Effect of Arsenic on Fluoride Tolerance in Microbacterium paraoxydans Strain IR-1.

Fluoride (F) and arsenic (As) are two major contaminants of water and soil systems around the globe, causing potential toxicity to humans, plants, animals, and microbes. These contaminated soil systems can be restored by microorganisms that can tolerate toxic stress and provide rapid mineralization of soil, organic matter, and contaminants, using various tolerance mechanisms. Thus, the present study was undertaken with the arsenic hyper-tolerant bacterium Microbacterium paraoxydans strain IR-1 to determine its tolerance and toxicity to increasing doses of fluoride, either individually or in combination with arsenic, in terms of growth inhibition using a toxicity unit model. The minimum inhibitory concentration (MIC)and half maximal inhibitory concentration (IC50) values for fluoride increased, from 9 g/L to 11 g/L and from 5.91 ± 0.1 g/L to 6.32 ± 0.028 g/L, respectively, in the combination (F + As) group. The statistical comparison of observed and expected additive toxicities, with respect to toxicity unit (TU difference), using Student's t-test, was found to be highly significant (p < 0.001). This suggests the antagonistic effect of arsenic on fluoride toxicity to the strain IR-1. The unique stress tolerance of IR-1 ensures its survival as well as preponderance in fluoride and arsenic co-contaminated sites, thus paving the way for its possible application in the natural or artificial remediation of toxicant-exposed degraded soil systems.

Role of High Frequency Ultrasound in Diagnosing Carpal Tunnel Syndrome as Compared with Conventional Nerve Conduction Studies.

OBJECTIVE: Our aim was to evaluate High Frequency Ultrasonography as a tool for diagnosis in patients withcarpal tunnel syndrome in comparison with electrophysiological study. METHODS: Thirty- one patients [56 hands] with CTS and twenty-five asymptomatic controls [50 hands] were assessed and underwent ultrasonography of the wrists and electrophysiological testing. Data from the patient and the control groups was compared for both the investigations to determine the CTS and the grade of severity. RESULTS: There was a high degree of correlation between the conduction abnormalities of the median nerve as detected by electrodiagnostic tests, historic and objective scale [Hi-Ob] and the measurement of the cross-sectional area of the nerve by US (P < 0.05). A cut-off point of 0.88 mm2 for the mean cross-sectional area of the median nerve was found to be the upper limit for normal values. Compared to Ultrasonography which found one hand negative, six hands (10%) were negative on the electrophysiological tests. Using critical CSA value of 1.0 mm2 in these CTS cases by US with sensitivity and specificity of 100% and 88%. Based on the results of this study, ultrasonography of wrist is another useful tool along with nerve conduction studies as per sensitivity and specificity patterns found in our study in diagnosis of carpal tunnel syndrome. CONCLUSION: High-frequency US examination of the median nerve and measurement of its cross-sectional area can be strongly considered as useful diagnostic diagnostic modality for the evaluation of CTS along with nerve conduction studies. In addition to its high diagnostic accuracy it is able to define the cause of nerve compression, aids treatment planning and provides a reliable method to follow response to therapy.

Genetic and antigenic variation of foot-and-mouth disease virus during persistent infection in naturally infected cattle and Asian buffalo in India.

The role of foot-and-mouth disease virus (FMDV) persistently infected ruminants in initiating new outbreaks remains controversial, and the perceived threat posed by such animals hinders international trade in FMD-endemic countries. In this study we report longitudinal analyses of genetic and antigenic variations of FMDV serotype O/ME-SA/Ind2001d sublineage during naturally occurring, persistent infection in cattle and buffalo at an organised dairy farm in India. The proportion of animals from which FMDV RNA was recovered was not significantly different between convalescent (post-clinical) and sub-clinically infected animals or between cattle and buffalo across the sampling period. However, infectious virus was isolated from a higher proportion of buffalo samples and for a longer duration compared to cattle. Analysis of the P1 sequences from recovered viruses indicated fixation of mutations at the rate of 1.816 x 10-2substitution/site/year (s/s/y) (95% CI 1.362-2.31 x 10-2 s/s/y). However, the majority of point mutations were transitional substitutions. Within individual animals, the mean dN/dS (ω) value for the P1 region varied from 0.076 to 0.357, suggesting the selection pressure acting on viral genomes differed substantially across individual animals. Statistical parsimony analysis indicated that all of the virus isolates from carrier animals originated from the outbreak virus. The antigenic relationship value as determined by 2D-VNT assay revealed fluctuation of antigenic variants within and between carrier animals during the carrier state which suggested that some carrier viruses had diverged substantially from the protection provided by the vaccine strain. This study contributes to understanding the extent of within-host and within-herd evolution that occurs during the carrier state of FMDV.

Comprehensive Validation of Cytology Specimens for Next-Generation Sequencing and Clinical Practice Experience.

Biopsy specimens are subjected to an expanding portfolio of assays that regularly include mutation profiling via next-generation sequencing (NGS). Specimens derived via fine-needle aspiration, a common biopsy technique, are subjected to a variety of cytopreparatory methods compared with surgical biopsies that are almost uniformly processed as formalin-fixed, paraffin-embedded tissue. Therefore, the fine-needle aspiration-derived specimens most commonly accepted for molecular analysis are cell blocks (CBs), because they are processed most similarly to surgical biopsy tissue. However, CB preparations are fraught with challenges that risk unsuccessful sequencing and repeat biopsies, with the potential to further increase health care costs and delay clinical care. The diversity of cytopreparations and the resource-intensive clinical validation of NGS pose significant challenges to more consistent use of non-CB (NCB) cytology specimens. As part of clinical validation of a targeted NGS assay, DNA subjected to nine cytopreparatory methods was evaluated for sequencing performance and was shown to be uniformly acceptable for clinical NGS. Of the 379 clinical cases analyzed after validation, the majority (56%) were derived from NCB cytology specimens. This specimen class had the lowest DNA insufficiency rate (1.5%) and showed equivalent sequencing performance to surgical and CB formalin-fixed, paraffin-embedded tissue. NCB cytology specimens are valuable sources of tumor nucleic acid and are the preferred specimen type for clinical NGS at our institution.

The role of Twist1 in mutant huntingtin-induced transcriptional alterations and neurotoxicity.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an abnormal expansion of polyglutamine repeats in the huntingtin protein (Htt). Transcriptional dysregulation is an early event in the course of HD progression and is thought to contribute to disease pathogenesis, but how mutant Htt causes transcriptional alterations and subsequent cell death in neurons is not well understood. RNA-Seq analysis revealed that expression of a mutant Htt fragment in primary cortical neurons leads to robust gene expression changes before neuronal death. Basic helix-loop-helix transcription factor Twist1, which is essential for embryogenesis and is normally expressed at low levels in mature neurons, was substantially up-regulated in mutant Htt-expressing neurons in culture and in the brains of HD mouse models. Knockdown of Twist1 by RNAi in mutant Htt-expressing primary cortical neurons reversed the altered expression of a subset of genes involved in neuronal function and, importantly, abrogated neurotoxicity. Using brain-derived neurotrophic factor (Bdnf), which is known to be involved in HD pathogenesis, as a model gene, we found that Twist1 knockdown could reverse mutant Htt-induced DNA hypermethylation at the Bdnf regulatory region and reactivate Bdnf expression. Together, these results suggest that Twist1 is an important upstream mediator of mutant Htt-induced neuronal death and may in part operate through epigenetic mechanisms.

Routine use of clinical exome-based next-generation sequencing for evaluation of patients with thrombotic microangiopathies.

Next-generation sequencing is increasingly used for clinical evaluation of patients presenting with thrombotic microangiopathies because it allows for simultaneous interrogation of multiple complement and coagulation pathway genes known to be associated with disease. However, the diagnostic yield is undefined in routine clinical practice. Historic studies relied on case-control cohorts, did not apply current guidelines for variant pathogenicity assessment, and used targeted gene enrichment combined with next-generation sequencing. A clinically enhanced exome, targeting ~54 Mb, was sequenced for 73 patients. Variant analysis and interpretation were performed on genes with biological relevance in thrombotic microangiopathy (C3,CD46, CFB, CFH, CFI, DGKE, and THBD). CFHR3-CFHR1 deletion status was also assessed using multiplex ligation-dependent probe amplification. Variants were classified using American College of Medical Genetics and Genomics guidelines. We identified 5 unique novel and 14 unique rare variants in 25% (18/73) of patients, including a total of 5 pathogenic, 4 likely pathogenic, and 15 variants of uncertain clinical significance. Nine patients had homozygous deletions in CFHR3-CFHR1. The diagnostic yield, defined as the presence of a pathogenic variant, likely pathogenic variant or homozygous deletion of CFHR3-CFHR1, was 25% for all patients tested. Variants of uncertain clinical significance were identified in 21% (15/73) of patients.These results illustrate the expected diagnositic yield in the setting of thrombotic microangiopathies through the application of standardized variant interpretation, and highlight the utility of such an approach. Sequencing a clinically enhanced exome to enable targeted, disease-specific variant analysis is a viable approach. The moderate rate of variants of uncertain clinical significance highlights the paucity of data surrounding the variants in our cohort and illustrates the need for expanded variant curation resources to aid in thrombotic microangiopathy-related disease variant classification.

caTissue Suite to OpenSpecimen: Developing an extensible, open source, web-based biobanking management system.

The National Cancer Institute (NCI) Cancer Biomedical Informatics Grid® (caBIG®) program established standards and best practices for biorepository data management by creating an infrastructure to propagate biospecimen resource sharing while maintaining data integrity and security. caTissue Suite, a biospecimen data management software tool, has evolved from this effort. More recently, the caTissue Suite continues to evolve as an open source initiative known as OpenSpecimen. The essential functionality of OpenSpecimen includes the capture and representation of highly granular, hierarchically-structured data for biospecimen processing, quality assurance, tracking, and annotation. Ideal for multi-user and multi-site biorepository environments, OpenSpecimen permits role-based access to specific sets of data operations through a user-interface designed to accommodate varying workflows and unique user needs. The software is interoperable, both syntactically and semantically, with an array of other bioinformatics tools given its integration of standard vocabularies thus enabling research involving biospecimens. End-users are encouraged to share their day-to-day experiences in working with the application, thus providing to the community board insight into the needs and limitations which need be addressed. Users are also requested to review and validate new features through group testing environments and mock screens. Through this user interaction, application flexibility and interoperability have been recognized as necessary developmental focuses essential for accommodating diverse adoption scenarios and biobanking workflows to catalyze advances in biomedical research and operations. Given the diversity of biobanking practices and workforce roles, efforts have been made consistently to maintain robust data granularity while aiding user accessibility, data discoverability, and security within and across applications by providing a lower learning curve in using OpenSpecimen. Iterative development and testing cycles provide continuous maintenance and up-to-date capabilities for this freely available, open-access, web-based software application that is globally-adopted at over 25 institutions.

Electrochemical DNA sensor for anthrax toxin activator gene atxA-detection of PCR amplicons.

We report the DNA probe functionalized electrochemical genosensor for the detection of Bacillus anthracis, specific towards the regulatory gene atxA. The DNA sensor is fabricated on electrochemically deposited gold nanoparticle on self assembled layer of (3-Mercaptopropyl) trimethoxysilane (MPTS) on GC electrode. DNA hybridization is monitored by differential pulse voltammogram (DPV). The modified GC electrode is characterized by atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) method. We also quantified the DNA probe density on electrode surface by the chronocoulometric method. The detection is specific and selective for atxA gene by DNA probe on the electrode surface. No report is available for the detection of B. anthracis by using atxA an anthrax toxin activator gene. In the light of real and complex sample, we have studied the PCR amplicons of 303, 361 and 568 base pairs by using symmetric and asymmetric PCR approaches. The DNA probe of atxA gene efficiently hybridizes with different base pairs of PCR amplicons. The detection limit is found to be 1.0 pM (S/N ratio=3). The results indicate that the DNA sensor is able to detect synthetic target as well as PCR amplicons of different base pairs.

Electrochemical immunosensor based on bismuth nanocomposite film and cadmium ions functionalized titanium phosphates for the detection of anthrax protective antigen toxin.

Bacillus anthracis is a bioterrorism agent classified by the Centers for Disease Control and Prevention (CDC). Herein, a novel electrochemical immunosensor for the sensitive, specific and easy detection of anthrax protective antigen (PA) toxin in picogram concentration was developed. The immunosensor consists of (i) a Nafion-multiwall carbon nanotubes-bismuth nanocomposite film modified glassy carbon electrodes (BiNPs/Nafion-MWCNTs/GCE) as a sensing platform and (ii) titanium phosphate nanoparticles-cadmium ion-mouse anti-PA antibodies (TiP-Cd(2+)-MαPA antibodies) as signal amplification tags. Scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), thermogravimmetric analysis (TGA), Fourier transform-infra red spectroscopy (FT-IR), zeta-potential analysis, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed to characterize the synthesized TiP nanoparticles and modified electrode surfaces. The immunosensing performance of BiNPs/Nafion-MWCNTs/GCE was evaluated based on sandwich immunoassay protocol. A square wave voltammetry (SWV) scan from -1.2 to -0.3 V in HAc-NaAc buffer solution (pH 4.6) without stripping process was performed to record the electrochemical responses at -0.75 V corresponding to high content of Cd(2+) ions loaded in TiP nanoparticles for the measurement of PA toxin. Under optimal conditions, the currents increased with increasing PA toxin concentrations in spiked human serum samples and showed a linear range from 0.1 ng/ml to 100 ng/ml. The limit of detection of developed immunosensor was found to be 50 pg/ml at S/N=3. The total time of analysis was 35 min.

Electrochemical immunosensor for botulinum neurotoxin type-E using covalently ordered graphene nanosheets modified electrodes and gold nanoparticles-enzyme conjugate.

In this work, a novel electrochemical immunosensor was developed for the detection of botulinum neurotoxin-E (BoNT/E). This method relied on graphene nanosheets-aryldiazonium salt modified glassy carbon electrodes (GCE) as sensing platform and enzyme induced silver nanoparticles (AgNPs) deposited on gold nanoparticles (AuNPs) as signal amplifier. Herein, a GCE was electrografted with mixed monolayer of phenyl and aminophenyl (Ph-PhNH2/GCE) by diazotization reaction. Further, graphene nanosheets (GNS) were covalently attached on electrode surface (GNS/Ph-PhNH2/GCE). Field emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed to characterize synthesized graphene oxide and modified electrode surfaces. In the sandwich immunoassay format, the sensitivity was amplified using rabbit anti-mouse IgG-alkaline phosphatase (RαMIgG-ALP) functionalized with gold nanoparticles (RαMIgG-ALP/AuNPs). In order to study the immunosensing performance of GNS/Ph-PhNH2/GCE, first the capturing antibody (rabbit-anti BoNT/E antibody) was covalently immobilized via EDC/NHS chemistry. Further, the electrode was sequentially subjected to sample containing spiked BoNT/E, revealing antibody (mouse-anti BoNT/E) followed by RαMIgG-ALP/AuNPs. 3-indoxyl phosphate (3-IP) was used as substrate which finally reduces the silver ions. The deposited AgNPs on electrode surface were determined by linear sweep voltammetry (LSV). The developed electrochemical immunosensor could detect BoNT/E with linear range from 10pg/ml to 10ng/ml with the minimum detection limit of 5.0pg/ml and total analysis time of 65min. In addition, the immunosensor was successfully evaluated against food samples (orange juice and milk).

Chemoenzymatic routes to enantiomerically enriched and polyoxygenated perhydro-3,5a-methanoindeno[4,5-c]furans related to the tashironin class of sesquiterpenes.

The alcohol 12, which is available in eight steps from the enzymatically derived cis-1,2-dihydrocatechol 8, engages in an intramolecular alkoxy radical-mediated remote functionalization reaction to form the tetrahydrofuran 13, thus establishing the perhydro-3,5a-methanoindeno[4,5-c]furan framework associated with the biologically active tashironins. Various manipulations of compound 13 and certain derivatives allow for the formation of compounds bearing strong structural resemblances to the title natural products.

An electrochemical genosensor for Salmonella typhi on gold nanoparticles-mercaptosilane modified screen printed electrode.

In this work, we fabricated a system of integrated self-assembled layer of organosilane 3-mercaptopropyltrimethoxy silane (MPTS) on the screen printed electrode (SPE) and electrochemically deposited gold nanoparticle for Salmonella typhi detection employing Vi gene as a molecular marker. Thiolated DNA probe was immobilized on a gold nanoparticle (AuNP) modified SPE for DNA hybridization assay using methylene blue as redox (electroactive) hybridization indicator, and signal was monitored by differential pulse voltammetry (DPV) method. The modified SPE was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) method. The DNA biosensor showed excellent performances with high sensitivity and good selectivity. The current response was linear with the target sequence concentrations ranging from 1.0 × 10(-11) to 0.5 × 10(-8)M and the detection limit was found to be 50 (± 2.1)pM. The DNA biosensor showed good discrimination ability to the one-base, two-base and three-base mismatched sequences. The fabricated genosensor could also be regenerated easily and reused for three to four times for further hybridization studies.

Clinical genomicist workstation.

The use of NextGen Sequencing clinically necessitates the need for informatics tools that support the complete workflow from sample accessioning to data analysis and reporting. To address this need we have developed Clinical Genomicist Workstation (CGW). CGW is a secure, n-tiered application where web browser submits requests to application servers that persist the data in a relational database. CGW is used by Washington University Genomic and Pathology Services for clinical genomic testing of many cancers. CGW has been used to accession, analyze and sign out over 409 cases since November, 2011. There are 22 ordering oncologists and 7 clinical genomicists that use the CGW. In summary, CGW a 'soup-to-nuts' solution to track, analyze, interpret, and report clinical genomic diagnostic tests.

Life sciences domain analysis model.

OBJECTIVE: Meaningful exchange of information is a fundamental challenge in collaborative biomedical research. To help address this, the authors developed the Life Sciences Domain Analysis Model (LS DAM), an information model that provides a framework for communication among domain experts and technical teams developing information systems to support biomedical research. The LS DAM is harmonized with the Biomedical Research Integrated Domain Group (BRIDG) model of protocol-driven clinical research. Together, these models can facilitate data exchange for translational research. MATERIALS AND METHODS: The content of the LS DAM was driven by analysis of life sciences and translational research scenarios and the concepts in the model are derived from existing information models, reference models and data exchange formats. The model is represented in the Unified Modeling Language and uses ISO 21090 data types. RESULTS: The LS DAM v2.2.1 is comprised of 130 classes and covers several core areas including Experiment, Molecular Biology, Molecular Databases and Specimen. Nearly half of these classes originate from the BRIDG model, emphasizing the semantic harmonization between these models. Validation of the LS DAM against independently derived information models, research scenarios and reference databases supports its general applicability to represent life sciences research. DISCUSSION: The LS DAM provides unambiguous definitions for concepts required to describe life sciences research. The processes established to achieve consensus among domain experts will be applied in future iterations and may be broadly applicable to other standardization efforts. CONCLUSIONS: The LS DAM provides common semantics for life sciences research. Through harmonization with BRIDG, it promotes interoperability in translational science.

Amperometric immunosensor based on gold nanoparticles/alumina sol-gel modified screen-printed electrodes for antibodies to Plasmodium falciparum histidine rich protein-2.

We report herein the amperometric immunosensor for antibodies to Plasmodium falciparum histidine rich protein-2 (PfHRP-2). Screen-printed electrodes (SPEs) were modified with alumina sol-gel (Al(2)O(3) sol-gel) derived film and gold nanoparticles i.e. AuNPs/Al(2)O(3)sol-gel/SPE. A thin film was formed by dripping Al(2)O(3) sol on SPE followed by electrochemical deposition of gold nanoparticles (AuNPs). The modified SPEs were characterized by scanning electron microscopy/energy dispersive X-ray analysis (SEM-EDAX), Raman spectra and voltammetric experiments. Antibodies in rabbit serum sample were allowed to react with the PfHRP-2 protein that was immobilized on the modified SPE to form antigen-antibody immune complex (PfHRP-2/anti-PfHRP-2). The bound antibodies were quantified by alkaline phosphatase (AP) enzyme labeled secondary antibodies (anti-rabbit immunoglobulins-AP conjugate). Enzymatic substrate, 1-naphthyl phosphate was converted to 1-naphthol by AP and an electroactive product was quantified using amperometry. The performances of the developed immunosensor and Dot-ELISA were tested against different dilutions of hyper immune serum (rabbit anti-PfHRP-2). Dot ELISA and the developed immunosensor (AuNPs/Al(2)O(3)sol-gel/SPE) results for the hyper immune serum containing anti-PfHRP-2 were distinctly positive when diluted upto 8 times (1 : 12800 dilution) and 11 times (1 : 102400 dilution), respectively. The developed immunosensor was applied for antibodies to PfHRP-2 in human clinical samples.

Trends in mitral valve surgery: a single practice experience.

BACKGROUND AND AIM OF THE STUDY: Mitral repair has evolved to a point where three methods can be used to address most pathologies: full ring annuloplasty (RA) for annular disease; Gore-Tex artificial chordal replacement (ACR) for chordal disease; and autologous pericardial augmentation (PA) for leaflet disease. The study aim was to assess the impact of the increasing application of these methods on operative results over time. METHODS: Of 328 consecutive mitral valve procedures, 34% involved myxomatous prolapse, 23% rheumatic, 13% ischemic, 12% pure annular dilatation, 7% prosthetic dysfunction, 6% endocarditis, 3% hypertrophic obstructive cardiomyopathy (HOCM), and 2% 'other'. All patients underwent RA. Myxomatous prolapse was repaired with ACR, and ischemic and annular dilatation with RA alone. Rheumatic, endocarditis, and HOCM etiologies were repaired with all three methods. Patients were allocated to two-year increments, and also to repair versus replacement groups. Operative outcomes over time were assessed with linear and binomial regression. RESULTS: Overall, 66% of mitral valves were repaired; the average operative mortality was 6% (2% for repair, 7% for replacement), and 18% involved multiple valve procedures (mortality 16%). The extent of repair increased over time, from 55% to 100% of all etiologies. Over the same period, operative mortality fell from 6% in 1994 to 0% over the past six years. Other variables, such as age, presentation status, left ventricular dysfunction and etiology were relatively constant over the period. Reoperation rates after repair have been only 2% over the past 10 years of follow up. CONCLUSION: With recent innovations, most mitral disease can be repaired with combinations of RA, ACR and PA. Today, operative mortality is approaching zero, and one factor may be the increasing application of repair to all mitral pathologies. These data support the trend of expanding valve repair across most mitral disorders.

Immobilization of acetylcholineesterase-choline oxidase on a gold-platinum bimetallic nanoparticles modified glassy carbon electrode for the sensitive detection of organophosphate pesticides, carbamates and nerve agents.

A novel, highly sensitive amperometric biosensor, based on electrodeposition of gold-platinum bimetallic nanoparticles onto 3-aminopropyltriethoxy silane modified glassy carbon electrode for the detection of paraoxon ethyl, aldicarb, and sarin has been developed. The biosensor consists of acetylcholineesterase (AChE)/choline oxidase (ChOx) immobilized by cross-linking with glutaraldehyde on a modified electrode. The properties of nanoparticles modified electrodes are characterized by scanning electron microscopy (SEM), energy dispersive X-ray (EDX), cyclic voltammograms (CVs) and electrochemical impedance spectroscopy (EIS). The synergistic action of Au and Pt nanoparticles showed excellent electrocatalytic activity with low applied potential for the detection of hydrogen peroxide (H(2)O(2)). The IC(50) and inhibition rate constant (K(i)) values were determined for the inhibitors using immobilized enzymes on modified electrode and the data were compared by spectrophotometric determination of these kinetic parameters using free enzymes in solution. Paraoxon ethyl, sarin, and aldicarb could be detected up to 150-200nM, 40-50nM, and 40-60 microM respectively at 30-40% inhibition level of AChE enzyme and followed linearity in wide range concentration.