Genome integration and excision by a new Streptomyces bacteriophage, ϕJoe

Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces, most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here we present the genome of a new Streptomyces phage, ϕJoe, and investigate the conditions for integration and excision of the φJoe genome. ϕJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from S. venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe int/attP locus. The attB site for ϕJoe integrase was occupied in several Streptomyces genomes, including S. coelicolor, by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified and its function was confirmed in vivo Both the integrase and RDF were active in in vitro recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. IMPORTANCE: Streptomyces spp. are prolific producers of secondary metabolites including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering as they enable integration of heterologous DNA into the Streptomyces chromosome with ease and high efficiency. Recently researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with different specificities for their integration sites. In order to provide a broad platform of integrases we identified and validated the integrase from a newly isolated Streptomyces phage, ϕJoe. ϕJoe integrase is active in vitro and in vivo The specific recognition site for integration is present in a wide range of different Actinobacteria, including Streptomyces venezuelae, an emerging model bacterium in Streptomyces research

Recombination directionality factor gp3 binds ϕC31 integrase via the zinc domain, potentially affecting the trajectory of the coiled-coil motif

To establish a prophage state, the genomic DNA of temperate bacteriophages normally becomes integrated into the genome of their host bacterium by integrase-mediated, site-specific DNA recombination. Serine integrases catalyse a single crossover between an attachment site in the host (attB) and a phage attachment site (attP) on the circularized phage genome to generate the integrated prophage DNA flanked by recombinant attachment sites, attL and attR. Exiting the prophage state and entry into the lytic growth cycle requires an additional phage-encoded protein, the recombination directionality factor or RDF, to mediate recombination between attL and attR and excision of the phage genome. The RDF is known to bind integrase and switch its activity from integration (attP x attB) to excision (attL x attR) but its precise mechanism is unclear. Here, we identify amino acid residues in the RDF, gp3, encoded by the Streptomyces phage ϕC31 and within the ϕC31 integrase itself that affect the gp3:Int interaction. We show that residue substitutions in integrase that reduce gp3 binding adversely affect both excision and integration reactions. The mutant integrase phenotypes are consistent with a model in which the RDF binds to a hinge region at the base of the coiled-coil motif in ϕC31 integrase

Ionisation and discharge in cloud-forming atmospheres of brown dwarfs and extrasolar planets

The authors highlight financial support of the European Community under the FP7 by the ERC starting grant 257431.Brown dwarfs and giant gas extrasolar planets have cold atmospheres with a rich chemical compositions from which mineral cloud particles form. Their properties, like particle sizes and material composition, vary with height, and the mineral cloud particles are charged due to triboelectric processes in such dynamic atmospheres. The dynamics of the atmospheric gas is driven by the irradiating host star and/or by the rotation of the objects that changes during its lifetime. Thermal gas ionisation in these ultra-cool but dense atmospheres allows electrostatic interactions and magnetic coupling of a substantial atmosphere volume. Combined with a strong magnetic field ≫ BEarth, a chromosphere and aurorae might form as suggested by radio and X-ray observations of brown dwarfs. Non-equilibrium processes like cosmic ray ionisation and discharge processes in clouds will increase the local pool of free electrons in the gas. Cosmic rays and lighting discharges also alter the composition of the local atmospheric gas such that tracer molecules might be identified. Cosmic rays affect the atmosphere through air showers which was modelled with a 3D Monte Carlo radiative transfer code to be able to visualise their spacial extent. Given a certain degree of thermal ionisation of the atmospheric gas, we suggest that electron attachment to charge mineral cloud particles is too inefficient to cause an electrostatic disruption of the cloud particles. Cloud particles will therefore not be destroyed by Coulomb explosion for the local temperature in the collisional dominated brown dwarf and giant gas planet atmospheres. However, the cloud particles are destroyed electrostatically in regions with strong gas ionisation. The potential size of such cloud holes would, however, be too small and might occur too far inside the cloud to mimic the effect of, e.g., magnetic field induced star spots.PostprintPeer reviewe

Latent regulatory potential of human-specific repetitive elements

At least half of the human genome is derived from repetitive elements, which are often lineage specific and silenced by a variety of genetic and epigenetic mechanisms. Using a transchromosomic mouse strain that transmits an almost complete single copy of human chromosome 21 via the female germline, we show that a heterologous regulatory environment can transcriptionally activate transposon-derived human regulatory regions. In the mouse nucleus, hundreds of locations on human chromosome 21 newly associate with activating histone modifications in both somatic and germline tissues, and influence the gene expression of nearby transcripts. These regions are enriched with primate and human lineage-specific transposable elements, and their activation corresponds to changes in DNA methylation at CpG dinucleotides. This study reveals the latent regulatory potential of the repetitive human genome and illustrates the species specificity of mechanisms that control it

RNA-binding protein CPEB1 remodels host and viral RNA landscapes.

Host and virus interactions occurring at the post-transcriptional level are critical for infection but remain poorly understood. Here, we performed comprehensive transcriptome-wide analyses revealing that human cytomegalovirus (HCMV) infection results in widespread alternative splicing (AS), shortening of 3' untranslated regions (3' UTRs) and lengthening of poly(A)-tails in host gene transcripts. We found that the host RNA-binding protein CPEB1 was highly induced after infection, and ectopic expression of CPEB1 in noninfected cells recapitulated infection-related post-transcriptional changes. CPEB1 was also required for poly(A)-tail lengthening of viral RNAs important for productive infection. Strikingly, depletion of CPEB1 reversed infection-related cytopathology and post-transcriptional changes, and decreased productive HCMV titers. Host RNA processing was also altered in herpes simplex virus-2 (HSV-2)-infected cells, thereby indicating that this phenomenon might be a common occurrence during herpesvirus infections. We anticipate that our work may serve as a starting point for therapeutic targeting of host RNA-binding proteins in herpesvirus infections

Inflammation mobilizes copper metabolism to promote colon tumorigenesis via an IL-17-STEAP4-XIAP axis.

Copper levels are known to be elevated in inflamed and malignant tissues. But the mechanism underlying this selective enrichment has been elusive. In this study, we report a axis by which inflammatory cytokines, such as IL-17, drive cellular copper uptake via the induction of a metalloreductase, STEAP4. IL-17-induced elevated intracellular copper level leads to the activation of an E3-ligase, XIAP, which potentiates IL-17-induced NFκB activation and suppresses the caspase 3 activity. Importantly, this IL-17-induced STEAP4-dependent cellular copper uptake is critical for colon tumor formation in a murine model of colitis-associated tumorigenesis and STEAP4 expression correlates with IL-17 level and XIAP activation in human colon cancer. In summary, this study reveals a IL-17-STEAP4-XIAP axis through which the inflammatory response induces copper uptake, promoting colon tumorigenesis

BigMouth: a multi-institutional dental data repository

Few oral health databases are available for research and the advancement of evidence-based dentistry. In this work we developed a centralized data repository derived from electronic health records (EHRs) at four dental schools participating in the Consortium of Oral Health Research and Informatics. A multi-stakeholder committee developed a data governance framework that encouraged data sharing while allowing control of contributed data. We adopted the i2b2 data warehousing platform and mapped data from each institution to a common reference terminology. We realized that dental EHRs urgently need to adopt common terminologies. While all used the same treatment code set, only three of the four sites used a common diagnostic terminology, and there were wide discrepancies in how medical and dental histories were documented. BigMouth was successfully launched in August 2012 with data on 1.1 million patients, and made available to users at the contributing institutions

On the Morphology and Chemical Composition of the HR 4796A Debris Disk

[abridged] We present resolved images of the HR 4796A debris disk using the Magellan adaptive optics system paired with Clio-2 and VisAO. We detect the disk at 0.77 \microns, 0.91 \microns, 0.99 \microns, 2.15 \microns, 3.1 \microns, 3.3 \microns, and 3.8 \microns. We find that the deprojected center of the ring is offset from the star by 4.76$\pm$1.6 AU and that the deprojected eccentricity is 0.06$\pm$0.02, in general agreement with previous studies. We find that the average width of the ring is 14$^{+3}_{-2}$, also comparable to previous measurements. Such a narrow ring precludes the existence of shepherding planets more massive than \about 4 \mj, comparable to hot-start planets we could have detected beyond \about 60 AU in projected separation. Combining our new scattered light data with archival HST/STIS and HST/NICMOS data at \about 0.5-2 \microns, along with previously unpublished Spitzer/MIPS thermal emission data and all other literature thermal data, we set out to constrain the chemical composition of the dust grains. After testing 19 individual root compositions and more than 8,400 unique mixtures of these compositions, we find that good fits to the scattered light alone and thermal emission alone are discrepant, suggesting that caution should be exercised if fitting to only one or the other. When we fit to both the scattered light and thermal emission simultaneously, we find mediocre fits (reduced chi-square \about 2). In general, however, we find that silicates and organics are the most favored, and that water ice is usually not favored. These results suggest that the common constituents of both interstellar dust and solar system comets also may reside around HR 4796A, though improved modeling is necessary to place better constraints on the exact chemical composition of the dust.Comment: Accepted to ApJ on October 27, 2014. 21 pages, 12 figures, 4 table

Fungal Levels in the Home and Allergic Rhinitis by 5 Years of Age

Studies have repeatedly demonstrated that sensitization to fungi, such as Alternaria, is strongly associated with allergic rhinitis and asthma in children. However, the role of exposure to fungi in the development of childhood allergic rhinitis is poorly understood. In a prospective birth cohort of 405 children of asthmatic/allergic parents from metropolitan Boston, Massachusetts, we examined in-home high fungal concentrations (> 90th percentile) measured once within the first 3 months of life as predictors of doctor-diagnosed allergic rhinitis in the first 5 years of life. In multivariate Cox regression analyses, predictors of allergic rhinitis included high levels of dust-borne Aspergillus [hazard ratio (HR) = 3.27; 95% confidence interval (CI), 1.50–7.14], Aureobasidium (HR = 3.04; 95% CI, 1.33–6.93), and yeasts (HR = 2.67; 95% CI, 1.26–5.66). The factors controlled for in these analyses included water damage or mild or mildew in the building during the first year of the child’s life, any lower respiratory tract infection in the first year, male sex, African-American race, fall date of birth, and maternal IgE to Alternaria > 0.35 U/mL. Dust-borne Alternaria and non-sporulating and total fungi were also predictors of allergic rhinitis in models excluding other fungi but adjusting for all of the potential confounders listed above. High measured fungal concentrations and reports of water damage, mold, or mildew in homes may predispose children with a family history of asthma or allergy to the development of allergic rhinitis

Proteinuria as a modifiable risk factor for the progression of non-diabetic renal disease

Proteinuria as a modifiable risk factor for the progression of non-diabetic renal disease.BackgroundAngiotensin-converting enzyme (ACE) inhibitors reduce urine protein excretion and slow the progression of renal disease. The beneficial effect in slowing the progression of renal disease is greater in patients with higher urine protein excretion at the onset of treatment. We hypothesized that the greater beneficial effect of ACE inhibitors on the progression of renal disease in patients with higher baseline levels of proteinuria is due to their greater antiproteinuric effect in these patients.MethodsData were analyzed from 1860 patients enrolled in 11 randomized controlled trials comparing the effect of antihypertensive regimens, including ACE inhibitors to regimens not including ACE inhibitors on the progression of non-diabetic renal disease. Multivariable linear regression analysis was used to assess the relationship between the level of proteinuria at baseline and changes in urine protein excretion during follow-up. The Cox proportional hazards analysis was used to assess the relationship between changes in urine protein excretion during follow-up and the effect of ACE inhibitors on the time to doubling of baseline serum creatinine values or onset of end-stage renal disease.ResultsMean (median) baseline urine protein excretion was 1.8 (0.94) g/day. Patients with higher baseline urine protein excretion values had a greater reduction in proteinuria during the follow-up in association with treatment with ACE inhibitors and in association with lowering systolic and diastolic blood pressures (interaction P < 0.001 for all). A higher level of urine protein excretion during follow-up (baseline minus change) was associated with a greater risk of progression [relative risk 5.56 (3.87 to 7.98) for each 1.0 g/day higher protein excretion]. After controlling for the current level of urine protein excretion, the beneficial effect of ACE inhibitors remained significant [relative risk for ACE inhibitors vs. control was 0.66 (0.52 to 0.83)], but there was no significant interaction between the beneficial effect of ACE inhibitors and the baseline level of urine protein excretion.ConclusionsThe antiproteinuric effects of ACE inhibitors and lowering blood pressure are greater in patients with a higher baseline urine protein excretion. The greater beneficial effect of ACE inhibitors on renal disease progression in patients with higher baseline proteinuria can be explained by their greater antiproteinuric effects in these patients. The current level of urine protein excretion is a modifiable risk factor for the progression of non-diabetic renal disease. ACE inhibitors provide greater beneficial effect at all levels of current urine protein excretion