Occurrence and determinants of postpartum maternal morbidities and disabilities among women in Matlab, Bangladesh.

The burden of maternal ill-health includes not only the levels of maternal mortality and complications during pregnancy and around the time of delivery but also extends to the standard postpartum period of 42 days with consequences of obstetric complications and poor management at delivery. There is a dearth of reliable data on these postpartum maternal morbidities and disabilities in developing countries, and more research is warranted to investigate these and further strengthen the existing safe motherhood programmes to respond to these conditions. This study aims at identifying the consequences of pregnancy and delivery in the postpartum period, their association with acute obstetric complications, the sociodemographic characteristics of women, mode and place of delivery, nutritional status of the mother, and outcomes of birth. From among women who delivered between 2007 and 2008 in the icddr,b service area in Matlab, we prospectively recruited all women identified with complicated births (n=295); a perinatal mortality (n=182); and caesarean-section delivery without any maternal indication (n=147). A random sample of 538 women with uncomplicated births, who delivered at home or in a facility, was taken as the control. All subjects were clinically examined at 6-9 weeks for postpartum morbidities and disabilities. Postpartum women who had suffered obstetric complications during birth and delivered in a hospital were more likely to suffer from hypertension [adjusted odds ratio (AOR)=3.44; 95% confidence interval (CI)=1.14-10.36], haemorrhoids (AOR=1.73; 95% CI=1.11-3.09), and moderate to severe anaemia (AOR=7.11; 95% CI=2.03-4.88) than women with uncomplicated normal deliveries. Yet, women who had complicated births were less likely to have perineal tears (AOR=0.05; 95% CI=0.02-0.14) and genital prolapse (AOR=0.22; 95% CI=0.06-0.76) than those with uncomplicated normal deliveries. Genital infections were more common amongst women experiencing a perinatal death than those with uncomplicated normal births (AOR=1.92; 95% CI=1.18-3.14). Perineal tears were significantly higher (AOR=3.53; 95% CI=2.32-5.37) among those who had delivery at home than those giving birth in a hospital. Any woman may suffer a postpartum morbidity or disability. The increased likelihood of having hypertension, haemorrhoids, or anaemia among women with obstetric complications at birth needs specific intervention. A higher quality of maternal healthcare services generally might alleviate the suffering from perineal tears and prolapse amongst those with a normal uncomplicated delivery.

Lipoarabinomannan induced cytotoxic effects in human mononuclear cells.

Mycobacteria produce large quantities of lipoarabinomannan, a cell wall associated glycolipid, which confers virulence in many of the disease causing members of the genus. We studied the lipoarabinomannan induced altered signaling mechanism in human peripheral mononuclear cells. Lipoarabinomannan isolated from Mycobacterium smegmatis (a non-pathogenic species) at concentrations of 2, 5 and 10 microg ml(-1) was used for the study. It was found that protein kinase C activity of human mononuclear cells was significantly inhibited by lipoarabinomannan at these concentrations in a dose dependent manner. Calcium, phosphatidyl serine and diglyceride dependent phosphorylation of endogenous proteins (mainly 90-, 80-, 66-, 38-, 36- and 34-kDa proteins) were also inhibited. Potentially cytotoxic superoxide anions generated by peripheral blood mononuclear cells were scavenged by lipoarabinomannan. It was also observed that incubation of peripheral blood mononuclear cells with lipoarabinomannan at concentrations of 5 and 10 microg ml(-1) for 6 h directed the cells towards apoptotic cell death, confirmed by DNA degradation analysis, microscopic observation of Wright-Giemsa as well as DAPI stained nuclei. These results clearly demonstrate that lipoarabinomannan from M. smegmatis may exert its cytotoxic activity via inhibition of protein kinase C, a key signaling molecule inside the mononuclear cells, which ultimately affects the phosphorylation of various proteins imperative for cellular defence and survival.

Expression systems for study of mycobacterial gene regulation and development of recombinant BCG vaccines.

Successful genetic engineering of mycobacteria is crucial for developing new approaches to combat tuberculosis as well as for dissecting out the molecular basis of pathogenesis of Mycobacterium tuberculosis. We have constructed a Mycobacterium-Escherichia coli shuttle expression vector pSD5. It carries a modular expression cassette which provides sites for cloning of promoters, a ribosome binding site (RBS) with an appropriately placed initiation codon and multiple cloning sites for cloning the genes of interest. We also constructed pDK20, an integration proficient derivative of pSD5, by incorporating mycobacteriophage L5 integration signals in lieu of the origin of DNA replication for mycobacteria. This vector permits stable expression of genes in M.bovis BCG, M.smegmatis, and M.tuberculosis under the transcriptional control of a mycobacterial promoter. These vectors enable the expression of a gene to be regulated by several hundred fold depending upon the strength of mycobacterial promoter. We propose that expression of protective antigens using an appropriate promoter derivative of pDK20 should help in development of recombinant BCG vaccines that can induce an optimal immune response from the host. We have further employed the integration proficient expression system for comparing the efficiency and specificity of transcriptional recognition in M.bovis BCG, M.tuberculosis, and M.smegmatis. We show that fast growing M.smegmatis and slow growing M.tuberculosis and M.bovis BCG recognize mycobacterial promoters with comparable efficiency inspite of differences in their growth rates.

Construction of shuttle vectors for genetic manipulation and molecular analysis of mycobacteria.

Two novel shuttle vectors for mycobacteria are described which have been derived from the expression system pSD5 developed in our laboratory. Plasmid pSD5B is a promoter-selection vector containing a promoterless lacZ gene and allows the identification of mycobacterial promoters by the blue colour of the colonies on solid media containing XGal. Moreover, the chronological order of appearance of blue colonies and intensity of colour provide a qualitative index of transcriptional strengths of the cloned promoters. Plasmid pSD5C has been designed to construct mycobacterial genomic libraries and express the cloned DNA inserts as fusion proteins with maltose binding protein in mycobacteria. Libraries in pSD5C provide feasibility for their screening with either DNA probes or specific antisera for identifying the genes of interest and for isolation of specific genetic loci by complementation of Escherichia coli and mycobacterial mutants. These vectors combine the ease of working in E. coli with the advantage of directly propagating them in mycobacteria without further manipulations. Finally, we demonstrate that these vectors function efficiently both in fast growing Mycobacterium smegmatis and slow growing mycobacteria including Mycobacterium tuberculosis and Mycobacterium bovis BCG.

A search for cholera toxin (CT), toxin coregulated pilus (TCP), the regulatory element ToxR and other virulence factors in non-01/non-0139 Vibrio cholerae.

Twenty-four selected non-O1/non-O139 Vibrio cholerae strains were examined for the presence of virulence associated genes like ctxA, tcpA, toxR and the repetitive sequence (RS element). Seventeen of these were isolated from diarrhoeal stool samples while the remaining seven were of local environmental origin. Nine and four respectively of these strains were positive for ctxA and tcpA by Multiplex PCR analysis. The majority (16 out of 18 tested) of the strains (including the four tcpA + strains) contained toxR sequences as determined by another PCR assay. The presence of RS element was demonstrable in ctxA+ strains only. Interestingly, three of these non-O1/non-O139 strains were shown to contain all the three virulence associated genes (ctxA, tcpA and toxR) as well as the RS element. Two of these belonged to serogroups 037 (V2) and 064 (CG15) while the third one (V315-1) was untypable. These three strains also produced cholera toxin, expressed toxin coregulated pilus (TCP) and/or TcpA related antigens when grown under appropriate culture conditions. Southern hybridization analysis of their chromosomal DNA fragments using DNA probes representing ctxA, zot, ace and RS element revealed that the strains V2 and CG15 contained, at least, two complete copies of the CTX genetic element, while the strain V315-1 had three or more copies of the same. Presence of the RS element in these strains led to tandem duplication of the CTX genetic element in the chromosome of V2 and V315-1, but not in CG15 where the copies were likely to be present at different loci. These results also indicate the presence of additional copies of incomplete "core region' with zot and ace genes, but not ctxA, in strains V2 and CG15. The significance of these results in terms of the pathogenic and epidemic potential of V. cholerae strains is discussed.

A study of mycobacterial transcriptional apparatus: identification of novel features in promoter elements.

Our earlier studies on transcriptional signals of mycobacteria had revealed that (i) strong promoters occur less frequently in the slowly growing pathogen Mycobacterium tuberculosis H37Rv than in the fast-growing saprophyte M. smegmatis and (ii) mycobacterial promoters function poorly in Escherichia coli. We now present evidence that RNA polymerases of M. smegmatis, M. tuberculosis, and M. bovis BCG recognize promoter elements with comparable efficiencies. Analysis of these randomly isolated mycobacterial promoters by DNA sequencing, primer extension, and deletion experiments revealed that their -10 regions are highly similar to those of E. coli promoters, in contrast to their -35 regions, which can tolerate a greater variety of sequences, owing presumably to the presence of multiple sigma factors with different or overlapping specificities for -35 regions, as reported earlier for the Streptomyces promoters. A comparison of the -10 and -35 binding domains of MysA, HrdB, and RpoD (the principal sigma factors of M. smegmatis, Streptomyces aureofaciens, and E. coli, respectively) showed that all three sigma factors have nearly identical -10 binding domains. However, the -35 binding domains of the principal mycobacterial and streptomycete sigma factors, although nearly identical to each other, are vastly different from the corresponding region of the sigma factor of E. coli. Thus, the transcriptional signals of mycobacteria have features in common with Streptomyces promoters but differ from those of E. coli because of major differences in the -35 regions of the promoters and the corresponding binding domain in the sigma factor.

Synthesis of isotopically labeled saturated fatty acids.

An approach to the synthesis of isotopically labeled saturated fatty acids is outlined which is based on the copper-catalyzed coupling of an omega-bromo acid with an isotopically labeled Grignard reagent. The method provides high yields of pure products and offers considerable flexibility in the type of isotopically enriched compound that can be prepared.-DasGupta, S. K., D. M. Rice, and R. G. Griffin. Synthesis of isotopically labeled saturated fatty acids.