Mutation studies of the gene encoding YuiC, a stationary phase survival protein in Bacillus subtilis

Aims: YuiC is a stationary phase survival (Sps) protein from the Firmicute Bacillus subtilis that possesses muralytic activity to cleave bacterial cell-wall peptidoglycan. It has a small lytic transglycosylase (MltA) fold analogous to the resuscitation promoting factors (Rpfs) of Actinobacteria which have a hybrid of a mini lysozyme and soluble lytic transglycosylase (Slt35/70) fold. The present study aimed at identifying key residues of YuiC/Sps that are catalytically active and studying the effect of B. subtilis cell growth upon sps/yuiC deletion. Methodology and results: Four forms of mutated yuiC were created through Site-directed, Ligase-Independent Mutagenesis Polymerase Chain Reaction (SLIM PCR) that include the substitutions of D129A, D151A, D162A and K102A. These individual mutated yuiC genes were cloned and expressed in the Escherichia coli BL21 (DE3) expression system and subsequently purified to homogeneity using affinity, cation exchange and size exclusion chromatography. The D129A variant was shown to be insoluble, indicating its role in maintaining the right protein folding of YuiC. The remaining three variants resulted in soluble proteins but were inactive on zymograms indicating that they may be responsible for catalysis. B. subtilis cells harbouring individual sps genes (yuiC, yabE, yocH and yorM) knocked out showed stationary phase defects and altered colony morphologies compared to the wild type. Conclusion, significance and impact of study: This study has identified the key residues involved in catalysis of YuiC, which are the D151, D162 and K102. These are conserved in Sps domains. The catalytic mechanism of YuiC is similar to the mechanism reported for Neisseria gonorrhoeae MltA. sps/yuiC knock outs have implied that each sps/yuiC has a significant role on B. subtilis late growth stage. The B. subtilis YuiC/Sps model has given an insight into Sps functions in the final growth stage of the Firmicutes, which members include etiologic agents of anthrax, botulism and listeriosis. Inhibition of Sps protein may inactivate pathogen replication and facilitate entrance into a non-contagious dormant sporulation stage

Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery

Essential residues for the enzyme activity of ATP-dependent MurE ligase from Mycobacterium tuberculosis

The emergence of total drug-resistant tuberculosis (TDRTB) has made the discovery of new therapies for tuberculosis urgent. The cytoplasmic enzymes of peptidoglycan biosynthesis have generated renewed interest as attractive targets for the development of new anti-mycobacterials. One of the cytoplasmic enzymes, uridine diphosphate (UDP)-MurNAc-tripeptide ligase (MurE), catalyses the addition of meso-diaminopimelic acid (m-DAP) into peptidoglycan in Mycobacterium tuberculosis coupled to the hydrolysis of ATP. Mutants of M. tuberculosis MurE were generated by replacing K157, E220, D392, R451 with alanine and N449 with aspartate, and truncating the first 24 amino acid residues at the N-terminus of the enzyme. Analysis of the specific activity of these proteins suggested that apart from the 24 Nterminal residues, the other mutated residues are essential for catalysis. Variations in K m values for one or more substrates were observed for all mutants, except the N-terminal truncation mutant, indicating that these residues are involved in binding substrates and form part of the active site structure. These mutant proteins were also tested for their specificity for a wide range of substrates. Interestingly, the mutations K157A, E220A and D392A showed hydrolysis of ATP uncoupled from catalysis. The ATP hydrolysis rate was enhanced by at least partial occupation of the uridine nucleotide dipeptide binding site. This study provides an insight into the residues essential for the catalytic activity and substrate binding of the ATP-dependent MurE ligase. Since ATP-dependent MurE ligase is a novel drug target, the understanding of its function may lead to development of novel inhibitors against resistant forms of M. tuberculosis

What is eHealth (6)? Development of a Conceptual Model for eHealth: Qualitative Study with Key Informants

©Tim Shaw, Deborah McGregor, Melissa Brunner, Melanie Keep, Anna Janssen, Stewart Barnet. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 24.10.2017. BACKGROUND: Despite rapid growth in eHealth research, there remains a lack of consistency in defining and using terms related to eHealth. More widely cited definitions provide broad understanding of eHealth but lack sufficient conceptual clarity to operationalize eHealth and enable its implementation in health care practice, research, education, and policy. Definitions that are more detailed are often context or discipline specific, limiting ease of translation of these definitions across the breadth of eHealth perspectives and situations. A conceptual model of eHealth that adequately captures its complexity and potential overlaps is required. This model must also be sufficiently detailed to enable eHealth operationalization and hypothesis testing.OBJECTIVE: This study aimed to develop a conceptual practice-based model of eHealth to support health professionals in applying eHealth to their particular professional or discipline contexts.METHODS: We conducted semistructured interviews with key informants (N=25) from organizations involved in health care delivery, research, education, practice, governance, and policy to explore their perspectives on and experiences with eHealth. We used purposeful sampling for maximum diversity. Interviews were coded and thematically analyzed for emergent domains.RESULTS: Thematic analyses revealed 3 prominent but overlapping domains of eHealth: (1) health in our hands (using eHealth technologies to monitor, track, and inform health), (2) interacting for health (using digital technologies to enable health communication among practitioners and between health professionals and clients or patients), and (3) data enabling health (collecting, managing, and using health data). These domains formed a model of eHealth that addresses the need for clear definitions and a taxonomy of eHealth while acknowledging the fluidity of this area and the strengths of initiatives that span multiple eHealth domains.CONCLUSIONS: This model extends current understanding of eHealth by providing clearly defined domains of eHealth while highlighting the benefits of using digital technologies in ways that cross several domains. It provides the depth of perspectives and examples of eHealth use that are lacking in previous research. On the basis of this model, we suggest that eHealth initiatives that are most impactful would include elements from all 3 domains

Cell wall peptidoglycan in Mycobacterium tuberculosis: An Achilles’ heel for the TB-causing pathogen

Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis, remains one of the leading causes of mortality across the world. There is an urgent requirement to build a robust arsenal of effective antimicrobials, targeting novel molecular mechanisms to overcome the challenges posed by the increase of antibiotic resistance in TB. Mycobacterium tuberculosis has a unique cell envelope structure and composition, containing a peptidoglycan layer that is essential for maintaining cellular integrity and for virulence. The enzymes involved in the biosynthesis, degradation, remodelling and recycling of peptidoglycan have resurfaced as attractive targets for anti-infective drug discovery. Here, we review the importance of peptidoglycan, including the structure, function and regulation of key enzymes involved in its metabolism. We also discuss known inhibitors of ATP-dependent Mur ligases, and discuss the potential for the development of pan-enzyme inhibitors targeting multiple Mur ligases

Preparedness for eHealth: Health sciences students' knowledge, skills, and confidence

There is increasing recognition of the role eHealth will play in the effective and efficient delivery of healthcare. This research challenges the assumption that students enter university as digital natives, able to confidently and competently adapt their use of information and communication technology (ICT) to new contexts. This study explored health sciences students' preparedness for working, and leading change, in eHealth-enabled environments. Using a cross-sectional study design, 420 undergraduate and postgraduate students participated in an online survey investigating their understanding of and attitude towards eHealth, frequency of online activities and software usage, confidence learning and using ICTs, and perceived learning needs. Although students reported that they regularly engaged with a wide range of online activities and software and were confident learning new ICT skills especially where they have sufficient time or support, their understanding of eHealth was uncertain or limited. Poor understanding of and difficulty translating skills learned in personal contexts to the professional context may impair graduates ability to confidently engage in the eHealth-enabled workplace. These results suggest educators need to scaf-fold the learning experience to ensure students build on their ICT knowledge to transfer this to their future workplaces

Giving Miss Marple a makeover : graduate recruitment, systems failure and the Scottish voluntary sector

The voluntary sector in Scotland, as across the globe, is becoming increasingly business like. Resultantly, there is an increasing demand for graduates to work in business and support functions. In Scotland, however, despite an oversupply of graduates in the labor market, the voluntary sector reports skills shortages for graduate-level positions; a leadership deficit was also reported in countries such as the United States. Through exploratory, mainly qualitative, case study and stakeholder research, this article proposes that one reason for this mismatch between the supply of and demand for graduates is a systems failure within the sector. Many graduates and university students remain unaware of potentially suitable paid job opportunities, in part because of the sector's voluntary label. To rectify this systems failure, thought needs to be given to the sector's nomenclature and the manner in which voluntary sector organizations attract graduate recruits, for example, through levering value congruence in potential recruits

PEPT2-Mediated Uptake of Neuropeptides in Rat Choroid Plexus

Purpose . The peptide transporter PEPT2 was recently shown to be functionally active in rat choroid plexus, suggesting that it may play a role in neuropeptide homeostasis in the cerebrospinal fluid. This study, therefore, examined the role of PEPT2 in mediating neuropeptide uptake into choroid plexus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41485/1/11095_2004_Article_302748.pd

Interdisciplinary eHealth practice in cancer care: A review of the literature

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This review aimed to identify research that described how eHealth facilitates interdisciplinary cancer care and to understand the ways in which eHealth innovations are being used in this setting. An integrative review of eHealth interventions used for interdisciplinary care for people with cancer was conducted by systematically searching research databases in March 2015, and repeated in September 2016. Searches resulted in 8531 citations, of which 140 were retrieved and scanned in full, with twenty-six studies included in the review. Analysis of data extracted from the included articles revealed five broad themes: (i) data collection and accessibility; (ii) virtual multidisciplinary teams; (iii) communication between individuals involved in the delivery of health services; (iv) communication pathways between patients and cancer care teams; and (v) health professional-led change. Use of eHealth interventions in cancer care was widespread, particularly to support interdisciplinary care. However, research has focused on development and implementation of interventions, rather than on long-term impact. Further research is warranted to explore design, evaluation, and long-term sustainability of eHealth systems and interventions in interdisciplinary cancer care. Technology evolves quickly and researchers need to provide health professionals with timely guidance on how best to respond to new technologies in the health sector