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.

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'.

A Wnt kinase network alters nuclear localization of TCF-1 in colon cancer.

Constitutive activation of the Wnt/beta-catenin pathway has been implicated as the primary cause of colon cancer. However, the major transducers of Wnt signaling in the intestine, T-cell factor 1 (TCF-1) and TCF-4, have opposing functions. Knockout of TCF-4 suppresses growth and maintenance of crypt stem cells, whereas knockout of TCF-1 leads to adenomas. These phenotypes suggest that TCF-4 is Wnt-promoting, whereas TCF-1 acts like a tumor suppressor. Our study of TCF expression in human colon crypts reveals a mechanistic basis for this paradox. In normal colon cells, a dominant-negative isoform of TCF-1 (dnTCF-1) is expressed that is equally distributed between nuclear and cytoplasmic compartments. In colon cancer cells, TCF-1 is predominantly cytoplasmic. Localization is because of active nuclear export and is directed by an autocrine-acting Wnt ligand that requires Ca2+/calmodulin-dependent kinase II (CaMKII) activity for secretion and a downstream step in the export pathway. TCF-4 remains nuclear; its unopposed activity is accompanied by downregulation of dnTCF-1 and increased expression of full-length isoforms. Thus, the dnTCF-1 and TCF-4 balance is corrupted in cancer by two mechanisms, a Wnt/CaMKII kinase signal for nuclear export and decreased dnTCF-1 expression. We propose that dnTCF-1 provides homeostatic regulation of Wnt signaling and growth in normal colon, and the alterations in nuclear export and promoter usage contribute to aberrant Wnt activity in colon cancer.

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.

Chasing Aleck: the story of a dorm.

A student raised a hand in class and asked, "Why is this dorm named after Alexander Graham Bell?" On a deaf campus, this was a loaded question. Bell was an oralist, opposed to sign language. He was a eugenicist, opposed to deaf marriages. Indeed, the more I thought about it, the better this question got. Why did the school name a dormin after him? Unfortunately, I hadn't the foggiest idea. With apologies to that student, I offer this article as a belated answer.

Impaired apoptosis, extended duration of immune responses, and a lupus-like autoimmune disease in IEX-1-transgenic mice.

Susceptibility of activated T cells to apoptosis must be tightly regulated to ensure sufficient T cell progeny for an effective response, while allowing a rapid depletion of them at the end of the immune response. We show here that a previously isolated, NF-kappa B/rel target gene IEX-1 (Immediate Early response gene X-1) is highly expressed in T cells at early stages of activation, but declines with a prolonged period of activation time, coincident with an increased susceptibility of T cells to apoptosis during the late phases of an immune response. Transgenic expression of IEX-1 specifically in lymphocytes impaired apoptosis in activated T cells, extended a duration of an effector-phase of a specific immune response, and increased the accumulation of effector/memory-like T cells and the susceptibility to a lupus-like autoimmune disease. Our study demonstrated an antiapoptotic effect of IEX-1 on T cell apoptosis triggered by ligation of Fas and T cell receptor (TCR)/CD3 complex. The ability of extending life expectancy of T effectors, in line with a decrease in its expression following prolonged T cell activation, suggests a key role for IEX-1 in regulating T cell homeostasis during immune responses.

Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13.

The ubiquitin conjugating enzyme complex Mms2-Ubc13 plays a key role in post-replicative DNA repair in yeast and the NF-kappaB signal transduction pathway in humans. This complex assembles novel polyubiquitin chains onto yet uncharacterized protein targets. Here we report the crystal structure of a complex between hMms2 (Uev1) and hUbc13 at 1.85 A resolution and a structure of free hMms2 at 1.9 A resolution. These structures reveal that the hMms2 monomer undergoes a localized conformational change upon interaction with hUbc13. The nature of the interface provides a physical basis for the preference of Mms2 for Ubc13 as a partner over a variety of other structurally similar ubiquitin-conjugating enzymes. The structure of the hMms2-hUbc13 complex provides the conceptual foundation for understanding the mechanism of Lys 63 multiubiquitin chain assembly and for its interactions with the RING finger proteins Rad5 and Traf6.

Phenotypic consequences of lung-specific inducible expression of FGF-3.

Members of the fibroblast growth factor (FGF) family play a critical role in embryonic lung development and adult lung physiology. The in vivo investigation of the role FGFs play in the adult lung has been hampered because the constitutive pulmonary expression of these factors often has deleterious effects and frequently results in neonatal lethality. To circumvent these shortcomings, we expressed FGF-3 in the lungs under the control of the progesterone antagonist-responsive binary transgenic system. Four binary transgenic lines were obtained that showed ligand-dependent induction of FGF-3 with induced levels of FGF-3 expression dependent on the levels of expression of the GLp65 regulator as well as the dose of the progesterone antagonist, RU486, administered. FGF-3 expression in the adult mouse lung resulted in two phenotypes depending on the levels of induction of FGF-3. Low levels of FGF-3 expression resulted in massive free alveolar macrophage infiltration. High levels of FGF-3 expression resulted in diffuse alveolar type II cell hyperplasia. Both phenotypes were reversible after the withdrawal of RU486. This system will be a valuable means of investigating the diverse roles of FGFs in the adult lung.

Removing a hydrogen bond in the dimer interface of Escherichia coli manganese superoxide dismutase alters structure and reactivity.

Among manganese superoxide dismutases, residues His30 and Tyr174 are highly conserved, forming part of the substrate access funnel in the active site. These two residues are structurally linked by a strong hydrogen bond between His30 NE2 from one subunit and Tyr174 OH from the other subunit of the dimer, forming an important element that bridges the dimer interface. Mutation of either His30 or Tyr174 in Escherichia coli MnSOD reduces the superoxide dismutase activity to 30--40% of that of the wt enzyme, which is surprising, since Y174 is quite remote from the active site metal center. The 2.2 A resolution X-ray structure of H30A-MnSOD shows that removing the Tyr174-->His30 hydrogen bond from the acceptor side results in a significant displacement of the main-chain segment containing the Y174 residue, with local rearrangement of the protein. The 1.35 A resolution structure of Y174F-MnSOD shows that disruption of the same hydrogen bond from the donor side has much greater consequences, with reorientation of F174 having a domino effect on the neighboring residues, resulting in a major rearrangement of the dimer interface and flipping of the His30 ring. Spectroscopic studies on H30A, H30N, and Y174F mutants show that (like the previously characterized Y34F mutant of E. coli MnSOD) all lack the high pH transition of the wt enzyme. This observation supports assignment of the pH sensitivity of MnSOD to coordination of hydroxide ion at high pH rather than to ionization of the phenolic group of Y34. Thus, mutations near the active site, as in the Y34F mutant, as well as at remote positions, as in Y174F, similarly affect the metal reactivity and alter the effective pK(a) for hydroxide ion binding. These results imply that hydrogen bonding of the H30 imidazole N--H group plays a key role in substrate binding and catalysis.

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.

Outer sphere mutations perturb metal reactivity in manganese superoxide dismutase.

Tyrosine 34 and glutamine 146 are highly conserved outer sphere residues in the mononuclear manganese active site of Escherichia coli manganese superoxide dismutase. Biochemical and spectroscopic characterization of site-directed mutants has allowed functional characterization of these residues in the wild-type (wt) enzyme. X-ray crystallographic analysis of three mutants (Y34F, Q146L, and Q146H) reveal subtle changes in the protein structures. The Y34A mutant, as well as the previously reported Y34F mutant, retained essentially the full superoxide dismutase activity of the wild-type enzyme, and the X-ray crystal structure of Y34F manganese superoxide dismutase shows that mutation of this strictly conserved residue has only minor effects on the positions of active site residues and the organized water in the substrate access funnel. Mutation of the outer sphere solvent pocket residue Q146 has more dramatic effects. The Q146E mutant is isolated as an apoprotein lacking dismutase activity. Q146L and Q146H mutants retain only 5-10% of the dismutase activity of the wild-type enzyme. The absorption and circular dichroism spectra of the Q146H mutant resemble corresponding data for the superoxide dismutase from a hyperthermophilic archaeon, Pyrobaculum aerophilum, which is active in both Mn and Fe forms. Interestingly, the iron-substituted Q146H protein also exhibits low dismutase activity, which increases at lower pH. Mutation of glutamine 146 disrupts the hydrogen-bonding network in the active site and has a greater effect on protein structure than does the Y34F mutant, with rearrangement of the tyrosine 34 and tryptophan 128 side chains.

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.

A role for Salmonella fimbriae in intraperitoneal infections.

Enteric bacteria possess multiple fimbriae, many of which play critical roles in attachment to epithelial cell surfaces. SEF14 fimbriae are only found in Salmonella enterica serovar Enteritidis (S. enteritidis) and closely related serovars, suggesting that SEF14 fimbriae may affect serovar-specific virulence traits. Despite evidence that SEF14 fimbriae are expressed by S. enteritidis in vivo, previous studies showed that SEF14 fimbriae do not mediate adhesion to the intestinal epithelium. Therefore, we tested whether SEF14 fimbriae are required for virulence at a stage in infection after the bacteria have passed the intestinal barrier. Polar mutations that disrupt the entire sef operon decreased virulence in mice more than 1,000-fold. Nonpolar mutations that disrupted sefA (encoding the major structural subunit) did not affect virulence, but mutations that disrupted sefD (encoding the putative adhesion subunit) resulted in a severe virulence defect. The results indicate that the putative SEF14 adhesion subunit is specifically required for a stage of the infection subsequent to transit across the intestinal barrier. Therefore, we tested whether SefD is required for uptake or survival in macrophages. The majority of wild-type bacteria were detected inside macrophages soon after i.p. infection, but the sefD mutants were not readily internalized by peritoneal macrophages. These results indicate that the potential SEF14 adhesion subunit is essential for efficient uptake or survival of S. enteritidis in macrophages. This report describes a role of fimbriae in intracellular infection, and indicates that fimbriae may be required for systemic infections at stages beyond the initial colonization of host epithelial surfaces.

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.

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.

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.