MOST: a modified MLST typing tool based on short read sequencing

Multilocus sequence typing (MLST) is an effective method to describe bacterial populations. Conventionally, MLST involves Polymerase Chain Reaction (PCR) amplification of housekeeping genes followed by Sanger DNA sequencing. Public Health England (PHE) is in the process of replacing the conventional MLST methodology with a method based on short read sequence data derived from Whole Genome Sequencing (WGS). This paper reports the comparison of the reliability of MLST results derived from WGS data, comparing mapping and assembly-based approaches to conventional methods using 323 bacterial genomes of diverse species. The sensitivity of the two WGS based methods were further investigated with 26 mixed and 29 low coverage genomic data sets from Salmonella enteridis and Streptococcus pneumoniae. Of the 323 samples, 92.9% (n = 300), 97.5% (n = 315) and 99.7% (n = 322) full MLST profiles were derived by the conventional method, assembly- and mapping-based approaches, respectively. The concordance between samples that were typed by conventional (92.9%) and both WGS methods was 100%. From the 55 mixed and low coverage genomes, 89.1% (n = 49) and 67.3% (n = 37) full MLST profiles were derived from the mapping and assembly based approaches, respectively. In conclusion, deriving MLST from WGS data is more sensitive than the conventional method. When comparing WGS based methods, the mapping based approach was the most sensitive. In addition, the mapping based approach described here derives quality metrics, which are difficult to determine quantitatively using conventional and WGS-assembly based approaches

Amyloid Plaques Beyond Aβ: A Survey of the Diverse Modulators of Amyloid Aggregation

Aggregation of the amyloid-β (Aβ) peptide is strongly correlated with Alzheimer’s disease (AD). Recent research has improved our understanding of the kinetics of amyloid fibril assembly and revealed new details regarding different stages in plaque formation. Presently, interest is turning toward studying this process in a holistic context, focusing on cellular components which interact with the Aβ peptide at various junctures during aggregation, from monomer to cross-β amyloid fibrils. However, even in isolation, a multitude of factors including protein purity, pH, salt content, and agitation affect Aβ fibril formation and deposition, often producing complicated and conflicting results. The failure of numerous inhibitors in clinical trials for AD suggests that a detailed examination of the complex interactions that occur during plaque formation, including binding of carbohydrates, lipids, nucleic acids, and metal ions, is important for understanding the diversity of manifestations of the disease. Unraveling how a variety of key macromolecular modulators interact with the Aβ peptide and change its aggregation properties may provide opportunities for developing therapies. Since no protein acts in isolation, the interplay of these diverse molecules may differentiate disease onset, progression, and severity, and thus are worth careful consideration

Design Principles for Ligand-Sensing, Conformation-Switching Ribozymes

Nucleic acid sensor elements are proving increasingly useful in biotechnology and biomedical applications. A number of ligand-sensing, conformational-switching ribozymes (also known as allosteric ribozymes or aptazymes) have been generated by some combination of directed evolution or rational design. Such sensor elements typically fuse a molecular recognition domain (aptamer) with a catalytic signal generator (ribozyme). Although the rational design of aptazymes has begun to be explored, the relationships between the thermodynamics of aptazyme conformational changes and aptazyme performance in vitro and in vivo have not been examined in a quantitative framework. We have therefore developed a quantitative and predictive model for aptazymes as biosensors in vitro and as riboswitches in vivo. In the process, we have identified key relationships (or dimensionless parameters) that dictate aptazyme performance, and in consequence, established equations for precisely engineering aptazyme function. In particular, our analysis quantifies the intrinsic trade-off between ligand sensitivity and the dynamic range of activity. We were also able to determine how in vivo parameters, such as mRNA degradation rates, impact the design and function of aptazymes when used as riboswitches. Using this theoretical framework we were able to achieve quantitative agreement between our models and published data. In consequence, we are able to suggest experimental guidelines for quantitatively predicting the performance of aptazyme-based riboswitches. By identifying factors that limit the performance of previously published systems we were able to generate immediately testable hypotheses for their improvement. The robust theoretical framework and identified optimization parameters should now enable the precision design of aptazymes for biotechnological and clinical applications

Exploring the use of internal and externalcontrols for assessing microarray technical performance

<p>Abstract</p> <p>Background</p> <p>The maturing of gene expression microarray technology and interest in the use of microarray-based applications for clinical and diagnostic applications calls for quantitative measures of quality. This manuscript presents a retrospective study characterizing several approaches to assess technical performance of microarray data measured on the Affymetrix GeneChip platform, including whole-array metrics and information from a standard mixture of external spike-in and endogenous internal controls. Spike-in controls were found to carry the same information about technical performance as whole-array metrics and endogenous "housekeeping" genes. These results support the use of spike-in controls as general tools for performance assessment across time, experimenters and array batches, suggesting that they have potential for comparison of microarray data generated across species using different technologies.</p> <p>Results</p> <p>A layered PCA modeling methodology that uses data from a number of classes of controls (spike-in hybridization, spike-in polyA+, internal RNA degradation, endogenous or "housekeeping genes") was used for the assessment of microarray data quality. The controls provide information on multiple stages of the experimental protocol (e.g., hybridization, RNA amplification). External spike-in, hybridization and RNA labeling controls provide information related to both assay and hybridization performance whereas internal endogenous controls provide quality information on the biological sample. We find that the variance of the data generated from the external and internal controls carries critical information about technical performance; the PCA dissection of this variance is consistent with whole-array quality assessment based on a number of quality assurance/quality control (QA/QC) metrics.</p> <p>Conclusions</p> <p>These results provide support for the use of both external and internal RNA control data to assess the technical quality of microarray experiments. The observed consistency amongst the information carried by internal and external controls and whole-array quality measures offers promise for rationally-designed control standards for routine performance monitoring of multiplexed measurement platforms.</p

Spread of Epidemic MRSA-ST5-IV Clone Encoding PVL as a Major Cause of Community Onset Staphylococcal Infections in Argentinean Children

BACKGROUND: Community-associated methicillin-resistant Staphylococcus aureus-(CA-MRSA) strains have emerged in Argentina. We investigated the clinical and molecular evolution of community-onset MRSA infections (CO-MRSA) in children of Córdoba, Argentina, 2005-2008. Additionally, data from 2007 were compared with the epidemiology of these infections in other regions of the country. METHODOLOGY/PRINCIPAL FINDINGS: Two datasets were used: i) lab-based prospective surveillance of CA-MRSA isolates from 3 Córdoba pediatric hospitals-(CBAH1-H3) in 2007-2008 (compared to previously published data of 2005) and ii) a sampling of CO-MRSA from a study involving both, healthcare-associated community-onset-(HACO) infections in children with risk-factors for healthcare-associated infections-(HRFs), and CA-MRSA infections in patients without HRFs detected in multiple centers of Argentina in 2007. Molecular typing was performed on the CA-MRSA-(n: 99) isolates from the CBAH1-H3-dataset and on the HACO-MRSA-(n: 51) and CA-MRSA-(n: 213) isolates from other regions. Between 2005-2008, the annual proportion of CA-MRSA/CA-S. aureus in Córdoba hospitals increased from 25% to 49%, P<0.01. Total CA-MRSA infections increased 3.6 fold-(5.1 to 18.6 cases/100,000 annual-visits, P<0.0001), associated with an important increase of invasive CA-MRSA infections-(8.5 fold). In all regions analyzed, a single genotype prevailed in both CA-MRSA (82%) and HACO-MRSA(57%), which showed pulsed-field-gel electrophoresis-(PFGE)-type-"I", sequence-type-5-(ST5), SCCmec-type-IVa, spa-t311, and was positive for PVL. The second clone, pulsotype-N/ST30/CC30/SCCmecIVc/t019/PVL(+), accounted for 11.5% of total CA-MRSA infections. Importantly, the first 4 isolates of Argentina belonging to South American-USA300 clone-(USA300/ST8/CC8/SCCmecIVc/t008/PVL(+)/ACME(-)) were detected. We also demonstrated that a HA-MRSA clone-(pulsotype-C/ST100/CC5) caused 2% and 10% of CA-MRSA and HACO-MRSA infections respectively and was associated with a SCCmec type closely related to SCCmecIV(2B&5). CONCLUSIONS/SIGNIFICANCE: The dissemination of epidemic MRSA clone, ST5-IV-PVL(+) was the main cause of increasing staphylococcal community-onset infections in Argentinean children (2003-2008), conversely to other countries. The predominance of this clone, which has capacity to express the h-VISA phenotype, in healthcare-associated community-onset cases suggests that it has infiltrated into hospital-settings

Universal Sequence Replication, Reversible Polymerization and Early Functional Biopolymers: A Model for the Initiation of Prebiotic Sequence Evolution

Many models for the origin of life have focused on understanding how evolution can drive the refinement of a preexisting enzyme, such as the evolution of efficient replicase activity. Here we present a model for what was, arguably, an even earlier stage of chemical evolution, when polymer sequence diversity was generated and sustained before, and during, the onset of functional selection. The model includes regular environmental cycles (e.g. hydration-dehydration cycles) that drive polymers between times of replication and functional activity, which coincide with times of different monomer and polymer diffusivity. Template-directed replication of informational polymers, which takes place during the dehydration stage of each cycle, is considered to be sequence-independent. New sequences are generated by spontaneous polymer formation, and all sequences compete for a finite monomer resource that is recycled via reversible polymerization. Kinetic Monte Carlo simulations demonstrate that this proposed prebiotic scenario provides a robust mechanism for the exploration of sequence space. Introduction of a polymer sequence with monomer synthetase activity illustrates that functional sequences can become established in a preexisting pool of otherwise non-functional sequences. Functional selection does not dominate system dynamics and sequence diversity remains high, permitting the emergence and spread of more than one functional sequence. It is also observed that polymers spontaneously form clusters in simulations where polymers diffuse more slowly than monomers, a feature that is reminiscent of a previous proposal that the earliest stages of life could have been defined by the collective evolution of a system-wide cooperation of polymer aggregates. Overall, the results presented demonstrate the merits of considering plausible prebiotic polymer chemistries and environments that would have allowed for the rapid turnover of monomer resources and for regularly varying monomer/polymer diffusivities

ICAR: endoscopic skull‐base surgery

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Molecular gated nanoporous anodic alumina for the detection of cocaine

[EN] We present herein the use of nanoporous anodic alumina (NAA) as a suitable support to implement molecular gates for sensing applications. In our design, a NAA support is loaded with a fluorescent reporter (rhodamine B) and functionalized with a short single-stranded DNA. Then pores are blocked by the subsequent hybridisation of a specific cocaine aptamer. The response of the gated material was studied in aqueous solution. In a typical experiment, the support was immersed in hybridisation buffer solution in the absence or presence of cocaine. At certain times, the release of rhodamine B from pore voids was measured by fluorescence spectroscopy. The capped NAA support showed poor cargo delivery, but presence of cocaine in the solution selectively induced rhodamine B release. By this simple procedure a limit of detection as low as 5 × 10&#8722;7 M was calculated for cocaine. The gated NAA was successfully applied to detect cocaine in saliva samples and the possible re-use of the nanostructures was assessed. Based on these results, we believe that NAA could be a suitable support to prepare optical gated probes with a synergic combination of the favourable features of selected gated sensing systems and NAA.We thank Projects MAT2015-64139-C4-1-R and TEC2015-71324-R (MINECO/FEDER), the Catalan Government (Project 2014 SGR 1344), the ICREA (ICREA2014 Academia Award) and the Generalitat Valenciana (Project PROMETEOII/2014/047) for support. We also thank to the Agencia Espanola del Medicamento y Productos Sanitarios for its concessions. A.R. thanks the UPV for her predoctoral fellowship. The authors also thank the Electron Microscopy Service at UPV for support.Ribes, À.; Xifre Perez, E.; Aznar, E.; Sancenón Galarza, F.; Pardo Vicente, MT.; Marsal, LF.; Martínez-Máñez, R. (2016). Molecular gated nanoporous anodic alumina for the detection of cocaine. Scientific Reports. 6. https://doi.org/10.1038/srep38649S386496Nadrah, P., Planinšek, O. & Gaberšček, M. Stimulus-responsive Mesoporous Silica Particles. J. Mater. Sci. 49, 481–495 (2014).Baeza, A., Colilla, M. & Vallet-Regí, M. Advances in Mesoporous Silica Nanoparticles for Targeted Stimuli-Responsive Drug Delivery. Expert Opin. Drug Deliv. 12, 319–337 (2015).Karimi, M., Mirshekari, H., Aliakbari, M., Zangabad, P. S. & Hamblin, M. R. Smart Mesoporous Silica Nanoparticles for Controlled-Release Drug Delivery. Nanotech. Rev. 5, 195–207 (2016).Aznar, E. et al. Gated Materials for On-Command Release of Guest Molecules. Chem. Rev. 116, 561−718 (2016).Sancenón, F., Pascual, Ll., Oroval, M., Aznar, E. & Martínez-Máñez, R. Gated Silica Mesoporous Materials in Sensing Applications. Chemistry Open. 4, 418–437 (2015).Lu, C.-H., Willner, B. & Willner, I. DNA nanotechnology: From sensing and DNA machines to drug-delivery systems. ACSNano 7, 8320–8332 (2013).Klajn, R., Stoddart, J. F. & Grzybowski, B. A. Nanoparticles Functionalized With Reversible Molecular And Supramolecular Switches. Chem. Soc. Rev. 39, 2203–2237 (2010).Wei, R., Martin, T. G., Rant, U. & Dietz, H. DNA Origami Gatekeepers for Solid-State Nanopores. Angew. Chem. Int. Ed. 51, 4864 4867 (2012).Zhu, C. L., Lu, C. H., Song, X. Y., Yang, H. H. & Wang, X. R. Bioresponsive Controlled Release Using Mesoporous Silica Nanoparticles Capped with Aptamer-Based Molecular Gate. J. Am. Chem. Soc. 133, 1278–1281 (2011).Özalp, V. C., Pinto, A., Nikulina, E., Chulivin, A. & Schäfer, T. In Situ Monitoring of DNA-Aptavalve Gating Function on Mesoporous Silica Nanoparticles. Part. Part. Sys. Charact. 31, 161–167 (2014).Choi, Y. L., Jaworski, J., Seo, M. L., Lee, S. J. & Jung, J. H. Controlled release using mesoporous silica nanoparticles functionalized with 18-crown-6 derivative. J. Mater. Chem. 21, 7882–7885 (2011).Zhang, Z., Wang, F., Balogh, D. & Willner, I. pH-controlled release of substrates from mesoporous SiO2 nanoparticles gated by metal ion-dependent DNAzymes. J. Mater. Chem. B. 2, 4449–4455 (2014).Fu, L. et al. Portable and Quantitative Monitoring of Heavy Metal Ions Using Dnazyme-Capped Mesoporous Silica Nanoparticles with a Glucometer Readout. J. Mater. Chem. B. 1, 6123–6128 (2013).Díez, P. et al. Toward the Design of Smart Delivery Systems Controlled by Integrated Enzyme-Based Biocomputing Ensembles. J. Am. Chem. Soc. 136, 9116–9123 (2014).Tang, D. et al. Low-Cost and Highly Sensitive lmmunosensing Platform for Aflatoxins Using One-Step Competitive Displacement Reaction Mode and Portable Glucometer-Based Detection. Anal. Chem. 86, 11451–11458 (2014).Hou, L., Zhu, C., Wu, X., Chen, G. & Tang, D. Bioresponsive Controlled Release from Mesoporous Silica Nanocontainers with Glucometer Readout. Chem. Commun. 50, 1441–1443 (2014).Chen, Z. et al. Stimulus-response mesoporous silica nanoparticle-based chemiluminescence biosensor for cocaine determination. Biosens. Bioelectro. 75, 8–14 (2016).Pascual, L. L. et al. Oligonucleotide-Capped Mesoporous Silica Nanoparticles as DNA-Responsive Dye Delivery Systems for Genomic DNA Detection. Chem. Commun. 51, 1414–1416 (2015).Qian, R., Ding, I. & Ju, H. Switchable Fluorescent Imaging of Intracellular Telomerase Activity Using Telomerase-Responsive Mesoporous Silica Nanoparticle. J. Am. Chem. Soc. 135, 13282–13285 (2013).Ren, K., Wu, J., Zhang, Y., Yan, F. & Ju, H. Proximity Hybridization Regulated DNA Biogate for Sensitive Electrochemical Immunoassay. Anal. Chem. 86, 7494–7499 (2014).Chen, Y., Santos, A., Wang, Y., Wang, C. & Losic, D. Biomimetic Nanoporous Anodic Alumina Distributed Bragg Reflectors in the Form of Films and Microsized Particles for Sensing Applications. ACS Appl Mater Interfaces. 7, 19816–19824 (2015).Aw, M. S., Bariana, M. & Losic, D. In Nanoporous Alumina. Fabrication, Structure, Properties and Applications (ed. Losic, D., Santos, A. ) 319–354 (Springer International Publishing, 2015).Urteaga, R. & Berli, C. L. In Nanoporous Alumina. Fabrication, Structure, Properties and Applications (ed. Losic, D., Santos, A. ) 249–269 (Springer International Publishing, 2015).Vojkuvka, L., Marsal, L. F., Ferré-Borrull, J., Formentin, P. & Pallarés, J. Self-Ordered Porous Alumina Membranes with Large Lattice Constant Fabricated by Hard Anodization. Superlattices Microstruct. 44, 577–582 (2008).De la Escosura-Muñiz, A. & Merkoçi, A. Nanochannels Preparation and Application in Biosensing. ACS Nano. 6, 7556–7583 (2012).Kumeria, T. et al. Nanoporous Anodic Alumina Rugate Filters for Sensing of Ionic Mercury: Toward Environmental Point-of-Analysis Systems. ACS Appl. Mater. Interfaces. 6, 12971−12978 (2014).Santos, A., Kumeria, T. & Losic, D. Nanoporous Anodic Alumina: A Versatile Platform for Optical Biosensors. Materials. 7, 4297–4320 (2014).Ferré-Borrull, J., Pallarès, J., Macías, G. & Marsal, L. F. Nanostructural Engineering of Nanoporous Anodic Alumina for Biosensing Applications. Materials. 7, 5225–5253 (2014).Gong, D., Yadavalli, V., Paulose, M., Pishko, M. & Grimes, C. A. Controlled Molecular Release Using Nanoporous Alumina Capsules. Biomed Microdevices. 5, 75–80 (2003).Alvarez, S. D., Li, C.-P., Chiang, C. E., Schuller, I. K. & Sailor, M. J. A Label-Free Porous Alumina Interferometric Immunosensor. ACSNano. 3, 3301–3307 (2009).Krismastuti, F. S. H., Bayat, H., Voelcker, N. H. & Schönherr, H. Real Time Monitoring of Layer-by-Layer Polyelectrolyte Deposition and Bacterial Enzyme Detection in Nanoporous Anodized Aluminum Oxide Anal. Chem. 87, 3856–3863 (2015).Ma, D.-L. et al. A Luminescent Cocaine Detection Platform Using a Split G-Quadruplex-Selective Iridium (III) Complex and a Three-Way DNA Junction Architecture. ACS Appl. Mater. Interfaces. 7, 19060−19067 (2015).Kohli, P. et al. DNA-Functionalized Nanotube Membranes with Single-Base Mismatch Selectivity. Science 305, 984–986 (2004).Abelow, A. E. et al. Biomimetic glass nanopores employing aptamer gates responsive to a small molecule. Chem. Commun. 46, 7984–7986 (2010).Ma, D.-L., Chan, D. S.-H. & Leung, C.-H. Group 9 Organometallic Compounds for Therapeutic and Bioanalytical Applications. Acc. Chem. Res. 47, 3614–3631 (2014).Wanga, G., Zhua, Y., Chena, L. & Zhanga, X. Photoinduced electron transfer (PET) based label-free aptasensor for platelet-derived growth factor-BB and its logic gate application. Biosens. Bioelectron. 63, 552–557 (2015).Laptenko, O. et al. The p53 C Terminus Controls Site-Specific DNA Binding and Promotes Structural Changes within the Central DNA Binding Domain. Molec. Cell. 57, 1034–1046 (2015).McKeague, M. & DeRosa, M. C. Challenges and Opportunities for Small Molecule Aptamer Development. J. Nucleic Acids. 2012, 1–20 (2012).McKeague, M. et al. Analysis of In Vitro Aptamer Selection Parameters, J. Mol. Evol. 81, 150–161 (2015).Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature. 346, 818–822 (1990).Wochner, A. et al. A DNA aptamer with high affinity and specificity for therapeutic anthracyclines. Anal Biochem. 373, 34–42 (2008).Song, K. M., Jeong, E., Jeon, W., Cho, M. & Ban, C. Aptasensor for ampicillin using gold nanoparticle based dual fluorescence-colorimetric methods. Anal. Bioanal. Chem. 402, 2153–2161 (2012).Özalp, V. C. & Schäfer, T. Aptamer-Based Switchable Nanovalves for Stimuli-Responsive Drug Delivery. Chem. Eur. J. 17, 9893–9896 (2011).Stojanovic, M. N., de Prada, P. & Landry, D. W. Aptamer-Based Folding Fluorescent Sensor for Cocaine. J. Am. Chem Soc. 123, 4928–4931 (2001).Wen, Y. et al. DNA-based intelligent logic controlled release systems. Chem. Commun. 48, 8410–8412 (2012).Chen, L. et al. Programmable DNA switch for bioresponsive controlled release. J. Mater. Chem. 21, 13811–13816 (2011).Oroval, M. et al. An aptamer-gated silica mesoporous material for thrombin detection. Chem. Commun. 49, 5480–5482 (2013).Barroso, M., Gallardo, E. & Queiroz, J. A. Bioanalytical methods for the determination of cocaine and metabolites in human biological samples. Bioanalysis. 1, 977–1000 (2009).Phan, H. M., Yoshizuka, K., Murry, D. J. & Perry, P. J. Drug testing in the workplace. Pharmacotherapy. 32, 649–656 (2012).Kidwell, D. A., Blanco, M. A. & P. Smith, F. P. Cocaine detection in a university population by hair analysis and skin swab testing. Forensic Sci. Int. 84, 75–86 (1997).Swensen, J. S. et al. Continuous, Real-Time Monitoring of Cocaine in Undiluted Blood Serum via a Microfluidic, Electrochemical Aptamer-Based Sensor. J. Am. Chem. Soc. 131, 4262–4266 (2009).Cai, Q. et al. Determination of cocaine on banknotes through an aptamer-based electrochemiluminescence biosensor. Anal. Bioanal. Chem. 400, 289–294 (2011).Zou, R. et al. Highly specific triple-fragment aptamer for optical detection of cocaine. RSC Adv. 2, 4636–4638 (2012).Qiu, L. et al. A novel label-free fluorescence aptamer-based sensor method for cocaine detection based on isothermal circular strand-displacement amplification and graphene oxide absorption. New J. Chem. 37, 3998 (2013).Marsal, L. F., Vojkuvka, L., Formentin, P., Pallarés, J. & Ferré-Borrull, J. Fabrication and Optical Characterization of Nanoporous Alumina Films Annealed at Different Temperatures. Optical Mater. 31, 860–864 (2009).Bosker, W. M. & Huestis, M. A. Oral Fluid Testing for Drugs of Abuse. Clinical Chem. 55, 1910–1931 (2009).Kolbrich, E. A. et al. Cozart® RapiScan Oral Fluid Drug Testing System: An Evaluation of Sensitivity, Specificity, and Efficiency for Cocaine Detection Compared with ELISA and GC-MS Following Controlled Cocaine Administration. J. Anal Toxicol. 27, 407–411 (2003).Cooper, G., Wilson, L., Reid, C., Main, L. & Hand, C. Evaluation of the Cozart® RapiScan drug test system for opiates and cocaine in oral fluid. Forensic Sci. Int. 150, 239–243 (2005).Chang, Y. H. et al. Cocaine detection by a mid-infrared waveguide integrated with a microfluidic chip. Lab Chip. 12, 3020–3023 (2012).Walczak, R. et al. Toward Portable Instrumentation for Quantitative Cocaine Detection with Lab-on-a-Paper and Hybrid Optical Readout. Procedia Chem. 1, 999–1002 (2009).Qiu, L. et al. A novel label-free fluorescence aptamer-based sensor method for cocaine detection based on isothermal circular strand-displacement amplification and graphene oxide absorption. New J. Chem. 37, 3998–4003 (2013)