Longitudinal genetic analyses of Staphylococcus aureus nasal carriage dynamics in a diverse population

Background: Staphylococcus aureus (SA) nasal colonization plays a critical role in the pathogenesis of staphylococcal infections and SA eradication from the nares has proven to be effective in reducing endogenous infections. To understand SA nasal colonization and its relation with consequent disease, assessment of nasal carriage dynamics and genotypic diversity among a diverse population is a necessity. Results: We have performed extensive longitudinal monitoring of SA nasal carriage isolates in 109 healthy individuals over a period of up to three years. Longitudinal sampling revealed that 24% of the individuals were persistent SA nasal carriers while 32% were intermittent. To assess the genetic relatedness between different SA isolates within our cohort, multi locus sequence typing (MLST) was performed. MLST revealed that not only were strains colonizing intermittent and persistent nasal carriers genetically similar, belonging to the same clonal complexes, but strain changes within the same host were also observed over time for both types of carriers. More highly discriminating genetic analyses using the hypervariable regions of staphylococcal protein A and clumping factor B virulence genes revealed no preferential colonization of specific SA strains in persistent or intermittent carriers. Moreover, we observed that a subset of persistent and intermittent carriers retained clinically relevant community-acquired methicillin-resistant SA (CA-MRSA) strains in their nares over time. Conclusions: The findings of this study provides added perspective on the nasal carriage dynamics between strains colonizing persistent and intermittent carriers; an area currently in need of assessment given that persistent carriers are at greater risk of autoinfection than intermittent carriers

2,4-dihydroxy benzaldehyde derived Schiff bases as small molecule Hsp90 inhibitors: rational identification of a new anticancer lead

Hsp90 is a molecular chaperone that heals diverse array of biomolecules ranging from multiple oncogenic proteins to the ones responsible for development of resistance to chemotherapeutic agents. Moreover they are over-expressed in cancer cells as a complex with co-chaperones and under-expressed in normal cells as a single free entity. Hence inhibitors of Hsp90 will be more effective and selective in destroying cancer cells with minimum chances of acquiring resistance to them. In continuation of our goal to rationally develop effective small molecule azomethines against Hsp90, we designed few more compounds belonging to the class of 2,4-dihydroxy benzaldehyde derived imines (1-13) with our validated docking protocol. The molecules exhibiting good docking score were synthesized and their structures were confirmed by IR, (1)H NMR and mass spectral analysis. Subsequently, they were evaluated for their potential to suppress Hsp90 ATPase activity by Malachite green assay. The antiproliferative effect of the molecules were examined on PC3 prostate cancer cell lines by adopting 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay methodology. Finally, schiff base 13 emerged as the lead molecule for future design and development of Hsp90 inhibitors as anticancer agents.Fil: Dutta Gupta, Sayan. Osmania University; India. Jawaharlal Nehru Technological University; IndiaFil: Revathi, B.. Osmania University; IndiaFil: Mazaira, Gisela Ileana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Galigniana, Mario Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Subrahmanyam, C. V. S.. Osmania University; IndiaFil: Gowrishankar, N. L.. Swami Vivekananda Institute of Pharmaceutical Sciences; IndiaFil: Raghavendra, N. M.. Osmania University; Indi

PET imaging of tumor glycolysis downstream of hexokinase through noninvasive measurement of pyruvate kinase M2

Cancer cells reprogram their metabolism to meet increased biosynthetic demands, commensurate with elevated rates of replication. Pyruvate kinase M2 (PKM2) catalyzes the final and rate-limiting step in tumor glycolysis, controlling the balance between energy production and the synthesis of metabolic precursors. We report here the synthesis and evaluation of a positron emission tomography (PET) radiotracer, [(11)C]DASA-23, that provides a direct noninvasive measure of PKM2 expression in preclinical models of glioblastoma multiforme (GBM). In vivo, orthotopic U87 and GBM39 patient-derived tumors were clearly delineated from the surrounding normal brain tissue by PET imaging, corresponding to exclusive tumor-associated PKM2 expression. In addition, systemic treatment of mice with the PKM2 activator TEPP-46 resulted in complete abrogation of the PET signal in intracranial GBM39 tumors. Together, these data provide the basis for the clinical evaluation of imaging agents that target this important gatekeeper of tumor glycolysis

Structure Prediction and Active Site Analysis of New H1N1 Neuraminidase:Target for Antiviral Drug Design

Abstract: The H1N1 viral envelope protein neuraminidase encoded by NA gene plays a key role in the pathogenesis of swine flu. The active site of the neuraminidase protein is targeted by presently available antiviral drugs. The influenza virus often proves to be resistant to currently available drugs, due single amino acid substitutions conferred by the mutations in the gene coding for neuraminidase protein. The latest Influenza A virus A/Perth/262/2009(H1N1) sequence with accession number ADJ67981 was selected from NCBI. The BLAST program was used to identify the best template structure, which was found to be 3NSS_A. Sequence alignment was carried out with the template and query sequence, the identity and similarity was found to be 81.9% and 82.6% respectively. Homology modeling was performed using Accelrys Discovery Studio 3.5 software, the model with the lowest energy was then assessed for stereochemical quality and side-chain environment. The PDF energy and DOPE score of the best modeled structure was 2090.1682 and -43752.3632 respectively. Further active site optimization of the modeled protein was performed by molecular dynamics. The key active site residues which are crucial for further docking studies were ascertained

Mechanical characterization of heat treated Al2219 hybrid composites

Aluminium alloy matrix composites with Al2O3 reinforcements exhibit superior mechanical properties and utilize in several demanding fields’ viz., automobile, aerospace, defense, sports equipment, electronics and bio-medical. The present work emphasizes on improvement of microstructure and mechanical properties of age hardened graphite and alumina reinforced Al alloy matrix hybrid composites. Different composites with a constant carbon content of 1 weight % and 0, 2, 4 and 6 weight % Al2O3 as reinforcements are fabricated by using stir casting technic and tested for hardness, tensile and impact strength. Scanning electron microscopy (SEM) is performed to analyse the failure mode under tensile load. All the composites are subjected to age hardening treatment with solutionising temperature of 530oC and aging temperatures of 100 and 200oC. The peak hardness of the composites at two aging temperatures are noted. Tensile and impact tests are conducted for the peak aged specimens. Results show substantial increase in the hardness of the age hardened specimens in the range of 34-44% in comparison with the as cast specimens. Result analysis shows increase in tensile strength (upto 40%) and decrease in impact resistance (upto 33%) with the increase in weight % of reinforcements. As the aging temperature increases a reduction in tensile strength and impact resistance is observed in each composites

Varicella zoster virus infection of highly pure terminally differentiated human neurons

In vitro analyses of varicella zoster virus (VZV) reactivation from latency in human ganglia have been hampered by the inability to isolate virus by explantation or cocultivation techniques. Furthermore, attempts to study interaction of VZV with neurons in experimentally infected ganglion cells in vitro have been impaired by the presence of nonneuronal cells, which become productively infected and destroy the cultures. We have developed an in vitro model of VZV infection in which highly pure (>95 %) terminally differentiated human neurons derived from pluripotent stem cells were infected with VZV. At 2 weeks post-infection, infected neurons appeared healthy compared to VZV-infected human fetal lung fibroblasts (HFLs), which developed a cytopathic effect (CPE) within 1 week. Tissue culture medium from VZV-infected neurons did not produce a CPE in uninfected HFLs and did not contain PCR-amplifiable VZV DNA, but cocultivation of infected neurons with uninfected HFLs did produce a CPE. The nonproductively infected neurons contained multiple regions of the VZV genome, as well as transcripts and proteins corresponding to VZV immediate-early, early, and late genes. No markers of the apoptotic caspase cascade were detected in healthy-appearing VZV-infected neurons. VZV infection of highly pure terminally differentiated human neurons provides a unique in vitro system to study the VZV-neuronal relationship and the potential to investigate mechanisms of VZV reactivation

Troubleshooting coupled in vitro transcription–translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins

Cell-free coupled transcription–translation systems with bacterial lysates are widely used to synthesize recombinant proteins in amounts of several mg per ml. By using reporter green fluorescence protein (GFP) we demonstrate that proteins are synthesized with an unsatisfyingly low-active fraction of (50 ± 20)%. One reason is probably the T7 polymerase used, being up to eight times faster than the intrinsic transcriptase and thus breaking the coupling between transcription and translation in bacterial systems. The active fraction of the synthesized protein was improved by using either a slower T7 transcriptase mutant or lowering the incubation temperature to 20°C. A drop of protein synthesis observed after 7 h incubation time was not due to a shortage of nucleotide triphosphates, but rather to a shortage of amino acids. Accordingly, a second addition of amino acids after 10 h during an incubation at 20°C led to synthesis of up to 4 mg/ml of GFP with virtually 100% activity

Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli

Now that many genomes have been sequenced and the products of newly identified genes have been annotated, the next goal is to engineer the desired phenotypes in organisms of interest. For the phenotypic engineering of microorganisms, we have developed novel artificial transcription factors (ATFs) capable of reprogramming innate gene expression circuits in Escherichia coli. These ATFs are composed of zinc finger (ZF) DNA-binding proteins, with distinct specificities, fused to an E. coli cyclic AMP receptor protein (CRP). By randomly assembling 40 different types of ZFs, we have constructed more than 6.4 × 104 ATFs that consist of 3 ZF DNA-binding domains and a CRP effector domain. Using these ATFs, we induced various phenotypic changes in E. coli and selected for industrially important traits, such as resistance to heat shock, osmotic pressure and cold shock. Genes associated with the heat-shock resistance phenotype were then characterized. These results and the general applicability of this platform clearly indicate that novel ATFs are powerful tools for the phenotypic engineering of microorganisms and can facilitate microbial functional genomic studies

Electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells

It has been reported previously that electric pulses of sufficiently high voltage and short duration can permeabilize the membranes of various organelles inside living cells. In this article, we describe electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells. The cells were exposed to short, high-voltage electric pulses (from 1 to 20 pulses, 60 ns, 50 kV/cm, repetition frequency 1 kHz). We observed that 10 and 20 such pulses induced permeabilization of membranes of endocytotic vesicles, detected by release of lucifer yellow from the vesicles into the cytosol. Simultaneously, we detected uptake of propidium iodide through plasma membrane in the same cells. With higher number of pulses permeabilization of the membranes of endocytotic vesicles by pulses of given parameters is accompanied by permeabilization of plasma membrane. However, with lower number of pulses only permeabilization of the plasma membrane was detected