Lytic to temperate switching of viral communities.

Microbial viruses can control host abundances via density-dependent lytic predator-prey dynamics. Less clear is how temperate viruses, which coexist and replicate with their host, influence microbial communities. Here we show that virus-like particles are relatively less abundant at high host densities. This suggests suppressed lysis where established models predict lytic dynamics are favoured. Meta-analysis of published viral and microbial densities showed that this trend was widespread in diverse ecosystems ranging from soil to freshwater to human lungs. Experimental manipulations showed viral densities more consistent with temperate than lytic life cycles at increasing microbial abundance. An analysis of 24 coral reef viromes showed a relative increase in the abundance of hallmark genes encoded by temperate viruses with increased microbial abundance. Based on these four lines of evidence, we propose the Piggyback-the-Winner model wherein temperate dynamics become increasingly important in ecosystems with high microbial densities; thus 'more microbes, fewer viruses'.

Charting the diversity of uncultured viruses of Archaea and Bacteria.

BACKGROUND: Viruses of Archaea and Bacteria are among the most abundant and diverse biological entities on Earth. Unraveling their biodiversity has been challenging due to methodological limitations. Recent advances in culture-independent techniques, such as metagenomics, shed light on the unknown viral diversity, revealing thousands of new viral nucleotide sequences at an unprecedented scale. However, these novel sequences have not been properly classified and the evolutionary associations between them were not resolved. RESULTS: Here, we performed phylogenomic analysis of nearly 200,000 viral nucleotide sequences to establish GL-UVAB: Genomic Lineages of Uncultured Viruses of Archaea and Bacteria. The pan-genome content of the identified lineages shed light on some of their infection strategies, potential to modulate host physiology, and mechanisms to escape host resistance systems. Furthermore, using GL-UVAB as a reference database for annotating metagenomes revealed elusive habitat distribution patterns of viral lineages and environmental drivers of community composition. CONCLUSIONS: These findings provide insights about the genomic diversity and ecology of viruses of prokaryotes. The source code used in these analyses is freely available at https://sourceforge.net/projects/gluvab/.

Comparison of an alcohol-based hand sanitation product with a traditional chlorhexidine hand scrub technique for hand hygiene preparation in an equine hospital.

AIMS: To investigate the efficacy of an alcohol gel-based hand antisepsis protocol compared with a traditional chlorhexidine-based protocol under conditions of routine clinical contamination, and following heavy faecal contamination. METHODS: Twelve adult participants were recruited and on four separate days completed a hand sanitation protocol using a chlorhexidine scrub or an alcohol-based gel, with hands that were grossly clean but contaminated or with faecal contamination. Bacterial samples were obtained from participants' hands before sanitation, immediately after and then 2 hours later. All samples were cultured on blood and MacConkey agar and bacterial colonies counted after 48 hours. RESULTS: for clean contaminated hands, the percentage reduction in bacterial colonies on blood agar immediately after hand sanitation was similar for both protocols (p=0.3), but was greater for the alcohol gel than chlorhexidine after 2 hours (p=0.005). For hands with faecal contamination, the percentage reduction in bacterial colonies on blood agar was similar for both protocols immediately and 2 hours after sanitation (p>0.2), but positive cultures were obtained on blood agar from samples collected after both protocols, for almost all participants. CONCLUSIONS: The results indicate equivalent efficacy of the alcohol-based gel and the pre-surgical chlorhexidine protocol. CLINICAL RELEVANCE: The alcohol-based gel protocol is an effective hand asepsis technique for grossly clean contaminated hands and those following faecal contamination, with comparable efficacy to chlorhexidine based scrub.

beta-Catenin associates with the actin-bundling protein fascin in a noncadherin complex.

Catenins were first characterized as linking the cytoplasmic domains of cadherin cell-cell adhesion molecules to the cortical actin cytoskeleton. In addition to their essential role in modulating cadherin adhesivity, catenins have more recently been indicated to participate in cell and developmental signaling pathways. beta-Catenin, for example, associates directly with at least two receptor tyrosine kinases and transduces developmental signals within the Wnt pathway. Catenins also complex with the tumor suppressor protein adenomatous polyposis coli (APC), which appears to have a role in regulating cell proliferation. We have used the yeast two-hybrid method to reveal that fascin, a bundler of actin filaments, binds to beta-catenin's central Armadillo repeat domain. Western blotting of immunoprecipitates from cell line and mouse and rat brain extracts indicate that this interaction exists in vivo. Fascin and beta-catenin's association was further substantiated in vitro using purified proteins isolated from recombinant bacterial and baculoviral sources. Immunoprecipitation analysis indicates that fascin additionally binds to plakoglobin, which is highly homologous to beta-catenin but not to p120cas, a newly described catenin which contains a more divergent Armadillo-repeat domain. Immunoprecipitation, in vitro competition, and domain-mapping experiments demonstrate that fascin and E-cadherin utilize a similar binding site within beta-catenin, such that they form mutually exclusive complexes with beta-catenin. Immunofluorescence microscopy reveals that fascin and beta-catenin colocalize at cell-cell borders and dynamic cell leading edges of epithelial and endothelial cells. In addition to cell-cell borders, cadherins were unexpectedly observed to colocalize with fascin and beta-catenin at cell leading edges. It is conceivable that beta-catenin participates in modulating cytoskeletal dynamics in association with the microfilament-bundling protein fascin, perhaps in a coordinate manner with its functions in cadherin and APC complexes.

The metagenomics RAST server - a public resource for the automatic phylogenetic and functional analysis of metagenomes.

BACKGROUND: Random community genomes (metagenomes) are now commonly used to study microbes in different environments. Over the past few years, the major challenge associated with metagenomics shifted from generating to analyzing sequences. High-throughput, low-cost next-generation sequencing has provided access to metagenomics to a wide range of researchers. RESULTS: A high-throughput pipeline has been constructed to provide high-performance computing to all researchers interested in using metagenomics. The pipeline produces automated functional assignments of sequences in the metagenome by comparing both protein and nucleotide databases. Phylogenetic and functional summaries of the metagenomes are generated, and tools for comparative metagenomics are incorporated into the standard views. User access is controlled to ensure data privacy, but the collaborative environment underpinning the service provides a framework for sharing datasets between multiple users. In the metagenomics RAST, all users retain full control of their data, and everything is available for download in a variety of formats. CONCLUSION: The open-source metagenomics RAST service provides a new paradigm for the annotation and analysis of metagenomes. With built-in support for multiple data sources and a back end that houses abstract data types, the metagenomics RAST is stable, extensible, and freely available to all researchers. This service has removed one of the primary bottlenecks in metagenome sequence analysis - the availability of high-performance computing for annotating the data. http://metagenomics.nmpdr.org.

Fimbrial expression in enteric bacteria: a critical step in intestinal pathogenesis.

The ability of species of enteric bacteria to recognize and colonize unique niches along the intestine is mainly based on receptor distribution and interpretation of a combination of environmental signals leading to the expression of specific adherence factors. Such elaborate orchestration of events is critical during the initial steps of pathogenesis.

Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression.

A series of vectors has been developed to provide improved positive and negative selection for allelic exchange. Based on homologous regions of DNA ranging in size from less than 200 bp to over 1 kb, we have successfully used these new plasmids to introduce or remove markers in chromosomal or plasmid DNA. Wild type fimbria genes were replaced both in Salmonella enteritidis (sefA, agfA and fimC) and Escherichia coli (fasA and fasH). Regulation of 987P fimbriation could be identified after replacement of fasA and fasH with allelic reporter fusions. The expression of fasA but not fasH is dependent upon the osmolarity of the growth medium in an HNS-dependent manner, but unlike some other fimbrial systems expression is not dependent on the exogenous iron concentration.

Metabolic effects of adding sucrose and aspartame to the diet of subjects with noninsulin-dependent diabetes mellitus.

This study compared the effects of adding sucrose and aspartame to the usual diet of individuals with well-controlled noninsulin-dependent diabetes mellitus (NIDDM). A double-blind, cross-over design was used with each 6-wk study period. During the sucrose period, 45 g sucrose (9% of total daily energy) was added, 10 g with each main meal and 5 g with each between-meal beverage. An equivalent sweetening quantity of aspartame (162 mg) was ingested during the aspartame period. The addition of sucrose did not have a deleterious effect on glycemic control, lipids, glucose tolerance, or insulin action. No differences were observed between sucrose and aspartame. Sucrose added as an integral part of the diabetic diet does not adversely affect metabolic control in well-controlled NIDDM subjects. Aspartame is an acceptable sugar substitute for diabetic individuals but no specific advantage over sucrose was demonstrated.

Versatile E. coli thioredoxin specific monoclonal antibodies afford convenient analysis and purification of prokaryote expressed soluble fusion protein.

A recently developed E. coli thioredoxin (Trx) gene fusion expression system has circumvented the difficulties associated with inclusion body formation. Although ample quantities of soluble recombinant protein can be expressed using this system, no universal means of quantifying or purifying the fusion product exists. To facilitate the study of Trx fusion proteins, anti-E. coli Trx monoclonal antibodies (mAb) were generated. Two distinct Trx epitopes were defined by competitive ELISA. Both mAb were capable of detecting Trx fusion proteins by sandwich ELISA, and by immunoblot analysis under reducing and non-reducing conditions. In addition, these mAb enabled purification of Trx fusion proteins by immunoprecipitation, as well as affinity chromatography. This report provides the first description of anti-Trx antibodies. These reagents represent a major advance in the isolation and analysis of prokaryote expressed recombinant Trx fusion proteins.

Hospital admissions through the emergency department: does insurance status matter?

PURPOSE: To assess the effect of insurance status on the probability of admission and subsequent health status of patients presenting to emergency departments. SUBJECTS AND METHODS: We performed a prospective cohort study of patients with common medical problems at five urban, academic hospital emergency departments in Boston and Cambridge, Massachusetts. The outcome measure for the study was admission to the hospital from the emergency department and functional health status at baseline and follow-up. RESULTS: During a 1-month period, 2,562 patients younger than 65 years of age presented with either abdominal pain (52%), chest pain (19%) or shortness of breath (29%). Of the 1,368 patients eligible for questionnaire, 1,162 (85%) completed baseline questionnaires, and of these, 964 (83%) completed telephone follow-up interviews 10 days later. Fifteen percent of patients were uninsured and 34% were admitted to the hospital from the emergency department. Uninsured patients were significantly less likely than insured patients to be admitted, both when adjusting for urgency, chief complaint, age, gender and hospital (odds ratio = 0.5, 95% confidence interval 0.3 to 0.7), and when additionally adjusting for comorbid conditions, lack of a regular physician, income, employment status, education and race (odds ratio = 0.4, 95% confidence interval 0.2 to 0.8). However, there were no differences in adjusted functional health status between admitted and nonadmitted patients by insurance status, either at baseline or at 10-day follow-up. CONCLUSIONS: Uninsured patients with one of three common chief complaints appear to be less frequently admitted to the hospital than are insured patients, although health status does not appear to be affected. Whether these results reflect underutilization among uninsured patients or overutilization among insured patients remains to be determined.

Inside or outside: detecting the cellular location of bacterial pathogens.

Salmonella are intracellular pathogens that infect and multiply inside macrophages. Although Salmonella are some of the best-studied pathogens, it is difficult to determine quickly and reliably whether the bacteria are intracellular or extracellular. We have developed a novel method using differential fluorescence of two fluorescent proteins to determine the cellular location of pathogenic bacteria in macrophage infection assays. Using the differential expression of two unique fluorescent proteins that are expressed under specific conditions, we have developed a real-time assay for macrophage infections. The critical advantages of this system are that it does not alter the bacterial surface, it is not toxic to either the bacteria or the host cell, and it may be used in real-time quantitative assays. This assay can be readily applied to any other model pathogenic systems such as Listeria, Mycobacteria, and Legionella in which intracellular gene expression has been characterized.

Increasing DNA transfer efficiency by temporary inactivation of host restriction.

E. coli and Salmonella typhimurium are widely used bacterial hosts for genetic manipulation of DNA from prokaryotes and eukaryotes. Introduction of foreign DNA by electroporation or transduction into E. coli and Salmonella is limited by host restriction of incoming DNA by the recipient cells. Here, we describe a simple method that temporarily inactivates host restriction, allowing high-frequency DNA transfer. This technique might be readily applied to a wide range of bacteria to increase DNA transfer between strains and species.

Brevetoxins bind to multiple classes of sites in rat brain synaptosomes.

The brevetoxins (PbTx series), neurotoxins produced by the marine dinoflagellate Ptychodiscus brevis, cause dose-dependent activation of the voltage-sensitive sodium channel (VSSC). Saturation binding studies employing adult rat brain synaptosomes suggest the existence of a high affinity/low capacity (HA/LC) and a second, lower affinity/higher capacity (LA/HC) class of binding site. LIGAND analysis of saxitoxin and brevetoxin saturation binding data yields a statistically identical Bmax for the brevetoxin high affinity/low capacity (HA/LC) site (1.9 +/- 0.98 pmol/mg protein) and for saxitoxin (1.72 +/- 0.78 pmol/mg protein; P less than 0.001). The stoichiometry of HA/LC brevetoxin binding and saxitoxin binding approaches 1:1. Covalent modification of synaptosomes with a brevetoxin photoaffinity probe preferentially blocks the HA/LC binding site. Hill plots of saturation binding data yield a coefficient of 1.0 +/- 0.02, demonstrating a lack of cooperativity between brevetoxin binding site classes. Kd and Bmax for toxin binding are independent of membrane polarity, intimating that the observed low affinity/high capacity (LA/HC) binding characteristics are not due to modification of the HA/LC site, and strongly argue for the presence of multiple brevetoxin binding site classes. Half-maximal binding at the LA/HC site, and strongly argue for the presence of multiple brevetoxin binding site classes. Half-maximal binding at the LA/HC site occurs at concentration ranges for which the brevetoxins allosterically modulate binding of other natural toxins to their specific sites.

Taxonomic studies on a psychrophilic Clostridium from vacuum-packed beef: description of Clostridium estertheticum sp. nov.

Taxonomic studies were performed on an anaerobic Gram-positive, spore-forming, psychrophilic bacterium originally isolated from spoiled vacuum-packed refrigerated beef. Based on the present finding it is proposed that this unknown psychrophilic bacterium be classified as a new species of the genus Clostridium, as Clostridium estertheticum sp. nov. The type strain is NCIMB 12511.

The substitution of digital images for dermatologic physical examination.

OBJECTIVE: To investigate the diagnostic accuracy of clinicians viewing a patient's history and static digital image set compared with clinicians who conducted office-based physical examinations of the same patients. DESIGN: Observational study. SETTING AND PATIENTS: One hundred sixteen adult patients presenting with dermatologic symptoms in a university-based practice who consented to have their skin conditions documented with a still digital camera according to a standardized protocol. MAIN OUTCOME MEASURES: Concordance between office-based dermatologists' diagnoses and 2 remote clinicians' diagnoses using still digital images (resolution, 92 dots per inch) and identical medical history data to render diagnoses. RESULTS: When photographic quality was high and office-based clinician certainty was high, remote clinicians were in agreement more than 75% of the time. Office-based and remote clinicians were in agreement 61% to 64% of the time for all cases. No specific disease category appeared to be more or less amenable to diagnosis based on still digital imagery. The diagnostic certainty of the office-based clinician (reported from 0-10) had the most impact on agreement. When cases with office-based clinician certainty of no more than 7 were compared with cases with certainty of at least 9, agreement increased 54% for remote clinician 1 and 111% for remote clinician 2. As an isolated variable, photographic quality had a modest impact on agreement. CONCLUSIONS: Still digital images can substitute for the dermatologic physical examination in up to 83% of cases. This study provides validation of the store-and-forward concept of telemedicine as applied to dermatology. These results serve as the foundation for field testing of the concept in primary care settings.

Brevetoxin binding: molecular pharmacology versus immunoassay.

Brevetoxin PbTx-3 isolated from Florida's red tide dinoflagellate Ptychodiscus brevis has been produced recently in tritiated form by reductive tritiation of brevetoxin PbTx-2. Tritiated PbTx-3 has been used as a specific probe in competitive radioimmunoassays developed to detect brevetoxins in food sources, and this probe has also been utilized to characterize the brevetoxin binding component in rat brain synaptosomes. Brevetoxins PbTx-2 and PbTx-3, possessing the same structural backbone (type-1) as the tritiated probe, and PbTx-1 and PbTx-7, possessing a second structural backbone (type-2), have been compared quantitatively in their individual abilities to competitively displace tritiated PbTx-3 from its specific binding site in each assay. Type-1 toxins displaced labeled probe with ED50 values of 20-22 nM and 12-17 nM in radioimmunoassay and synaptosomes, respectively. Type-2 toxins displaced labeled probe with ED50 values of 92-93 nM and 3.5-4.1 nM in RIA and synaptosomes, respectively. Synaptosome assays reflect potency of each toxin examined, while radioimmunoassay reflects structural similarities to the immunizing toxin PbTx-3.

A double-blind comparison of moclobemide and fluoxetine in the treatment of depressive disorders.

Moclobemide and fluoxetine were tested in a six-week trial involving 122 patients with major depressive illness. Patients initially received moclobemide, 150 mg three times daily, or fluoxetine 20 mg/day, but during weeks 3, 4, 5 and 6 the doses could be altered, giving a range of 300-600 mg/day for moclobemide or 20-40 mg/day for fluoxetine. No dietary restrictions were imposed on the patients. The trial was completed by 49 patients receiving moclobemide, and 43 patients receiving fluoxetine. The efficacies of these two agents, as determined on the Hamilton Depression Rating Scale and from Clinical Global Assessments, were found not to differ significantly. The frequencies of occurrence of adverse reactions were also similar, but sedation, nausea and vomiting were reported more frequently with fluoxetine, and insomnia was experienced with moclobemide. Tolerance of both drugs was judged to be high.

Unilocular retroperitoneal cyst of mesothelial origin presenting as a renal mass.

We report the first 2 cases, to our knowledge, of retroperitoneal cysts with features of mesothelial differentiation that clinically mimic renal masses. The first lesion occurred in a 71-year-old man who presented with flank pain. Ultrasound and magnetic resonance imaging studies showed a unilocular cystic structure arising from the upper pole of the left kidney. The second lesion was in a 44-year-old woman who presented with left flank pain. Imaging studies revealed an 8-cm hemorrhagic cyst at the lower pole of the left kidney. Histologic examination of the nephrectomy specimens in each case revealed a unilocular cyst with intracystic and pericystic hemorrhage. In each case, the cyst was lined by a single layer of cells with ample eosinophilic cytoplasm and benign nuclear features without mucinous or müllerian differentiation. Histochemical staining showed Alcian blue positivity on the cell surface, which was sensitive to hyaluronidase digestion. Intracytoplasmic mucin, however, was not detected. Immunostaining showed that the cyst lining cells were positive for keratin, vimentin, HBME-1, WT1, and thrombomodulin but negative for carcinoembryonic antigen, B72.3, Leu-M1, and BerEP4. The first case was positive for calretinin, whereas the second was negative. These findings support the mesothelial nature of the cysts.

Advantages and disadvantages of benzodiazepine prescription.

There is little evidence to warrant the long term prescribing of benzodiazepine as anxiolytics and there is an urgent need for research into the long term effects of these drugs. Benzodiazepine abuse with dependence and serious withdrawal symptoms is increasingly recognised as a major problem. Various studies show that the benzodiazepines can release aggressive impulses and that serious disinhibition can result from their use. Their suicidogenic potential in depressed patients is part of this disinhibiting effect. There is strong evidence that the benzodiazepines especially in combination with alcohol render individuals accident prone. Cognitive defects are being increasingly recognised in benzodiazepine users. While these are reversible in short term users the question as to whether long term users suffer an increasing dementia urgently requires further research.