Adapting the Finetech-Brindley Sacral Anterior Root Stimulator for Bioelectronic Medicine.

The Finetech-Brindley Sacral Anterior Root Stimulator (SARS) is a low cost and reliable system. The architecture has been used for various bioelectric treatments, including several thousand implanted systems for restoring bladder function following spinal cord injury (SCI). Extending the operational frequency range would expand the capability of the system; enabling, for example, the exploration of eliminating the rhizotomy through an electrical nerve block. The distributed architecture of the SARS system enables stimulation parameters to be adjusted without modifying the implant design or manufacturing. To explore the design degrees-of-freedom, a circuit simulation was created and validated using a modified SARS system that supported stimulation frequencies up to 600 Hz. The simulation was also used to explore high frequency (up to 30kHz) behaviour, and to determine the constraints on charge delivered at the higher rates. A key constraint found was the DC blocking capacitors, designed originally for low frequency operation, not fully discharging within a shortened stimulation period. Within these current implant constraints, we demonstrate the potential capability for higher frequency operation that is consistent with presynaptic stimulation block, and also define targeted circuit improvements for future extension of stimulation capability.

Discovery of a Series of Indole-2 Carboxamides as Selective Secreted Phospholipase A2 Type X (sPLA2-X) Inhibitors.

In order to assess the potential of sPLA2-X as a therapeutic target for atherosclerosis, novel sPLA2 inhibitors with improved type X selectivity are required. To achieve the objective of identifying such compounds, we embarked on a lead generation effort that resulted in the identification of a novel series of indole-2-carboxamides as selective sPLA2-X inhibitors with excellent potential for further optimization.

Divergence between the Highly Virulent Zoonotic Pathogen Helicobacter heilmannii and Its Closest Relative, the Low-Virulence "Helicobacter ailurogastricus" sp. nov.

Helicobacter heilmannii naturally colonizes the stomachs of dogs and cats and has been associated with gastric disorders in humans. Nine feline Helicobacter strains, classified as H. heilmannii based on ureAB and 16S rRNA gene sequences, were divided into a highly virulent and a low-virulence group. The genomes of these strains were sequenced to investigate their phylogenetic relationships, to define their gene content and diversity, and to determine if the differences in pathogenicity were associated with the presence or absence of potential virulence genes. The capacities of these helicobacters to bind to the gastric mucosa were investigated as well. Our analyses revealed that the low-virulence strains do not belong to the species H. heilmannii but to a novel, closely related species for which we propose the name Helicobacter ailurogastricus. Several homologs of H. pylori virulence factors, such as IceA1, HrgA, and jhp0562-like glycosyltransferase, are present in H. heilmannii but absent in H. ailurogastricus. Both species contain a VacA-like autotransporter, for which the passenger domain is remarkably larger in H. ailurogastricus than in H. heilmannii. In addition, H. ailurogastricus shows clear differences in binding to the gastric mucosa compared to H. heilmannii. These findings highlight the low-virulence character of this novel Helicobacter species.

Comparative genomic analysis of Helicobacter pylori from Malaysia identifies three distinct lineages suggestive of differential evolution.

The discordant prevalence of Helicobacter pylori and its related diseases, for a long time, fostered certain enigmatic situations observed in the countries of the southern world. Variation in H. pylori infection rates and disease outcomes among different populations in multi-ethnic Malaysia provides a unique opportunity to understand dynamics of host-pathogen interaction and genome evolution. In this study, we extensively analyzed and compared genomes of 27 Malaysian H. pylori isolates and identified three major phylogeographic lineages: hspEastAsia, hpEurope and hpSouthIndia. The analysis of the virulence genes within the core genome, however, revealed a comparable pathogenic potential of the strains. In addition, we identified four genes limited to strains of East-Asian lineage. Our analyses identified a few strain-specific genes encoding restriction modification systems and outlined 311 core genes possibly under differential evolutionary constraints, among the strains representing different ethnic groups. The cagA and vacA genes also showed variations in accordance with the host genetic background of the strains. Moreover, restriction modification genes were found to be significantly enriched in East-Asian strains. An understanding of these variations in the genome content would provide significant insights into various adaptive and host modulation strategies harnessed by H. pylori to effectively persist in a host-specific manner.

Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates.

Genome sequencing of disease and carriage isolates of nontypeable Haemophilus influenzae identifies discrete population structure.

One of the main hurdles for the development of an effective and broadly protective vaccine against nonencapsulated isolates of Haemophilus influenzae (NTHi) lies in the genetic diversity of the species, which renders extremely difficult the identification of cross-protective candidate antigens. To assess whether a population structure of NTHi could be defined, we performed genome sequencing of a collection of diverse clinical isolates representative of both carriage and disease and of the diversity of the natural population. Analysis of the distribution of polymorphic sites in the core genome and of the composition of the accessory genome defined distinct evolutionary clades and supported a predominantly clonal evolution of NTHi, with the majority of genetic information transmitted vertically within lineages. A correlation between the population structure and the presence of selected surface-associated proteins and lipooligosaccharide structure, known to contribute to virulence, was found. This high-resolution, genome-based population structure of NTHi provides the foundation to obtain a better understanding, of NTHi adaptation to the host as well as its commensal and virulence behavior, that could facilitate intervention strategies against disease caused by this important human pathogen.

Comparative analysis of the full genome of Helicobacter pylori isolate Sahul64 identifies genes of high divergence.

Isolates of Helicobacter pylori can be classified phylogeographically. High genetic diversity and rapid microevolution are a hallmark of H. pylori genomes, a phenomenon that is proposed to play a functional role in persistence and colonization of diverse human populations. To provide further genomic evidence in the lineage of H. pylori and to further characterize diverse strains of this pathogen in different human populations, we report the finished genome sequence of Sahul64, an H. pylori strain isolated from an indigenous Australian. Our analysis identified genes that were highly divergent compared to the 38 publically available genomes, which include genes involved in the biosynthesis and modification of lipopolysaccharide, putative prophage genes, restriction modification components, and hypothetical genes. Furthermore, the virulence-associated vacA locus is a pseudogene and the cag pathogenicity island (cagPAI) is not present. However, the genome does contain a gene cluster associated with pathogenicity, including dupA. Our analysis found that with the addition of Sahul64 to the 38 genomes, the core genome content of H. pylori is reduced by approximately 14% (∼170 genes) and the pan-genome has expanded from 2,070 to 2,238 genes. We have identified three putative horizontally acquired regions, including one that is likely to have been acquired from the closely related Helicobacter cetorum prior to speciation. Our results suggest that Sahul64, with the absence of cagPAI, highly divergent cell envelope proteins, and a predicted nontransportable VacA protein, could be more highly adapted to ancient indigenous Australian people but with lower virulence potential compared to other sequenced and cagPAI-positive H. pylori strains.

Comparing the genomes of Helicobacter pylori clinical strain UM032 and Mice-adapted derivatives.

BACKGROUND: Helicobacter pylori is a Gram-negative bacterium that persistently infects the human stomach inducing chronic inflammation. The exact mechanisms of pathogenesis are still not completely understood. Although not a natural host for H. pylori, mouse infection models play an important role in establishing the immunology and pathogenicity of H. pylori. In this study, for the first time, the genome sequences of clinical H. pylori strain UM032 and mice-adapted derivatives, 298 and 299, were sequenced using the PacBio Single Molecule, Real-Time (SMRT) technology. RESULT: Here, we described the single contig which was achieved for UM032 (1,599,441 bp), 298 (1,604,216 bp) and 299 (1,601,149 bp). Preliminary analysis suggested that methylation of H. pylori genome through its restriction modification system may be determinative of its host specificity and adaptation. CONCLUSION: Availability of these genomic sequences will aid in enhancing our current level of understanding the host specificity of H. pylori.

Transcriptional response in the peripheral blood of patients infected with Salmonella enterica serovar Typhi.

We used microarrays and transcriptional profiling of peripheral blood to investigate the host response of 29 individuals who contracted typhoid fever in the Mekong Delta region of Vietnam. Samples were taken over a nine month period encompassing acute disease, convalescence, and recovery. We found that typhoid fever induced a distinct and highly reproducible signature in the peripheral blood that changed during treatment and convalescence, returning in the majority of cases to the "normal" profile as measured in healthy uninfected controls. Unexpectedly, there was a strong, distinct signature of convalescence present at day 9 after infection that remained virtually unchanged one month after acute infection and in some cases persisted as long as nine months despite a complete clinical recovery in all patients. Patients who retain the convalescent signature may be genetically or temporarily incapable of developing an effective immune response and may be more susceptible to reinfection, relapse, or the establishment of a carrier state.

Design of an implant for preventing incontinence after spinal cord injury.

An implanted device is being designed and tested which has the main function of suppressing hyperreflexic bladder contractions by stimulating the pudendal afferent pathway. The concept is that the contractions will be detected by recording natural nerve signals. This is challenging because the changes in neural signal are very small (sub-microvolt), and the device must run 24 h per day, which means that for convenience it must be battery-powered. The energy budget is therefore tight. Furthermore, because the patient must be able to intervene to occasionally empty the bladder, a radio link is needed to the device. Within the EU project Healthy Aims, most aspects of the design have been made and tested. This includes the battery, battery charger, neural amplifier, and the package incorporating the Medical Implant Communication System (MICS) antenna, which are briefly described here. This article is a progress report.

Long-term intensive electrically stimulated cycling by spinal cord-injured people: effect on muscle properties and their relation to power output.

Inactivity and muscular adaptations following spinal cord injury (SCI) result in secondary complications such as cardiovascular disease, obesity, and pressure sores. Functional electrically stimulated (FES) cycling can potentially reduce these complications, but previous studies have provided inconsistent results. We studied the effect of intensive long-term FES cycle training on muscle properties in 11 SCI subjects (mean +/- SEM: 41.8 +/- 2.3 years) who had trained for up to 1 hour/day, 5 days/week, for 1 year. Comparative measurements were made in 10 able-bodied (AB) subjects. Quadriceps maximal electrically stimulated torque increased fivefold (n = 5), but remained lower than in AB individuals. Relative force response at 1 HZ decreased, relaxation rate remained unchanged, and fatigue resistance improved significantly. Power output (PO) improved to a lesser extent than quadriceps torque and not to a greater extent than has been reported previously. We need to understand the factors that limit PO in order to maximize the benefits of FES cycling.

A linear plasmid truncation induces unidirectional flagellar phase change in H:z66 positive Salmonella Typhi.

The process by which bacteria regulate flagellar expression is known as phase variation and in Salmonella enterica this process permits the expression of one of two flagellin genes, fliC or fljB, at any one time. Salmonella Typhi (S. Typhi) is normally not capable of phase variation of flagellar antigen expression as isolates only harbour the fliC gene (H:d) and lacks an equivalent fljB locus. However, some S. Typhi isolates, exclusively from Indonesia, harbour an fljB equivalent encoded on linear plasmid, pBSSB1 that drives the expression of a novel flagellin named H:z66. H:z66+S. Typhi isolates were stimulated to change flagellar phase and genetically analysed for the mechanism of variation. The phase change was demonstrated to be unidirectional, reverting to expression from the resident chromosomal fliC gene. DNA sequencing demonstrated that pBSSB1 linear DNA was still detectable but that these derivatives had undergone deletion and were lacking fljA(z66) (encoding a flagellar repressor) and fljB(z66). The deletion end-point was found to involve one of the plasmid termini and a palindromic repeat sequence within fljB(z66), distinct to that found at the terminus of pBSSB1. These data demonstrate that, like some Streptomyces linear elements, at least one of the terminal inverted repeats of pBSSB1 is non-essential, but that a palindromic repeat sequence may be necessary for replication.

Structure-based virtual screening for low molecular weight chemical starting points for dipeptidyl peptidase IV inhibitors.

Structure-based virtual screening was performed against the target dipeptidyl peptidase IV (DPP-IV) to identify good chemical starting points for medicinal chemistry. A database of available compounds was filtered by calculated physical properties and undesired chemistry. This database was matched against two in-house designed DPP-IV pharmacophores, and the hits from these pharmacophore searches were docked into a DPP-IV crystal structure. Compounds were then selected for testing and 51 active compounds were identified from a list of 4000 compounds tested. These had activities ranging from 30% to 82% when tested at a concentration of 30 microM in an enzyme inhibition assay.

Control of leg-powered paraplegic cycling using stimulation of the lumbo-sacral anterior spinal nerve roots.

We investigated leg-powered cycling in a recumbent tricycle for a paraplegic using functional electrical stimulation (FES) with the lumbo-sacral anterior root stimulator implant (LARSI). A female complete T9 paraplegic had a stimulator for the anterior L2 to S2 spinal roots (bilaterally) implanted in 1994. She was provided with equipment for daily FES cycling exercise at home. The cycling controller applies a pattern of stimulation in each of 16 crank angle phases. A 7-bit shaft encoder measures the crank angle with adequate precision. Each pattern was originally chosen to give the greatest propulsive force in that position when there was no motion. However, dynamically, some reduction in co-contraction is needed; also the patterns are applied with a preset advance time. Maximal power is obtained with an advance of 250 ms, which compensates for muscle response delay and accommodates changes in cadence (from about 25 to 85 rpm). With this system, she has cycled 1.2 km at a time on gently undulating road. We found that spinal root stimulation gives sufficient control over the muscles in the legs to produce a fluid cycling gait. We propose that root stimulation for leg cycling exercise may be a practicable and valuable function for paraplegics following spinal cord injury.