Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

Tempo and mode of early gene loss in endosymbiotic bacteria from insects

Background  

Understanding evolutionary processes that drive genome reduction requires determining the tempo (rate) and the mode (size and types of deletions) of gene losses. In this study, we analysed five endosymbiotic genome sequences of the gamma-proteobacteria (three different Buchnera aphidicola strains, Wigglesworthia glossinidia, Blochmannia floridanus) to test if gene loss could be driven by the selective importance of genes. We used a parsimony method to reconstruct a minimal ancestral genome of insect endosymbionts and quantified gene loss along the branches of the phylogenetic tree. To evaluate the selective or functional importance of genes, we used a parameter that measures the level of adaptive codon bias in E. coli (i.e. codon adaptive index, or CAI), and also estimates of evolutionary rates (Ka) between pairs of orthologs either in free-living bacteria or in pairs of symbionts.     

Structural requirements of carbohydrates to bind Agaricus bisporus lectin

Galbeta1-3GalNAc (T-disaccharide) and related molecules were assayed to describe the structural requirements of carbohydrates to bind Agaricus bisporus lectin (ABL). Results provide insight into the most relevant regions of T-disaccharide involved in the binding of ABL. It was found that monosaccharides bind ABL weakly indicating a more extended carbohydrate-binding site as compared to those involvedin the T- disaccharide specific lectins such as jacalin and peanut agglutinin. Lacto-N-biose (Galbeta1-3GlcNAc) unlike T-disaccharide, is unable to inhibit the ABL interaction, thus showing the great importance of the position of the axial C-4 hydroxyl group of GalNAc in T-disaccharide. This finding could explain the inhibitory ability of Galbeta1-6GlcNAc and lactose because C-4 and C-3 hydroxyl groups of reducing Glc, respectively, occupy a similar position as reported by conformational analysis. From the comparison of different glycolipids bearing terminal T-disaccharide bound to different linkages, it can be seen than ABL binding is even more impaired by an adjacent C-6 residual position than by the anomeric influence of T-disaccharide. Furthermore, the addition of beta-GlcNAc to the terminal T-disaccharide in C-3 position of Gal does not affect the ABL binding whereas if an anionic group such as glucuronic acid is added to C-3, the binding is partially affected. These findings demonstrate that ABL holds a particular binding nature different from that of other T-disaccharide specific lectins.      

Immunoreactivity of the <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> 19-kDa lipoprotein

Background  

The Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed.     

An open real-time tele-stethoscopy system

Background

The detection of change in magnitude of directional coupling between two non-linear time series is a common subject of interest in the biomedical domain, including studies involving the respiratory chemoreflex system. Although transfer entropy is a useful tool in this avenue, no study to date has investigated how different transfer entropy estimation methods perform in typical biomedical applications featuring small sample size and presence of outliers.

Methods

With respect to detection of increased coupling strength, we compared three transfer entropy estimation techniques using both simulated time series and respiratory recordings from lambs. The following estimation methods were analyzed: fixed-binning with ranking, kernel density estimation (KDE), and the Darbellay-Vajda (D-V) adaptive partitioning algorithm extended to three dimensions. In the simulated experiment, sample size was varied from 50 to 200, while coupling strength was increased. In order to introduce outliers, the heavy-tailed Laplace distribution was utilized. In the lamb experiment, the objective was to detect increased respiratory-related chemosensitivity to O ₂ and CO ₂ induced by a drug, domperidone. Specifically, the separate influence of end-tidal PO ₂ and PCO ₂ on minute ventilation before and after administration of domperidone was analyzed.

Results

In the simulation, KDE detected increased coupling strength at the lowest SNR among the three methods. In the lamb experiment, D-V partitioning resulted in the statistically strongest increase in transfer entropy post-domperidone for . In addition, D-V partitioning was the only method that could detect an increase in transfer entropy for , in agreement with experimental findings.

Conclusions

Transfer entropy is capable of detecting directional coupling changes in non-linear biomedical time series analysis featuring a small number of observations and presence of outliers. The results of this study suggest that fixed-binning, even with ranking, is too primitive, and although there is no clear winner between KDE and D-V partitioning, the reader should note that KDE requires more computational time and extensive parameter selection than D-V partitioning. We hope this study provides a guideline for selection of an appropriate transfer entropy estimation method.     

An open real-time tele-stethoscopy system

ABSTRACT: BACKGROUND: Acute respiratory infections are the leading cause of childhood mortality. The lack of physicians in rural areas of developing countries makes difficult their correct diagnosis and treatment. The staff of rural health facilities (health-care technicians) may not be qualified to distinguish respiratory diseases by auscultation. For this reason, the goal of this project is the development of a tele-stethoscopy system that allows a physician to receive real-time cardio-respiratory sounds from a remote auscultation, as well as video images showing where the technician is placing the stethoscope on the patient's body. METHODS: A real-time wireless stethoscopy system was designed. The initial requirements were: 1) The system must send audio and video synchronously over IP networks, not requiring an Internet connection; 2) It must preserve the quality of cardiorespiratory sounds, allowing to adapt the binaural pieces and the chestpiece of standard stethoscopes, and; 3) Cardiorespiratory sounds should be recordable at both sides of the communication. In order to verify the diagnostic capacity of the system, a clinical validation with eight specialists has been designed. In a preliminary test, twelve patients have been auscultated by all the physicians using the tele-stethoscopy system, versus a local auscultation using traditional stethoscope. The system must allow listen the cardiac (systolic and diastolic murmurs, gallop sound, arrhythmias) and respiratory (rhonchi, rales and crepitations, wheeze, diminished and bronchial breath sounds, pleural friction rub) sounds. RESULTS: The design, development and initial validation of the real-time wireless tele-stethoscopy system are described in detail. The system was conceived from scratch as open-source, low-cost and designed in such a way that many universities and small local companies in developing countries may manufacture it. Only free open-source software has been used in order to minimize manufacturing costs and look for alliances to support its improvement and adaptation. The microcontroller firmware code, the computer software code and the PCB schematics are available for free download in a subversion repository hosted in SourceForge. CONCLUSIONS: It has been shown that real-time tele-stethoscopy, together with a videoconference system that allows a remote specialist to oversee the auscultation, may be a very helpful tool in rural areas of developing countries.     

Nonequilibrium kinetics of a cyclic GMP-binding protein in dictyostelium discoideum

Chemoattractants added to cells of the cellular slime mold dictyostelium discoideum induce a transient elevation of cyclic GMP levels, with a maximum at 10 s and a recovery of basal levels at approximately 25 s after stimulation. We analyzed the kinetics of an intracellular cGMP binding protein in vitro and in vivo. The cyclic GMP binding protein in vitro at 0 degrees C can be described by its kinetic constants K(1)=2.5 x 10(6) M(- 1)s(-1), k(-1)=3.5 x 10(-3)s(-1), K(d)=1.4 x 10(-9) M, and 3,000 binding sites/cell. In computer simulation experiments the occupancy of the cGMP binding protein was calculated under nonequilibrium conditions by making use of the kinetic constants of the binding protein and of the shape of the cGMP accumulations. These experiments show that under nonequilibrium conditions by making use of the kinetic constants of the binding protein and the shape of the cGMP accumulations. These experiments show that under nonequilibrium conditions the affinity of the binding protein for cGMP is determined by the rate constant of association (k(1)) and not by the dissociation constant (k(d)). Experiments in vivo were performed by stimulation of aggregative cells with the chemoattractant cAMP, which results in a transient cGMP accumulation. At different times after stimulation with various cAMP concentrations, the cells were homogenized and immediately thereafter the number of binding proteins which were not occupied with native cGMP were determined. The results of these experiments in vivo are in good agreement with the results of the computer experiments. This may indicate that: (a) The cGMP binding protein in vivo at 22 degrees C can be described by its kinetic constants: K(1)=4x10(6)M(-1)s(-1) and K(-1)=6x10(-3)s(-1). (b) Binding the cGMP to its binding protein is transient with a maximum at about 20-30 s after chemotactic stimulation, followed by a decay to basal levels, with a half-life of approximately 2 min. (c) The cGMP to its binding proteins get half maximally occupied at a cGMP accumulation of δ[cGMP](10)=2x10(-8) M, which corresponds to an extracellular stimulation of aggregative cells by 10(-10) M cAMP. (d) Since the mean basal cGMP concentration is approximately 2x10(-7) M, the small increase of cGMP cannot be detected accurately. Therefore the absence of a measurable cGMP accumulation does not argue against a cGMP function. (e) There may exist two compartments of cGMP: one contains almost all the cGMP of unstimulated cells, and the other contains cGMP binding proteins and the cGMP which accumulates after chemotactic stimulation. (f) From the kinetics of binding, the cellular responses to the chemoattractant can be divided into two classes: responses which can be mediated by this binding protein (such as light scattering, proton extrusion, PDE induction, and chemotaxis) and responses which cannot be (solely) mediated by this binding protein such as rlay, refractoriness, phospholipids methylation, and protein methylation.     

Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS

Background

CCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively.

Results

Compared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362), a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs) residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure.

Conclusion

Enhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.