Arshad Khan

Bacterial FHA domains: neglected players in the phospho-threonine signalling game?

Forkhead-associated (FHA) domains bind phospho-threonine peptides and are known to mediate phosphorylation-dependent protein-protein interactions in a variety of eukaryotic settings. However, their role in bacterial physiology and signalling has been largely neglected. We have surveyed bacterial FHA domains and discovered that they are implicated in many bacterial processes, including regulation of cell shape, type III secretion, sporulation, pathogenic and symbiotic host-bacterium interactions, carbohydrate storage and transport, signal transduction and ethambutol resistance. The way is now open to identify the targets of each FHA domain, and their roles in cellular physiology, and perhaps even to develop novel FHA-blocking antibacterial agents.

coliBASE: an online database for Escherichia coli, Shigella and Salmonella comparative genomics.

We have constructed coliBASE, a database for Escherichia coli, Shigella and Salmonella comparative genomics available online at http://colibase. bham.ac.uk. Unlike other E.coli databases, which focus on the laboratory model strain K12, coliBASE is intended to reflect the full diversity of E.coli and its relatives. The database contains comparative data including whole genome alignments and lists of putative orthologous genes, together with numerous analytical tools and links to existing online resources. The data are stored in a relational database, accessible by a number of user-friendly search methods and graphical browsers. The database schema is generic and can easily be applied to other bacterial genomes. Two such databases, CampyDB (for the analysis of Campylobacter spp.) and ClostriDB (for Clostridium spp.) are also available at http://campy.bham.ac.uk and http://clostri. bham.ac.uk, respectively. An example of the power of E.coli comparative analyses such as those available through coliBASE is presented.

Bactericidal activity of 2-nitroimidazole against the active replicating stage of Mycobacterium bovis BCG and Mycobacterium tuberculosis with intracellular efficacy in THP-1 macrophages

This study evaluated the antituberculous potential of 2-nitroimidazole under in vitro conditions. Minimal bactericidal concentrations of the compound against actively replicating Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra were found to be 0.226 microg/mL and 0.556 microg/mL in enriched and minimal medium, respectively. Minimal inhibitory concentrations were >100 times lower than reported antituberculous nitroimidazoles such as nitrofurantoin and furaltadone, indicating the greater potential of 2-nitroimidazole. No discernible effect of 2-nitroimidazole was seen on saprophytic Mycobacterium smegmatis and the representative bacterial strain Escherichia coli DH5alpha, indicating the specificity of the molecule against tuberculous mycobacteria. The compound was also found to be effective against M. tuberculosis in the intracellular environment of the human monocytic cell line THP-1, with a reduction in viability of bacilli by 2.5 log after 144 h of incubation at a concentration of 0.113 microg/mL. A five-fold higher concentration (0.565 microg/mL) of 2-nitroimidazole sterilised the macrophages of intracellular pathogens within 192 h, without affecting the host. However, 2-nitroimidazole was unable to affect significantly the viability of dormant non-replicating bacilli of M. bovis BCG and M. tuberculosis in Waynes in vitro model. Overall, the results indicate that 2-nitroimidazole is a potent antituberculous agent active against the organisms active replicating stage, with promising intracellular efficacy as well.

Models for distinguishing innovative and noninnovative small firms

The importance of innovation is underscored by the fact that some authors blame U.S. industrys competitive decline on the excessively short-term focus of managers as opposed to longer-term innovative product or process development. At the same time the contribution of small firms to research and innovation continues to rise. Thus from 1945 to 1980 they introduced an average 48% share of innovations, a figure that rose to 56% for the 1980–1985 period. This article studies innovation in small manufacturing firms in Texas. The study sample was drawn from the 1985 Directory of Texas Manufacturers and covered SIC codes 34–39. These include Metal Fabrication, Nonelectrical Machinery, Electrical and Electronic Machinery, Transportation Equipment, Instrumentation, and Miscellaneous Manufacturing. A total of 50 usable responses were received of which the CEO was the responding executive in 38. Prior literature on innovation provided the relevant variables. In broad groupings, these covered strategy, structure, function, environment, the firm, and the responding executive plus his role in innovation. Product differentiation and risk taking were also included. Each broad group often consisted of several variables. For example, the environment grouping comprised dynamism, heterogeneity, and hostility. In all there were 31 independent variables. Their data values were based on either the responses to individual questions or the means of responses to groups of questions. The dependent variable, product-service innovation, averaged the responses to questions on technological leadership and quantity and quality of innovations. A stepwise regression procedure yields an eight-variable model with an R2 of 0.66. A runs test on the residuals confirms the randomness of errors. The computed discriminant function classifies the sample firms correctly in 43 of 50 cases. While the modeling process is successful, the models have not been retested on fresh data because of the small sample and so the caveat of sample specificity remains. The recurring variables in the two models are integrated decision making, environmental heterogeneity, percentage of research expenditure to cost of goods sold, and the responding executives role in technical development of innovations. For the latter, the answer seems to be that less is more or “hands-off” is best. The coefficients for the others are all positive. For investors or lenders the consequence of being able to evaluate the innovative potential of small firms is likely to attenuate the risk. Secondarily, it is also likely to enhance the efficient allocation of resources. The data obtained lacked sufficient variation in structural variables like centralization—the sample firms tended to be centralized. Thus future research exploring centralization and its effect on innovation in small firms would be most interesting, particularly as prior results are somewhat ambiguous.

The fbpA/sapM Double Knock Out Strain of Mycobacterium tuberculosis Is Highly Attenuated and Immunogenic in Macrophages

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine.

Nitrite Reductase NirBD Is Induced and Plays an Important Role during In Vitro Dormancy of Mycobacterium tuberculosis

Mycobacterium tuberculosis is one of the strongest reducers of nitrate among all mycobacteria. Reduction of nitrate to nitrite, mediated by nitrate reductase (NarGHJI) of M. tuberculosis, is induced during the dormant stage, and the enzyme has a respiratory function in the absence of oxygen. Nitrite reductase (NirBD) is also functional during aerobic growth when nitrite is the sole nitrogen source. However, the role of NirBD-mediated nitrite reduction during the dormancy is not yet characterized. Here, we analyzed nitrite reduction during aerobic growth as well as in a hypoxic dormancy model of M. tuberculosis in vitro. When nitrite was used as the sole nitrogen source in the medium, the organism grew and the reduction of nitrite was evident in both hypoxic and aerobic cultures of M. tuberculosis. Remarkably, the hypoxic culture of M. tuberculosis, compared to the aerobic culture, showed 32- and 4-fold-increased expression of nitrite reductase (NirBD) at the transcription and protein levels, respectively. More importantly, a nirBD mutant of M. tuberculosis was unable to reduce nitrite and compared to the wild-type (WT) strain had a >2-log reduction in viability after 240 h in the Wayne model of hypoxic dormancy. Dependence of M. tuberculosis on nitrite reductase (NirBD) was also seen in a human macrophage-based dormancy model where the nirBD mutant was impaired for survival compared to the WT strain. Overall, the increased expression and essentiality of nitrite reductase in the in vitro dormancy models suggested that NirBD-mediated nitrite reduction could be critical during the persistent stage of M. tuberculosis.

BCG vaccine mediated reduction in the MHC-II expression of macrophages and dendritic cells is reversed by activation of Toll-like receptors 7 and 9.

Tuberculosis is a major cause of death in mankind and BCG vaccine protects against childhood but not adult tuberculosis. BCG avoids lysosomal fusion in macrophages decreasing peptides required for activating CD4 T cells and Th1 immunity while suppressing the expression of MHC-II by antigen presenting cells (APCs). An in vitro model of antigen presentation showed that ligands for TLR-9, 7, 4 and 1/2 increased the ability of APCs to present antigen-85B of BCG to CD4 T cells, which correlated with an increase in MHC-II expression. TLR-activation led to a down-regulation of MARCH1 ubiquitin ligase which prevents the degradation of MHC-II and decreased IL-10 also contributed to an increase in MHC-II. TLR-activation induced up-regulation of MHC-II was inhibited by the blockade of IRAK, NF-kB, and MAPKs. TLR-7 and TLR-9 ligands had the most effective adjuvant like effect on MHC-II of APCs which allowed BCG vaccine mediated activation of CD4 T cells.

Nitrate reduction pathways in mycobacteria and their implications during latency

Mycobacterial persistence has gained a lot of attention with respect to developing novel antitubercular drugs, which could drastically reduce the duration of tuberculosis (TB) therapy. A better understanding of the physiology of Mycobacterium tuberculosis, and of the metabolic state of the bacillus during the latent period, is a primary need in finding drug targets against persistent TB. Recent biochemical and genetic studies of nitrate reduction in mycobacteria have revealed the roles of distinct proteins and enzymes involved in the pathway. The differential degree of nitrate reduction among pathogenic and non-pathogenic mycobacterial species, and its regulation during oxygen and nutrient limitation, suggest a link between nitrate reduction pathways and latency. The respiratory and assimilatory reduction of nitrate in mycobacteria may be interconnected to facilitate rapid adaptation to changing oxygen and/or nitrogen conditions, increasing metabolic flexibility for survival in the hostile host environment. This review summarizes the nitrate metabolic pathways operative in mycobacteria to provide an insight into the mechanisms that M. tuberculosis has evolved to adapt successfully to the host environment.

The Capital Asset Pricing Model in Project Selection: A Case Study

In this case study four actual energy efficiency projects are analyzed with two traditional engineering economy capital budgeting methods - internal rate of return and payback period - and the results compared with those obtained using the capital asset pricing model (CAPM). The alternative scenarios of the future states-of-the-world necessary for the latter are based on information available in Department of Energy reports. The traditional and CAPM approaches result in different economic conclusions for some of the projects. These differences are analyzed and the implications for certain types of improvement projects and equipment replacement problems as well as financial decision-making in general are discussed.

Neural input is critical for arcuate hypothalamic neurons to mount intracellular signaling responses to systemic insulin and deoxyglucose challenges in male rats: Implications for communication within feeding and metabolic control networks

The hypothalamic arcuate nucleus (ARH) controls rat feeding behavior in part through peptidergic neurons projecting to the hypothalamic paraventricular nucleus (PVH). Hindbrain catecholaminergic (CA) neurons innervate both the PVH and ARH, and ablation of CA afferents to PVH neuroendocrine neurons prevents them from mounting cellular responses to systemic metabolic challenges such as insulin or 2-deoxy-d-glucose (2-DG). Here, we asked whether ablating CA afferents also limits their ARH responses to the same challenges or alters ARH connectivity with the PVH. We examined ARH neurons for three features: (1) CA afferents, visualized by dopamine-β-hydroxylase (DBH)- immunoreactivity; (2) activation by systemic metabolic challenge, as measured by increased numbers of neurons immunoreactive (ir) for phosphorylated ERK1/2 (pERK1/2); and (3) density of PVH-targeted axons immunoreactive for the feeding control peptides Agouti-related peptide and α-melanocyte-stimulating hormone (αMSH). Loss of PVH DBH immunoreactivity resulted in concomitant ARH reductions of DBH-ir and pERK1/2-ir neurons in the medial ARH, where AgRP neurons are enriched. In contrast, pERK1/2 immunoreactivity after systemic metabolic challenge was absent in αMSH-ir ARH neurons. Yet surprisingly, axonal αMSH immunoreactivity in the PVH was markedly increased in CA-ablated animals. These results indicate that (1) intrinsic ARH activity is insufficient to recruit pERK1/2-ir ARH neurons during systemic metabolic challenges (rather, hindbrain-originating CA neurons are required); and (2) rats may compensate for a loss of CA innervation to the ARH and PVH by increased expression of αMSH. These findings highlight the existence of a hierarchical dependence for ARH responses to neural and humoral signals that influence feeding behavior and metabolism.