Characterization of Multi-Drug Resistant Enterococcus faecalis Isolated from Cephalic Recording Chambers in Research Macaques (Macaca spp.).

Nonhuman primates are commonly used for cognitive neuroscience research and often surgically implanted with cephalic recording chambers for electrophysiological recording. Aerobic bacterial cultures from 25 macaques identified 72 bacterial isolates, including 15 Enterococcus faecalis isolates. The E. faecalis isolates displayed multi-drug resistant phenotypes, with resistance to ciprofloxacin, enrofloxacin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, bacitracin, and erythromycin, as well as high-level aminoglycoside resistance. Multi-locus sequence typing showed that most belonged to two E. faecalis sequence types (ST): ST 4 and ST 55. The genomes of three representative isolates were sequenced to identify genes encoding antimicrobial resistances and other traits. Antimicrobial resistance genes identified included aac(6')-aph(2"), aph(3')-III, str, ant(6)-Ia, tetM, tetS, tetL, ermB, bcrABR, cat, and dfrG, and polymorphisms in parC (S80I) and gyrA (S83I) were observed. These isolates also harbored virulence factors including the cytolysin toxin genes in ST 4 isolates, as well as multiple biofilm-associated genes (esp, agg, ace, SrtA, gelE, ebpABC), hyaluronidases (hylA, hylB), and other survival genes (ElrA, tpx). Crystal violet biofilm assays confirmed that ST 4 isolates produced more biofilm than ST 55 isolates. The abundance of antimicrobial resistance and virulence factor genes in the ST 4 isolates likely relates to the loss of CRISPR-cas. This macaque colony represents a unique model for studying E. faecalis infection associated with indwelling devices, and provides an opportunity to understand the basis of persistence of this pathogen in a healthcare setting.

Antibacterial and Solubility Optimization of Thiomuracin A.

Synthetic studies of the antimicrobial secondary metabolite thiomuracin A (1) provided access to analogues in the Northern region (C2-C10). Selective hydrolysis of the C10 amide of lead compound 2 and subsequent derivatization led to novel carbon- and nitrogen-linked analogues (e.g., 3) which improved antibacterial potency across a panel of Gram-positive organisms. In addition, congeners with improved physicochemical properties were identified which proved efficacious in murine sepsis and hamster C. difficile models of disease. Optimal efficacy in the hamster model of C. difficile was achieved with compounds that possessed both potent antibacterial activity and high aqueous solubility.

Design and implementation of a genomics field trip program aimed at secondary school students.

With the rapid pace of advancements in biological research brought about by the application of computer science and information technology, we believe the time is right for introducing genomics and bioinformatics tools and concepts to secondary school students. Our approach has been to offer a full-day field trip in our research facility where secondary school students carry out experiments at the laboratory bench and on a laptop computer. This experience offers benefits for students, teachers, and field trip instructors. In delivering a wide variety of science outreach and education programs, we have learned that a number of factors contribute to designing a successful experience for secondary school students. First, it is important to engage students with authentic and fun activities that are linked to real-world applications and/or research questions. Second, connecting with a local high school teacher to pilot programs and linking to curricula taught in secondary schools will enrich the field trip experience. Whether or not programs are linked directly to local teachers, it is important to be flexible and build in mechanisms for collecting feedback in field trip programs. Finally, graduate students can be very powerful mentors for students and should be encouraged to share their enthusiasm for science and to talk about career paths. Our experiences suggest a real need for effective science outreach programs at the secondary school level and that genomics and bioinformatics are ideal areas to explore.

Antibiotic optimization and chemical structure stabilization of thiomuracin A.

Synthetic studies of the antimicrobial secondary metabolite thiomuracin A (1) were initiated to improve chemical stability and physicochemical properties. Functional group modifications of 1 included removing the C2-C7 side chain, derivatizing the C84 epoxide region, and altering the C44 hydroxyphenylalanine motif. The resulting derivatives simplified and stabilized the chemical structure and were evaluated for antibacterial activity relative to 1. The simplified structure and improved organic solubility of the derivatives facilitated isolation yields from fermentation broths and simplified the procedures involved for the process. These advancements increased material supply for continued medicinal chemistry optimization and culminated in the identification of 2, a structurally simplified and chemically stable analogue of 1 which retained potent antibiotic activity.

Discovery of LFF571: an investigational agent for Clostridium difficile infection.

Clostridium difficile (C. difficile) is a Gram positive, anaerobic bacterium that infects the lumen of the large intestine and produces toxins. This results in a range of syndromes from mild diarrhea to severe toxic megacolon and death. Alarmingly, the prevalence and severity of C. difficile infection are increasing; thus, associated morbidity and mortality rates are rising. 4-Aminothiazolyl analogues of the antibiotic natural product GE2270 A (1) were designed, synthesized, and optimized for the treatment of C. difficile infection. The medicinal chemistry effort focused on enhancing aqueous solubility relative to that of the natural product and previous development candidates (2, 3) and improving antibacterial activity. Structure-activity relationships, cocrystallographic interactions, pharmacokinetics, and efficacy in animal models of infection were characterized. These studies identified a series of dicarboxylic acid derivatives, which enhanced solubility/efficacy profile by several orders of magnitude compared to previously studied compounds and led to the selection of LFF571 (4) as an investigational new drug for treating C. difficile infection.

Management of gastroenteritis over 10 years: changing culture and maintaining the change.

OBJECTIVE: To examine the impact of an implementation program on adherence to a guideline for the management of acute gastroenteritis. DESIGN: Using four retrospective audits over a 10-year period, the authors examined the change in practice and maintenance of that change following a targeted implementation program for the clinical guideline. SETTING: Tertiary children's hospital in South Wales. PARTICIPANTS: 447 children aged less than 5 years, admitted to hospital with acute gastroenteritis, comprising four cross-sectional samples (106 in 1999, 153 in 2002, 99 in 2004, 89 in 2009). MAIN OUTCOME MEASURES: Age of child, hydration status, method of rehydration and duration of admission, for each audit, with an implementation strategy delivered after the second audit. RESULTS: In 1999 and 2002, intravenous rehydration was used in 20% and 15% of cases, respectively. After the implementation program in 2004, compared to 1999, there was a significant decrease in the intravenous rehydration rate to 4% in 2004 (p<0.001); in 2009 the intravenous rehydration rate was maintained at a low level of 6% (p<0.001). CONCLUSION: It was only after the implementation program that a change in practice was achieved. Once change had been accepted, it was maintained even in the absence of targeted training. Audit does not improve clinical practice unless, in addition, there is a clear, succinct guideline with an implementation programme in place.

Antibacterial optimization of 4-aminothiazolyl analogues of the natural product GE2270 A: identification of the cycloalkylcarboxylic acids.

4-Aminothiazolyl analogues of the antibiotic natural product GE2270 A (1) were designed, synthesized, and optimized for their activity against Gram positive bacterial infections. Optimization efforts focused on improving the physicochemical properties (e.g., aqueous solubility and chemical stability) of the 4-aminothiazolyl natural product template while improving the in vitro and in vivo antibacterial activity. Structure-activity relationships were defined, and the solubility and efficacy profiles were improved over those of previous analogues and 1. These studies identified novel, potent, soluble, and efficacious elongation factor-Tu inhibitors, which bear cycloalkylcarboxylic acid side chains, and culminated in the selection of development candidates amide 48 and urethane 58.

4-Aminothiazolyl analogs of GE2270 A: design, synthesis and evaluation of imidazole analogs.

Imidazole analogs of the antibiotic natural product GE2270 A (1) were designed, synthesized, and evaluated for gram positive bacteria growth inhibition. A recently reported, copper-mediated synthesis was exploited to prepare 4-thiazolyl imidazole analogs of 1. The synthesis described represents a structurally complex, natural product-based application of this recently reported synthetic methodology. In addition, the biological evaluation of the imidazole-based analogs further define the SAR of the 4-aminothiazolyl-based antibacterial template.

4-Aminothiazolyl analogues of GE2270 A: antibacterial lead finding.

4-Aminothiazolyl analogues of the antibacterial natural product GE2270 A (1) were designed, synthesized, and evaluated for gram positive bacteria growth inhibition. The aminothiazole-based chemical template was evaluated for chemical stability, and its decomposition revealed a novel, structurally simplified, des-thiazole analogue of 1. Subsequent stabilization of the 4-aminothiazolyl functional motif was achieved and initial structure activity relationships defined.

Keeping pace with the data: 2008 update on the Bioinformatics Links Directory.

The Bioinformatics Links Directory, http://bioinformatics.ca/links_directory/, is an online resource for public access to all of the life science research web servers published in this and previous issues of Nucleic Acids Research, together with other useful tools, databases and resources for bioinformatics and molecular biology research. Dependent on community input and development, the Bioinformatics Links Directory exemplifies an open access research tool and resource. The 2008 update includes the 94 web servers featured in the July 2008 Web Server issue of Nucleic Acids Research, bringing the total number of servers listed in the Bioinformatics Links Directory to over 1200 links. A complete list of all links listed in this Nucleic Acids Research 2008 Web Server issue can be accessed online at http://bioinfomatics.ca/links_directory/narweb2008/. The 2008 update of the Bioinformatics Links Directory, which includes the Web Server list and summaries, is also available online at the Nucleic Acids Research website, http://nar.oxfordjournals.org/.

Conducting research on the web: 2007 update for the bioinformatics links directory.

The Bioinformatics Links Directory, http://bioinformatics.ca/links_directory, is an actively maintained compilation of servers published in this and previous issues of Nucleic Acids Research issues together with many other useful tools, databases and resources for life sciences research. The 2007 update includes the 130 websites highlighted in the July 2007 Web Server issue of Nucleic Acids Research and brings the total number of servers listed in the Bioinformatics Links Directory to just under 1200 links. In addition to the updated content, the 2007 update of the Bioinformatics Links Directory includes new features for improved navigation, accessibility and open data exchange. A complete listing of all links listed in this Nucleic Acids Research 2007 Web Server issue can be accessed online at, http://bioinformatics.ca/links_directory/narweb2007. The 2007 update of the Bioinformatics Links Directory, which includes the Web Server list and summaries is also available online, at the Nucleic Acids Research web site, http://nar.oupjournals.org.

A compilation of molecular biology web servers: 2006 update on the Bioinformatics Links Directory.

The Bioinformatics Links Directory is a public online resource that lists the servers published in this and all previously published Nucleic Acids Research Web Server issues together with other useful tools, databases and resources for bioinformatics and molecular biology research. This rich directory of tools and websites can be browsed and searched with all listed links freely accessible to the public. The 2006 update includes the 149 websites highlighted in the July 2006 issue of Nucleic Acids Research and brings the total number of servers listed in the Bioinformatics Links Directory to over 1000 links. To aid navigation through this growing resource, all link entries contain a brief synopsis, a citation list and are classified by function in descriptive biological categories. The most up-to-date version of this actively maintained listing of bioinformatics resources is available at the Bioinformatics Links Directory website, http://bioinformatics.ubc.ca/resources/links_directory/. A complete list of all links listed in this Nucleic Acids Research 2006 Web Server issue can be accessed online at http://bioinformatics.ubc.ca/resources/links_directory/narweb2006/. The 2006 update of the Bioinformatics Links Directory, which includes the Web Server list and summaries, is also available online at the Nucleic Acids Research website, http://nar.oupjournals.org/.

The Bioinformatics Links Directory: a compilation of molecular biology web servers.

The Bioinformatics Links Directory is an online community resource that contains a directory of freely available tools, databases, and resources for bioinformatics and molecular biology research. The listing of the servers published in this and previous issues of Nucleic Acids Research together with other useful tools and websites represents a rich repository of resources that are openly provided to the research community using internet technologies. The 166 servers highlighted in the 2005 90002 are included in the more than 700 links to useful online resources that are currently contained within the descriptive biological categories of the Bioinformatics Links Directory. This curated listing of bioinformatics resources is available online at the Bioinformatics Links Directory web site, http://bioinformatics.ubc.ca/resources/links_directory/. A complete listing of the 2005 Nucleic Acids Research 90002 servers is available online at the Nucleic Acids web site, http://nar.oupjournals.org/, and on the Bioinformatics Links Directory web site, http://bioinformatics.ubc.ca/resources/links_directory/narweb2005/.

Phylogeny of Na+/Ca2+ exchanger (NCX) genes from genomic data identifies new gene duplications and a new family member in fish species.

The Na+/Ca2+ exchanger (NCX) is a member of the cation/Ca2+ antiporter (CaCA) family and plays a key role in maintaining cellular Ca2+ homeostasis in a variety of cell types. NCX is present in a diverse group of organisms and exhibits high overall identity across species. To date, three separate genes, i.e., NCX1, NCX2, and NCX3, have been identified in mammals. However, phylogenetic analysis of the exchanger has been hindered by the lack of nonmammalian NCX sequences. In this study, we expand and diversify the list of NCX sequences by identifying NCX homologs from whole-genome sequences accessible through the Ensembl Genome Browser. We identified and annotated 13 new NCX sequences, including 4 from zebrafish, 4 from Japanese pufferfish, 2 from chicken, and 1 each from honeybee, mosquito, and chimpanzee. Examination of NCX gene structure, together with construction of phylogenetic trees, provided novel insights into the molecular evolution of NCX and allowed us to more accurately annotate NCX gene names. For the first time, we report the existence of NCX2 and NCX3 in organisms other than mammals, yielding the hypothesis that two serial NCX gene duplications occurred around the time vertebrates and invertebrates diverged. In addition, we have found a putative new NCX protein, named NCX4, that is related to NCX1 but has been observed only in fish species genomes. These findings present a stronger foundation for our understanding of the molecular evolution of the NCX gene family and provide a framework for further NCX phylogenetic and molecular studies.

Structure, functions, and activities of a research support informatics section.

The authors describe a research group that supports the needs of investigators seeking data from an electronic medical record system. Since its creation in 1972, the Regenstrief Medical Records System has captured and stored more than 350 million discrete coded observations on two million patients. This repository has become a central data source for prospective and retrospective research. It is accessed by six data analysts--working closely with the institutional review board--who provide investigators with timely and accurate data while protecting patient and provider privacy and confidentiality. From January 1, 1999, to July 31, 2002, data analysts tracked their activities involving 47,559 hours of work predominantly for physicians (54%). While data retrieval (36%) and analysis (25%) were primary activities, data analysts also actively collaborated with researchers. Primary objectives of data provided to investigators were to address disease-specific (35.4%) and drug-related (12.2%) questions, support guideline implementation (13.1%), and probe various aspects of clinical epidemiology (5.7%). Outcomes of these endeavors included 117 grants (including 300,000 US dollars per year salary support for data analysts) and 139 papers in peer-reviewed journals by investigators who rated the support provided by data analysts as extremely valuable.

Disruption of a single Pten allele augments the chemotactic response of B lymphocytes to stromal cell-derived factor-1.

The tumor suppressor, Pten, has emerged as a critical negative regulator of phosphatidylinositol-3-kinase-dependent intracellular signaling pathways responsible for phenomena such as cellular adhesion, proliferation, and apoptosis. Herein, we present evidence that Pten regulates chemokine-dependent events in B lymphocytes. Primary B cells isolated from Pten(+/-) mice demonstrated increased responsiveness to stromal cell-derived factor-1-induced chemotaxis. This was accompanied by an elevated level of protein kinase B phosphorylation on Ser(473). Our results suggest not only that Pten may be an important regulator of stromal cell-derived factor-1-directed chemotaxis, but also that Pten heterozygosity is associated with increased cellular sensitivity to this chemokine, likely via dysregulation of events lying downstream of phosphatidylinositol-3-kinase. These observations suggest a mechanism by which loss of a single Pten allele may confer a selective advantage on cells during multistep tumor progression.