Synthesis and biological evaluation of novel sulfonyl-naphthalene-1,4-diols as FabH inhibitors

A series of analogs of 2-tosylnaphthalene-1,4-diol were prepared and were found to be potent 10-20 nM reversible inhibitors of the Escherichia coli FabH enzyme. The inhibitors were also effective but to a lesser degree (30 nM-5 microM), against the Mycobacterium tuberculosis and Plasmodium falciparum FabH enzymes. Preliminary SAR studies demonstrated that the sulfonyl group and naphthalene-1,4 diol were required for activity against all enzymes but the toluene portion could be significantly altered and leads to either modest increases or decreases in activity against the three enzymes. The in vitro activity of the analogs against E. coli FabH parallel the in vivo activity against E. coli TolC strain and many of the compounds were also shown to have antimalarial activity against P. falciparum.     

Interpreting voltage-sensitivity of gap junctions as a mechanism of cardiac memory

Memory in the nervous system is essentially a network effect, resulting from activity-dependent synaptic modification in a network of neurons. Like the nervous system, the heart is a network of cardiac cells electrically coupled by gap junctions. The heart too has memory, termed cardiac memory, whereby the effect of an external electrical activation persists long after the presentation of stimulus is terminated. We have earlier proposed that adaptation of gap junctions, as a function of membrane voltages of the cells that are coupled by the gap junctions, is related to cardiac memory [V.S. Chakravarthy, J. Ghosh, On Hebbian-like adaption in heart muscle: a proposal for "Cardiac Memory", Biol. Cybern. 76 (1997) 207, J. Krishnan, V.S. Chakravarthy, S. Radhakrishnan, On the role of gap junctions on cardiac memory effect, Comput. Cardiol. 32 (2005) 13]. Using the proposed mechanism, we demonstrate memory effect using computational models of interacting cell pairs. In this paper, we address the biological validity of the proposed mechanism of gap junctional adaptation. It is known from electrophysiology of gap junctions that the conductance of these channels adapts as a function of junctional voltage. At a first sight, this form of voltage dependence seems to be at variance with the form required by our mechanism. But we show, with the help of a theoretical model, that the proposed mechanism of voltage-dependent adaptation of gap junctions, is compatible with the known voltage-sensitivity of gap junctions observed in electrophysiological studies. Our analysis suggests a new significance of the voltage-sensitivity of gap junctions and its possible link to the phenomenon of cardiac memory.     

Muscle-specific MicroRNA1 (miR1) Targets Heat Shock Protein 70 (HSP70) during Dexamethasone-mediated Atrophy

High doses of dexamethasone (Dex) or myostatin (Mstn) induce severe atrophy of skeletal muscle. Here we show a novel microRNA1 (miR1)-mediated mechanism through which Dex promotes skeletal muscle atrophy. Using both C2C12 myotubes and mouse models of Dex-induced atrophy we show that Dex induces miR1 expression through glucocorticoid receptor (GR). We further show that Mstn treatment facilitates GR nuclear translocation and thereby induces miR1 expression. Inhibition of miR1 in C2C12 myotubes attenuated the Dex-induced increase in atrophy-related proteins confirming a role for miR1 in atrophy. Analysis of miR1 targets revealed that HSP70 is regulated by miR1 during atrophy. Our results demonstrate that increased miR1 during atrophy reduced HSP70 levels, which resulted in decreased phosphorylation of AKT, as HSP70 binds to and protects phosphorylation of AKT. We further show that loss of pAKT leads to decreased phosphorylation, and thus, enhanced activation of FOXO3, up-regulation of MuRF1 and Atrogin-1, and progression of skeletal muscle atrophy. Based on these results, we propose a model whereby Dex- and Mstn-mediated atrophic signals are integrated through miR1, which then either directly or indirectly, inhibits the proteins involved in providing protection against atrophy.     

Use of Xpert MTB/RIF in Decentralized Public Health Settings and Its Effect on Pulmonary TB and DR-TB Case Finding in India

BackgroundXpert MTB/RIF, the first automated molecular test for tuberculosis, is transforming the diagnostic landscape in high-burden settings. This study assessed the impact of up-front Xpert MTB/RIF testing on detection of pulmonary tuberculosis (PTB) and rifampicin-resistant PTB (DR-TB) cases in India.MethodsThis demonstration study was implemented in 18 sub-district level TB programme units (TUs) in India in diverse geographic and demographic settings covering a population of 8.8 million. A baseline phase in 14 TUs captured programmatic baseline data, and an intervention phase in 18 TUs had Xpert MTB/RIF offered to all presumptive TB patients. We estimated changes in detection of TB and DR-TB, the former using binomial regression models to adjust for clustering and covariates.ResultsIn the 14 study TUs, which participated in both phases, 10,675 and 70,556 presumptive TB patients were enrolled in the baseline and intervention phase, respectively, and 1,532 (14.4%) and 14,299 (20.3%) bacteriologically confirmed PTB cases were detected. The implementation of Xpert MTB/RIF was associated with increases in both notification rates of bacteriologically confirmed TB cases (adjusted incidence rate ratio [aIRR] 1.39; CI 1.18-1.64), and proportion of bacteriological confirmed TB cases among presumptive TB cases (adjusted risk ratio (aRR) 1.33; CI 1.6-1.52). Compared with the baseline strategy of selective drug-susceptibility testing only for PTB cases at high risk of drug-resistant TB, Xpert MTB/RIF implementation increased rifampicin resistant TB case detection by over fivefold. Among, 2765 rifampicin resistance cases detected, 1055 were retested with conventional drug susceptibility testing (DST). Positive predictive value (PPV) of rifampicin resistance detected by Xpert MTB/RIF was 94.7% (CI 91.3-98.1), in comparison to conventional DST.ConclusionIntroduction of Xpert MTB/RIF as initial diagnostic test for TB in public health facilities significantly increased case-notification rates of all bacteriologically confirmed TB by 39% and rifampicin-resistant TB case notification by fivefold.     

Marine bacterial,archaeal and protistan association networks reveal ecological linkages

Microbes have central roles in ocean food webs and global biogeochemical processes, yet specific ecological relationships among these taxa are largely unknown. This is in part due to the dilute, microscopic nature of the planktonic microbial community, which prevents direct observation of their interactions. Here, we use a holistic (that is, microbial system-wide) approach to investigate time-dependent variations among taxa from all three domains of life in a marine microbial community. We investigated the community composition of bacteria, archaea and protists through cultivation-independent methods, along with total bacterial and viral abundance, and physico-chemical observations. Samples and observations were collected monthly over 3 years at a well-described ocean time-series site of southern California. To find associations among these organisms, we calculated time-dependent rank correlations (that is, local similarity correlations) among relative abundances of bacteria, archaea, protists, total abundance of bacteria and viruses and physico-chemical parameters. We used a network generated from these statistical correlations to visualize and identify time-dependent associations among ecologically important taxa, for example, the SAR11 cluster, stramenopiles, alveolates, cyanobacteria and ammonia-oxidizing archaea. Negative correlations, perhaps suggesting competition or predation, were also common. The analysis revealed a progression of microbial communities through time, and also a group of unknown eukaryotes that were highly correlated with dinoflagellates, indicating possible symbioses or parasitism. Possible ‘keystone'' species were evident. The network has statistical features similar to previously described ecological networks, and in network parlance has non-random, small world properties (that is, highly interconnected nodes). This approach provides new insights into the natural history of microbes.     

How Do Patients Who Fail First-Line TB Treatment but Who Are Not Placed on an MDR-TB Regimen Fare in South India?

Setting

Seven districts in Andhra Pradesh, South India

Objectives

To a) determine treatment outcomes of patients who fail first line anti-TB treatment and are not placed on an multi-drug resistant TB (MDR-TB) regimen, and b) relate the treatment outcomes to culture and drug susceptibility patterns (C&DST).

Design

Retrospective cohort study using routine programme data and Mycobacterium TB Culture C&DST between July 2008 and December 2009.

Results

There were 202 individuals given a re-treatment regimen and included in the study. Overall treatment outcomes were: 68 (34%) with treatment success, 84 (42%) failed, 36 (18%) died, 13 (6.5%) defaulted and 1 transferred out. Treatment success for category I and II failures was low at 37%. In those with positive cultures, 81 had pan-sensitive strains with 31 (38%) showing treatment success, while 61 had drug-resistance strains with 9 (15%) showing treatment success. In 58 patients with negative cultures, 28 (48%) showed treatment success.

Conclusion

Treatment outcomes of patients who fail a first-line anti-TB treatment and who are not placed on an MDR-TB regimen are unacceptably poor. The worst outcomes are seen among category II failures and those with negative cultures or drug-resistance. There are important programmatic implications which need to be addressed.     

A Genomic Insight into Diversity Among Tribal and Nontribal Population Groups of Manipur, India

Twenty autosomal markers, including linked markers at two gene markers, are used to understand the genomic similarity and diversity among three tribal (Paite, Thadou, and Kom) and one nontribal communities of Manipur (Northeast India). Two of the markers (CD4 and HB9) are monomorphic in Paite and one (the CD4 marker) in Kom. Data suggest the Meitei (nontribal groups) stand apart from the three tribal groups with respect to higher heterozygosity (0.366) and presence of the highest ancestor haplotypes of DRD2 markers (0.228); this is also supported by principal co-ordinate analysis. These populations are found to be genomically closer to the Chinese population than to other Indian populations.     

Assembly and Disassembly of the Capsid-Like External Scaffold of Immature Virions during Vaccinia Virus Morphogenesis

Infectious poxvirus particles are unusual in that they are brick shaped and lack symmetry. Nevertheless, an external honeycomb lattice comprised of a capsid-like protein dictates the spherical shape and size of immature poxvirus particles. In the case of vaccinia virus, trimers of 63-kDa D13 polypeptides form the building blocks of the lattice. In the present study, we addressed two questions: how D13, which has no transmembrane domain, associates with the immature virion (IV) membrane to form the lattice structure and how this scaffold is removed during the subsequent stage of morphogenesis. Interaction of D13 with the A17 membrane protein was demonstrated by immunoaffinity purification and Western blot analysis. In addition, the results of immunogold electron microscopy indicated a close association of A17 and D13 in crescents, as well as in vesicular structures when crescent formation was prevented. Further studies indicated that binding of A17 to D13 was abrogated by truncation of the N-terminal segment of A17. The N-terminal region of A17 was also required for the formation of crescent and IV structures. Disassembly of the D13 scaffold correlated with the processing of A17 by the I7 protease. When I7 expression was repressed, D13 was retained on aberrant virus particles. Furthermore, the morphogenesis of IVs to mature virions was blocked by mutation of the N-terminal but not the C-terminal cleavage site on A17. Taken together, these data indicate that A17 and D13 interactions regulate the assembly and disassembly of the IV scaffold.The assembly and morphogenesis of vaccinia virus (VACV) and other poxviruses occurs in specialized regions of the cytoplasm called factories. The first distinctive viral forms discerned by transmission electron microscopy are spherical immature virions (IVs) and their membrane crescent precursors, which appear to be covered by a layer of spicules (14). More-recent studies employing three-dimensional deep-etch electron microscopy revealed that the “spicule coat” of IVs is actually a continuous honeycomb lattice (20). The IVs enclose dense granular material comprising the core precursors and a DNA nucleoid. The “spicule coat” is lost as the IVs undergo a remarkable transition into dense, brick-shaped infectious mature virions (MVs).Several studies led to the identification of D13 protein trimers as the building blocks of the scaffold: (i) single amino acid changes in D13 are responsible for VACV mutants that are resistant to the drug rifampin (rifampicin) (4, 11, 42), which causes reversible formation of irregular membranes lacking the “spicule coat” (18, 29, 30); (ii) repression of D13 expression results in a phenotype identical to that caused by the drug rifampin (50); (iii) antibody to D13 labels IVs (40) on the outer surface (28, 41); (iv) in the presence of rifampin, D13 antibodies label cytoplasmic inclusions that are distinct from aberrant viral membranes (40); and (v) the results of physical and microscopic studies indicate that D13 exists as trimers of 63-kDa subunits arranged mostly in hexagons on the surface of IVs (41).Poxviruses are thought to share a common origin with members of the asfarvirus, iridovirus, phycodnavirus, and mimivirus families (23). These large DNA viruses, except for the poxviruses, have an icosahedral capsid surrounding an internal membrane (31, 47-49). Interestingly, a domain of VACV D13 has homology with the capsid proteins of these related large DNA viruses (24). Moreover, a parapoxvirus ortholog of D13 was shown to self-assemble in vitro and to have structural similarities with the capsid proteins (22). These findings, together with the honeycomb lattice structure of the IV scaffold, suggest that the infectious form of the ancestor of poxviruses may have had an icosahedral capsid and that the stages of morphogenesis recapitulate evolution (41).In the present study, we addressed two questions: how D13, which has no transmembrane domain, associates with the IV membrane to form the lattice structure and how the scaffold is removed during morphogenesis.