Perceptions of HIV/AIDS Leaders about Faith-Based Organisations' Influence on HIV/AIDS Stigma in South Africa

The extent of the HIV pandemic - particularly in the hardest-hit countries; including South Africa - has prompted a call for greater engagement of all groups; including faith-based organisations (FBOs). Although FBOs are known to play a substantial role in providing care and support to those affected by HIV and AIDS; empirical evidence in regard to their actions in the broader context of stigma is limited. A qualitative; key-informant survey was conducted in South Africa as part of a six-country international study to examine perceptions of how FBOs have contributed to reduction in HIV risk; vulnerability and related impacts. The special emphasis of this paper is the influence of FBOs on stigma and discrimination. In-depth interviews were held with 34 senior-level key informants who act as key decision-makers in the response to HIV and AIDS in South Africa. Secular and faith-based respondents shared their perceptions of the faith-based response; including FBOs' actions in relation to HIV/AIDS stigma and discrimination. Our study revealed that while FBOs were perceived as taking some action to address stigma in South Africa; FBOs were also thought to contribute to HIV/AIDS- discrimination through conflating issues of sexuality and morality; and through associating HIV and AIDS with sin. The interviewees indicated a number of internal and external challenges faced by FBOs to deal effectively with stigma; including lack of information and skills; the difficulty of maintaining confidentiality in health services; and self-stigmatisation which prevents HIV-infected persons from revealing their status. Findings from this study may help both faith-based and secular groups capitalise on the perceived strengths of FBOs as well as to elucidate their perceived weaknesses so that these areas of concern can be further explored and addressed

Genome-wide analysis of the general stress response in Bacillus subtilis.

Bacteria respond to diverse growth-limiting stresses by producing a large set of general stress proteins. In Bacillus subtilis and related Gram-positive pathogens, this response is governed by the sigma(B) transcription factor. To establish the range of cellular functions associated with the general stress response, we compared the transcriptional profiles of wild and mutant strains under conditions that induce sigma(B) activity. Macroarrays representing more than 3900 annotated reading frames of the B. subtilis genome were hybridized to (33)P-labelled cDNA populations derived from (i) wild-type and sigB mutant strains that had been subjected to ethanol stress; and (ii) a strain in which sigma(B) expression was controlled by an inducible promoter. On the basis of their significant sigma(B)-dependent expression in three independent experiments, we identified 127 genes as prime candidates for members of the sigma(B) regulon. Of these genes, 30 were known previously or inferred to be sigma(B) dependent by other means. To assist in the analysis of the 97 new genes, we constructed hidden Markov models (HMM) that identified possible sigma(B) recognition sequences preceding 21 of them. To test the HMM and to provide an independent validation of the hybridization experiments, we mapped the sigma(B)-dependent messages for seven representative genes. For all seven, the 5' end of the message lay near typical sigma(B) recognition sequences, and these had been predicted correctly by the HMM for five of the seven examples. Lastly, all 127 gene products were assigned to functional groups by considering their similarity to known proteins. Notably, products with a direct protective function were in the minority. Instead, the general stress response increased relative message levels for known or predicted regulatory proteins, for transporters controlling solute influx and efflux, including potential drug efflux pumps, and for products implicated in carbon metabolism, envelope function and macromolecular turnover.

Identifying diagnostic errors with induced decision trees.

OBJECTIVE: The purpose of this article is to compare the diagnostic accuracy of induced decision trees with that of pruned neural networks and to improve the accuracy and interpretation of breast cancer diagnosis from readings of thin-needle aspirate by identifying cases likely to be misclassified by induced decision rules. METHOD: Using an online database consisting of 699 cases of suspected breast cancer and their corresponding readings of fine-needle aspirate, decision trees were induced from half of the cases, randomly selected. Accuracy was determined for the remaining cases in successive partitions. The pattern of errors in the multiple decision trees was examined. A smaller data set was created with 2 classes: (1) correctly classified and (2) misclassified by a decision tree, rather than the original benign and malignant classes. From this data set, decision trees that describe the misclassified cases were induced. RESULTS: Larger, less severely pruned decision trees were more accurate in breast cancer diagnosis for both training and test data. The accuracy of the induced decision trees exceeded that reported for the smaller pruned neural networks. Combining classifications from 2 trees was effective in identifying malignancies missed by a single tree. Induced decision trees were able to identify patterns associated with misclassified cases, but the identification of errors inductively did not improve the overall error rate. CONCLUSION: In this application, a model that is too compact identifies fewer cases of the minority class, malignancy. New methods that combine models and examine classification errors can improve diagnosis by identifying more malignancies and by describing ambiguous cases.

Observations on the persistence and vertical transmission of Salmonella enterica serovars Pullorum and Gallinarum in chickens: effect of bacterial and host genetic background.

Commercial laying hens inoculated with a strain of Salmonella enterica ser. Pullorum when they were 4 days old showed no morbidity, but harboured infection until they came into lay, and then produced S. Pullorum-contaminated eggs and infected progeny. There was limited evidence of transmission of maternal immunity to the progeny. Attempts were made to set up similar infections in hens with Salmonella Gallinarum, but without success. Infection either resulted in clinical disease or elimination of the pathogen. Infection of birds when in lay produced a similar result. The possibility of eggs becoming contaminated with S. Gallinarum after they were laid in the nest box was evaluated but there was no evidence for this. In-bred chicken lines with a SalI-susceptible phenotype showed greater localization of S. Pullorum in the reproductive tract than did a SalI-resistant line. In addition, in-bred birds, which were SalI resistant but showed greater susceptibility to intestinal colonization by Salmonella, infected with S. Gallinarum when they were 1 week old, showed longer term persistence in the liver and spleen than did a resistant line.

Further studies on vertical transmission and persistence of Salmonella enterica serovar Enteritidis phage type 4 in chickens.

One-week-old commercial layers were infected orally with 10(8) colony forming units of Salmonella enterica serovar Enteritidis phage type 4. No mortality was observed. The inoculated organism was isolated in decreasing viable numbers from a number of tissues, particularly the spleen, liver and caeca. Organisms present in the spleen were primarily localized within macrophages. No Salmonella Enteritidis organisms were isolated between 10 and 24 weeks of age, when the experiment was terminated after several weeks of lay. When two groups of adult hens, housed with males, were infected, contaminated eggs were found within 2 weeks of infection in one of the experiments only. Progeny hatched from these eggs showed no mortality unless they were infected artificially with the S. Enteritidis strain. In this case, the percentage mortality fell as the hatches progressed, indicating increasing immunity to infection. The faecal excretion of the inoculated phage type 4 strain by infected but healthy progeny was followed. Although most birds ceased to excrete by 11 to 12 weeks of age, a small number of the birds continued to excrete until they themselves came into lay. The small numbers of birds in which this occurred indicates that tolerance to infection does not occur readily following infection of hens laying fertile eggs or in progeny birds infected before or within hours of hatching. Birds infected when they were less than 24 h old remained persistently infected until they were well into lay. However, control birds infected when 1 week old, on this occasion, showed a high level of excretion until the birds began to lay at 18 weeks. Inbred lines of chickens showing differences in their susceptibility to systemic salmonellosis did not show significant differences in the extent to which S. Enteritidis localized in the organs of the reproductive tract or in the number of infected eggs produced.

The enoyl-[acyl-carrier-protein] reductases FabI and FabL from Bacillus subtilis.

Enoyl-[acyl-carrier-protein] (ACP) reductase is a key enzyme in type II fatty-acid synthases that catalyzes the last step in each elongation cycle. The FabI component of Bacillus subtilis (bsFabI) was identified in the genomic data base by homology to the Escherichia coli protein. bsFabI was cloned and purified and exhibited properties similar to those of E. coli FabI, including a marked preference for NADH over NADPH as a cofactor. Overexpression of the B. subtilis fabI gene complemented the temperature-sensitive growth phenotype of an E. coli fabI mutant. Triclosan was a slow-binding inhibitor of bsFabI and formed a stable bsFabI.NAD(+). triclosan ternary complex. Analysis of the B. subtilis genomic data base revealed a second open reading frame (ygaA) that was predicted to encode a protein with a relatively low overall similarity to FabI, but contained the Tyr-Xaa(6)-Lys enoyl-ACP reductase catalytic architecture. The purified YgaA protein catalyzed the NADPH-dependent reduction of trans-2-enoyl thioesters of both N-acetylcysteamine and ACP. YgaA was reversibly inhibited by triclosan, but did not form the stable ternary complex characteristic of the FabI proteins. Expression of YgaA complemented the fabI(ts) defect in E. coli and conferred complete triclosan resistance. Single knockouts of the ygaA or fabI gene in B. subtilis were viable, but double knockouts were not obtained. The fabI knockout was as sensitive as the wild-type strain to triclosan, whereas the ygaA knockout was 250-fold more sensitive to the drug. YgaA was renamed FabL to denote the discovery of a new family of proteins that carry out the enoyl-ACP reductase step in type II fatty-acid synthases.

Salmonella infection in a commercial line of ducks; experimental studies on virulence, intestinal colonization and immune protection.

Experimental infections of different salmonella serotypes were established in a commercial line of ducks to provide baseline information on which control measures might be based. The ducks were very resistant to systemic infection with Salmonella typhimurium, S. enteritidis and S. gallinarum within 36 h of hatching. This was associated with an inherent inability of the strains to multiply in the reticulo-endothelial system. The resistance was not associated with poor invasiveness or serum sensitivity. Individual strains of S. typhimurium, S. enteritidis, S. heidelberg and S. orion colonized the gut well and were excreted in the faeces for at least 6 weeks by ducks when they were infected orally within 2 days of hatching. The main sites of colonization were the caeca and, to a lesser extent, the crop. Viable counts of each inoculated strain in the caeca remained in excess of 10(6) c.f.u. 3 weeks after infection although the organisms had been cleared from the spleen by this time. Much less excretion occurred when the birds were infected at 3 weeks of age. When infected ducks, which had cleared themselves of infection, were challenged orally with the homologous strain expressing a different genetic marker, very low levels of excretion of the challenge strain were detected when compared with a control group. After infection low titres of circulating lipopolysaccharide-specific IgG antibodies were detected by an ELISA. Intestinal colonization of newly hatched ducks with an aroA strain of S. enteritidis resulted in extensive colonization which exerted an exclusion effect on the parent strain inoculated 24 h later.

Effect of enrofloxacin administration on excretion of Salmonella ententidis by experimentally infected chickens and on quinolone resistance of their Escherichia coli flora.

Chickens which had been experimentally-infected with a strain of Salmonella ententidis and treated by administration of enrofloxacin at commercially recommended concentrations in the drinking water, virtually eliminated this organism from the alimentary tract. However, an initially quinolone-sensitive Escherichia coli flora present in the birds' faeces was rapidly replaced by a quinolone-resistant flora which persisted after withdrawal of the medication. Resistance to quinolone in the form of nalidixic acid was transducible from a strain of S. typhimurium to S. enterinais with bacteriophage P22.

Multicopy suppression of cold-sensitive sec mutations in Escherichia coli.

Mutations in the secretory (sec) genes in Escherichia coli compromise protein translocation across the inner membrane and often confer conditional-lethal phenotypes. We have found that overproduction of the chaperonins GroES and GroEL from a multicopy plasmid suppresses a wide array of cold-sensitive sec mutations in E. coli. Suppression is accompanied by a stimulation of precursor protein translocation. This multicopy suppression does not bypass the Sec pathway because a deletion of secE is not suppressed under these conditions. Surprisingly, progressive deletion of the groE operon does not completely abolish the ability to suppress, indicating that the multicopy suppression of cold-sensitive sec mutations is not dependent on a functional groE operon. Indeed, overproduction of proteins unrelated to the process of protein export suppresses the secE501 cold-sensitive mutation, suggesting that protein overproduction, in and of itself, can confer mutations which compromise protein synthesis and the observation that low levels of protein synthesis inhibitors can suppress as well. In all cases, the mechanism of suppression is unrelated to the process of protein export. We suggest that the multicopy plasmids also suppress the sec mutations by compromising protein synthesis.

A double counter-selection system for the study of null alleles of essential genes in Escherichia coli.

The ability to analyze null alleles of genes can be an important means of studying both a protein's function and its interactions with other proteins involved in a particular process. However, if the gene encodes a protein that is essential to the viability of the cell, such analysis becomes complicated because a complementing copy of the gene must be present in the cell. In order to study the effects caused by the null allele, the complementing copy must be inactivated or lost. We report the development of a system in Escherichia coli which facilitates the manipulation of null alleles of essential genes. It consists of a strain deleted chromosomally for the essential gene and complemented for its function by a wild-type (wt) copy expressed from a plasmid counter-selectable for two markers bracketing the gene. Using this system, we have (i) searched for bypass suppressors of a deletion of the essential secE gene, (ii) ascertained the ability of various mutant secE genes to complement a deletion of the wt copy and (iii) isolated intragenic pseudorevertants of a null missense allele of secE. This methodology should be widely applicable to other cases in which essential genes are to be studied genetically.

Comparison of multiplex PCR and standard bacteriological methods of detecting Salmonella on chicken skin.

A multiplex PCR technique was compared with standard bacteriological techniques for the detection of Salmonella, and Salm. enteritidis in particular, in samples of chicken skin obtained from retail outlets. Five of 68 samples were positive by both systems (7.4%), 58 of 68 were negative by both systems (85%), five were positive by PCR only (7.4%) but none were positive by culture only. The PCR was quicker and more sensitive than the bacteriological methods, finding 15% of samples positive for Salmonella compared to 7.4%, and taking less than 24 h to reach a result as opposed to 2-3 d.

Residues essential for the function of SecE, a membrane component of the Escherichia coli secretion apparatus, are located in a conserved cytoplasmic region.

Protein export in Escherichia coli is absolutely dependent on two integral membrane proteins, SecY and SecE. Previous deletion mutagenesis of the secE gene showed that only the third of three membrane-spanning segments and a portion of the second cytoplasmic region are necessary for its function in protein export. Here we further define the residues important for SecE function. Alignment of the SecE homologues of various eubacteria reveals that they all contain one membrane-spanning segment, compared with three in E. coli SecE, and that the most conserved region among them lies in their putative cytoplasmic amino termini; little homology exists in their membrane-spanning segments. The SecE homologue of the extreme thermophilic bacterium Thermotoga maritima was cloned and found to complement a deletion of secE in E. coli. Deletion or replacement of the cytoplasmic region of E. coli SecE eliminated SecE function, indicating that this sequence is essential for a functional secretion machinery. Mutant analysis suggests that the most important function of the third membrane-spanning segment is to maintain the proper topological arrangement of the conserved cytoplasmic domain.

Mechanisms of TonB-catalyzed iron transport through the enteric bacterial cell envelope.

The recent solution of enteric bacterial porin structure, and new insights into the mechanism by which outer membrane receptor proteins recognize and internalize specific ligands, advocates the re-evaluation of TonB-dependent transport physiology. In this minireview we discuss the potential structural features of siderophore receptors and TonB, and use this analysis to evaluate both existing and new models of energy and signal transduction from the inner membrane to the outer membrane of gram-negative bacteria.

Protein export in Escherichia coli.

The export of protein from Escherichia coli has been studied by genetic, biochemical and biophysical techniques. These studies have defined a number of steps in the export pathway and have identified the cellular components required for the translocation process. New information is presented on the function of some of these components.

Surface topology of the Escherichia coli K-12 ferric enterobactin receptor.

Monoclonal antibodies (MAb) were raised to the Escherichia coli K-12 ferric enterobactin receptor, FepA, and used to identify regions of the polypeptide that are involved in interaction with its ligands ferric enterobactin and colicins B and D. A total of 11 distinct FepA epitopes were identified. The locations of these epitopes within the primary sequence of FepA were mapped by screening MAb against a library of FepA::PhoA fusion proteins, a FepA deletion mutant, and proteolytically modified FepA. These experiments localized the 11 epitopes to seven different regions within the FepA polypeptide, including residues 2 to 24, 27 to 37, 100 to 178, 204 to 227, 258 to 290, 290 to 339, and 382 to 400 of the mature protein. Cell surface-exposed epitopes of FepA were identified and discriminated by cytofluorimetry and by the ability of MAb that recognize them to block the interaction of FepA with its ligands. Seven surface epitopes were defined, including one each in regions 27 to 37, 204 to 227, and 258 to 290 and two each in regions 290 to 339 and 382 to 400. One of these, within region 290 to 339, was recognized by MAb in bacteria containing intact (rfa+) lipopolysaccharide (LPS); all other surface epitopes were susceptible to MAb binding only in a strain containing a truncated (rfaD) LPS core, suggesting that they are physically shielded by E. coli K-12 LPS core sugars. Antibody binding to FepA surface epitopes within region 290 to 339 or 382 to 400 inhibited killing by colicin B or D and the uptake of ferric enterobactin. In addition to the FepA-specific MAb, antibodies that recognized other outer membrane components, including Cir, OmpA, TonA, and LPS, were identified. Immunochemical and biochemical characterization of the surface structures of FepA and analysis of its hydrophobicity and amphilicity were used to generate a model of the ferric enterobactin receptor's transmembrane strands, surface peptides, and ligand-binding domains.

Export of FepA::PhoA fusion proteins to the outer membrane of Escherichia coli K-12.

A library of fepA::phoA gene fusions was generated in order to study the structure and secretion of the Escherichia coli K-12 ferric enterobactin receptor, FepA. All of the fusion proteins contained various lengths of the amino-terminal portion of FepA fused in frame to the catalytic portion of bacterial alkaline phosphatase. Localization of FepA::PhoA fusion proteins in the cell envelope was dependent on the number of residues of mature FepA present at the amino terminus. Hybrids containing up to one-third of the amino-terminal portion of FepA fractionated with their periplasm, while those containing longer sequences of mature FepA were exported to the outer membrane. Outer membrane-localized fusion proteins expressed FepA sequences on the external face of the outer membrane and alkaline phosphatase moieties in the periplasmic space. From sequence determinations of the fepA::phoA fusion joints, residues within FepA which may be exposed on the periplasmic side of the outer membrane were identified.