been a worldwide resurgence of severe disease from group A streptococcus (GAS), with clonal clusters implicated in Europe and the United States. However GAS associated sepsis and rheumatic fever have always remained at high levels in many less developed countries. In this context we aimed to study GAS necrotising fasciitis (NF) in a region where there are high background rates of GAS carriage and disease. METHODS: We describe the epidemiology, clinical and laboratory features of 14 consecutive cases of GAS NF treated over a seven year period from tropical northern Australia. RESULTS: Incidence rates of GAS NF in the Aboriginal population were up to five times those previously published from other countries. Clinical features were similar to those described elsewhere, with 7/14 (50%) bacteremic and 9/14 (64%) having associated streptococcal toxic shock syndrome. 11/14 (79%) had underlying chronic illnesses, including all four fatalities (29% mortality overall). Important laboratory differences from other series were that leukocytosis was absent in 9/14 (64%) but all had substantial lymphopenia. Sequence typing of the 14 NF-associated GAS isolates showed no clonality, with only one emm type 1 and two emm type 3 strains. CONCLUSIONS: While NF clusters can occur from a single emergent GAS clone, this was not evident in our tropical region, where high rates of NF parallel high overall rates of GAS infection from a wide diversity of strains. The specific virulence factors of GAS strains which do cause NF and the basis of the inadequate host response in those patients who develop NF on infection with these GAS require further elucidation

emm gene diversity, superantigen gene profiles and presence of SlaA among clinical isolates of group A, C and G streptococci from western Norway

In order to investigate molecular characteristics of beta-hemolytic streptococcal isolates from western Norway, we analysed the entire emm gene sequences, obtained superantigen gene profiles and determined the prevalence of the gene encoding streptococcal phospholipase A2 (SlaA) of 165 non-invasive and 34 contemporary invasive group A, C and G streptococci (GAS, GCS and GGS). Among the 25 GAS and 26 GCS/GGS emm subtypes identified, only emm3.1 was significantly associated with invasive disease. M protein size variation within GAS and GCS/GGS emm types was frequently identified. Two non-invasive and one invasive GGS possessed emm genes that translated to truncated M proteins as a result of frameshift mutations. Results suggestive of recombinations between emm or emm-like gene segments were found in isolates of emm4 and stG485 types. One non-invasive GGS possessed speC, speG, speH, speI and smeZ, and another non-invasive GGS harboured SlaA. speA and SlaA were over-represented among invasive GAS, probably because they were associated with emm3. speGdys was identified in 83% of invasive and 63% of non-invasive GCS/GGS and correlated with certain emm subtypes. Our results indicate the invasive potential of isolates belonging to emm3, and show substantial emm gene diversity and possible lateral gene transfers in our streptococcal population

Antibodies against a Surface Protein of Streptococcus pyogenes Promote a Pathological Inflammatory Response

Streptococcal toxic shock syndrome (STSS) caused by Streptococcus pyogenes is a clinical condition with a high mortality rate despite modern intensive care. A key feature of STSS is excessive plasma leakage leading to hypovolemic hypotension, disturbed microcirculation and multiorgan failure. Previous work has identified a virulence mechanism in STSS where M1 protein of S. pyogenes forms complexes with fibrinogen that activate neutrophils to release heparin-binding protein (HBP), an inducer of vascular leakage. Here, we report a marked inter-individual difference in the response to M1 protein–induced HBP release, a difference found to be related to IgG antibodies directed against the central region of the M1 protein. To elicit massive HBP release, such antibodies need to be part of the M1 protein–fibrinogen complexes. The data add a novel aspect to bacterial pathogenesis where antibodies contribute to the severity of disease by promoting a pathologic inflammatory response

Staphylococcal Enterotoxins

Staphylococcus aureus (S. aureus) is a Gram positive bacterium that is carried by about one third of the general population and is responsible for common and serious diseases. These diseases include food poisoning and toxic shock syndrome, which are caused by exotoxins produced by S. aureus. Of the more than 20 Staphylococcal enterotoxins, SEA and SEB are the best characterized and are also regarded as superantigens because of their ability to bind to class II MHC molecules on antigen presenting cells and stimulate large populations of T cells that share variable regions on the β chain of the T cell receptor. The result of this massive T cell activation is a cytokine bolus leading to an acute toxic shock. These proteins are highly resistant to denaturation, which allows them to remain intact in contaminated food and trigger disease outbreaks. A recognized problem is the emergence of multi-drug resistant strains of S. aureus and these are a concern in the clinical setting as they are a common cause of antibiotic-associated diarrhea in hospitalized patients. In this review, we provide an overview of the current understanding of these proteins