ALBERT

Beschreibung: Background: Single-stranded DNA binding proteins (SSB) are essential for DNA replication, repair, and recombination in all organisms. SSB works in concert with a variety of DNA metabolizing enzymes such as DNA polymerase. Results: We have cloned and purified SSB from Bacillus anthracis (SSBBA). In the absence of DNA, at concentrations [less than or equal to]100 mug/ml, SSBBA did not form a stable tetramer and appeared to resemble bacteriophage T4 gene 32 protein. Fluorescence anisotropy studies demonstrated that SSBBA bound ssDNA with high affinity comparable to other prokaryotic SSBs. Thermodynamic analysis indicated both hydrophobic and ionic contributions to ssDNA binding. FRET analysis of oligo(dT)70 binding suggested that SSBBA forms a tetrameric assembly upon ssDNA binding. This report provides evidence of a bacterial SSB that utilizes a novel mechanism for DNA binding through the formation of a transient tetrameric structure. Conclusions: Unlike other prokaryotic SSB proteins, SSBBA from Bacillus anthracis appeared to be monomeric at concentrations [less than or equal to]100 mug/ml as determined by SE-HPLC. SSBBA retained its ability to bind ssDNA with very high affinity, comparable to SSB proteins which are tetrameric. In the presence of a long ssDNA template, SSBBA appears to form a transient tetrameric structure. Its unique structure appears to be due to the cumulative effect of multiple key amino acid changes in its sequence during evolution, leading to perturbation of stable dimer and tetramer formation. The structural features of SSBBA could promote facile assembly and disassembly of the protein-DNA complex required in processes such as DNA replication.

Beschreibung: BackgroundIn recent years, the emergence of multidrug resistant hypervirulent K. pneumoniae (MDR hvKp) isolates poses severe therapeutic challenge to global public health. The present study used the complete genome sequence of two MDR hvKp isolates belonging to ST23 to characterize the phylogenetic background and plasmid diversity.MethodsTwo hvKp isolates from patients with bacteremia were sequenced using Ion Torrent PGM and Oxford Nanopore MinION platforms and assembled by hybrid genome assembly approach. Comparative genomics approaches were used to investigate the population structure, evolution, virulence, and antimicrobial resistance of MDR hvKp strains.ResultsThe study isolates exhibited typical features of hvKp phenotypes associated with ST23. The convergence of multidrug resistance and hypervirulence were attributed by the presence of multiple plasmids including a 216 kb virulence plasmid and MDR plasmids belonging to IncA/C2, IncFIB, IncX3, and ColKP3 groups. The insertion of catA1 gene into virulence plasmid was observed along with genetic factors such as aerobactin, salmochelin, and rmpA2 that confer hvKp’s hypervirulent phenotype. The core genome single nucleotide polymorphism (SNP) phylogenetic analyses of the isolates showed the evolution of ST23 hvKp was predominantly driven by ICEKp acquisitions.ConclusionTo the best of our knowledge, this is the first report of MDR hvKp isolates of ST23 with insertion of catA1 gene into the virulence plasmid which presents the possibility of hotspot integration sites on the plasmids to aid acquisition of AMR genes. ST23 is no longer confined to susceptible strains of hvKp. Our findings emphasize the need for more studies on recombinant events, plasmid transmission dynamics and evolutionary process involving hvKp.

Beschreibung: Background: Elevated glucose concentrations lead to increased insulin secretion and suppression of glucagon secretion. In fact, insulin is a physiological inhibitor of glucagon secretion. Type 2 diabetes mellitus (T2DM) patients have defects in insulin secretion. In addition to this, lack of suppression of glucagon secretion under elevated glucose concentrations is also observed in T2DM patients. We have earlier shown that GPR40 activation by CNX-011-67 stimulates glucose stimulated insulin secretion (GSIS). Here we extended our studies to examine the impact of GPR40 activation by CNX-011-67 on glucagon secretion from intact islets under both normal and glucolipotoxic conditions.FindingsGlucagon secretion from intact rat islets was suppressed under elevated glucose concentration. Activation of GPR40 by CNX-011-67 further suppressed glucagon secretion. Culturing islets under chronic glucolipotoxic (GL) conditions, we have observed increased high glucose mediated glucagon secretion and content which were reduced with GPR40 activation by CNX-011-67. Interestingly, expression of pre-proglucagon gene (GCG) remained unchanged under glucolipotoxicity in the presence or absence of GPR40 activation. Conclusion: Activation of GPR40 by CNX-011-67 can reduce glucagon secretion from pancreatic islets.

Beschreibung: Background: Heat stress leads to accelerated production of reactive oxygen species (ROS) which causes a huge amount of oxidative damage to the cellular components of plants. A large number of heat stress related genes as HSPs, catalases, peroxidases are overexpressed at the time of stress. A potent stress responsive gene peroxisomal ascorbate peroxidase (TapAPX) obtained from heat stress (42[degree sign]C) responsive subtractive cDNA library from a thermo tolerant wheat cv. Raj3765 at anthesis stage was cloned, characterized and its role was validated under heat stress by proteomics and in-silico studies.. In the present study we report the characterization at molecular and in-silico level of peroxisomal TapAPX gene isolated from heat tolerant wheat cultivar of India. Results: qPCR studies of TapAPX gene displayed up to 203 fold level of expression at 42[degree sign]C heat stress exposure. A full length cDNA of 876 bp obtained by RACE deduced a protein of 292 amino acid residues which gives a complete 3D structure of pAPX by homology modeling. TapAPX cDNA was cloned in expression vector pET28 (a+) and the recombinant protein over-expressed in E. coli BL21 showed highest homology with APX protein as deduced by peptide mass fingerprinting. Conclusions: TapAPX gene from wheat cv Raj3765 has a distinct role in conferring thermo tolerance to the plants and thus can be used in crop improvement programmes for development of crops tolerant to high temperature.

Beschreibung: Background: Procalcitonin is useful for the diagnosis of sepsis but its prognostic value regarding mortality is unclear. This prospective observational study was designed to study the prognostic value of procalcitonin in prediction of 28 day mortality in patients of sepsis. Fifty-four consecutive patients of sepsis, severe sepsis and septic shock defined using the 2001 Consensus Conference SCCM/ESICM/ACCP/ATS/SIS criteria from medical Intensive Care Unit (ICU) of a tertiary care center in New Delhi, India were enrolled from July 2011 to June 2013. Procalcitonin (PCT), C-reactive protein (CRP) measurements were recorded on day 1, day 7 and day 28 of follow up. Results: Procalcitonin value was a better predictor of all-cause short-term mortality than C-reactive protein. Those patients with Procalcitonin levels

Beschreibung: Background: In the progression towards diabetes, glucolipotoxicity is one of the main causes of pancreatic beta cell pathology. The aim of this study was to examine the in vitro effects of chronic glucolipotoxic conditions on cellular responses in pancreatic islets, including glucose and fat metabolism, Calcium mobilization, insulin secretion and insulin content. Results: Exposure of islets to chronic glucolipotoxic conditions decreased glucose stimulated insulin secretion in vitro. Reduced protein levels of Glut2/slc2a2, and decreased glucokinase and pyruvate carboxylase mRNA levels indicated a significant lowering in glucose sensing. Concomitantly, both fatty acid uptake and triglyceride accumulation increased significantly while fatty acid oxidation decreased. This general suppression in glucose metabolism correlated well with a decrease in mitochondrial number and activity, reduction in cellular ATP content and dampening of the TCA cycle. Further, we also observed a decrease in IP3 levels and lower Calcium mobilization in response to glucose. Importantly, chronic glucolipotoxic conditions in vitro decreased insulin gene expression, insulin content, insulin granule docking (to the plasma membrane) and insulin secretion. Conclusions: Our results present an integrated view of the effects of chronic glucolipotoxic conditions on known and novel signaling events, in vitro, that results in reduced glucose responsiveness and insulin secretion.

Beschreibung: Background; With an ever-growing ageing population, dementia is fast becoming the chronic disease of the 21stcentury. Elderly people affected with dementia progressively lose their autonomy as they encounterproblems in their Activities of Daily Living (ADLs). Hence, they need supervision and assistancefrom their family members or professional caregivers, which can often lead to underestimated psychologicaland financial stress for all parties. The use of Ambient Assistive Living (AAL) technologiesaims to empower people with dementia and relieve the burden of their caregivers.The aim of this paper is to present the approach we have adopted to develop and deploy a systemfor ambient assistive living in an operating nursing home, and evaluate its performance and usabilityin real conditions. Based on this approach, we emphasise on the importance of deployments in realworld settings as opposed to prototype testing in laboratories.Methods; We chose to conduct this work in close partnership with end-users (dementia patients) and specialistsin dementia care (professional caregivers). Our trial was conducted during a period of 14 monthswithin three rooms in a nursing home in Singapore, and with the participation of eight dementiapatients and two caregivers. A technical ambient assistive living solution, consisting of a set of sensorsand devices controlled by a software platform, was deployed in the collaborating nursing home. Thetrial was preceded by a pre-deployment period to organise several observation sessions with dementiapatients and focus group discussions with professional caregivers. A process of ground truth andsystem's log data gathering was also planned prior to the trial and a system performance evaluationwas realised during the deployment period with the help of caregivers. An ethical approval wasobtained prior to real life deployment of our solution.Results; Patients' observations and discussions allowed us to gather a set of requirements that a system forelders with mild-dementia should fulfil. In fact, our deployment has exposed more concrete requirementsand problems that need to be addressed, and which cannot be identified in laboratory testing.Issues that were neither forecasted during the design phase nor during the laboratory testing surfacedduring deployment, thus affecting the effectiveness of the proposed solution. Results of the systemperformance evaluation show the evolution of system precision and uptime over the deploymentphases, while data analysis demonstrates the ability to provide early detection of the degradation ofpatients' conditions. A qualitative feedback was collected from caregivers and doctors and a set oflessons learned emerged from this deployment experience.Conclusion; Lessons learned from this study were very useful for our research work and can serve as inspirationfor developers and providers of assistive living services. They confirmed the importance of realdeployment to evaluate assistive solutions especially with the involvement of professional caregivers.They also asserted the need for larger deployments. Larger deployments will allow to conduct surveyson assistive solutions social and health impact, even though they are time and manpower consumingduring their first phases.

Beschreibung: Background Mutation of amino acid sequences in a protein may have diverse effects on its structure and function. Point mutations ofeven a single amino acid residue in the helices of thenon-redundant database may lead to sequentially identical peptides whichadopt different secondary structures in different proteins. However, variousphysico-chemical factors which govern the formation of these ambivalent helices generated by point mutationsof a sequence are not clearly known.Results Sequences generated by point mutations of helices are mapped on to theirnon-helical counterparts in the SCOP database. The results show thatshort helices are prone to transform into non-helical conformations upon pointmutations. Mutation of amino acid residues by helix breakerspreferentially yield non-helical conformations, while mutation withresidues of intermediate helix propensity display least preferences fornon-helical conformations. Differences in the solvent accessibility of themutating/mutated residues are found to be a major criteria for these sequencesto conform to non-helical conformations. Even with minimal differencesin the amino acid distributions of the sequences flanking the helicaland non-helical conformations, helix-flanking sequences are found bemore solvent accessible.Conclusions All types of mutations from helicalto non-helical conformations are investigated. The primary factorsattributing such changes in conformation can be: i) type/propensity ofthe mutating and mutant residues ii) solvent accessibility of the residue at the mutation siteiii) context/environment dependence of the flanking sequences. Theresults from the present study may be used to design de novoproteins via point mutations.