Reconstructing Viral Genomes from the Environment Using Fosmid Clones: The Case of Haloviruses

Background: Metaviriomes, the viral genomes present in an environment, have been studied by direct sequencing of the viral DNA or by cloning in small insert libraries. The short reads generated by both approaches make it very difficult to assemble and annotate such flexible genomic entities. Many environmental viruses belong to unknown groups or prey on uncultured and little known cellular lineages, and hence might not be present in databases. Methodology and Principal Findings: Here we have used a different approach, the cloning of viral DNA into fosmids before sequencing, to obtain natural contigs that are close to the size of a viral genome. We have studied a relatively low diversity extreme environment: saturated NaCl brines, which simplifies the analysis and interpretation of the data. Forty-two different viral genomes were retrieved, and some of these were almost complete, and could be tentatively identified as head-tail phages (Caudovirales). Conclusions and Significance: We found a cluster of phage genomes that most likely infect Haloquadratum walsbyi, the square archaeon and major component of the community in these hypersaline habitats. The identity of the prey could be confirmed by the presence of CRISPR spacer sequences shared by the virus and one of the available strain genomes. Other viral clusters detected appeared to prey on the Nanohaloarchaea and on the bacterium Salinibacter ruber, covering most of the diversity of microbes found in this type of environment. This approach appears then as a viable alternative to describe metaviriomes in a much more detailed and reliable way than by the more common approaches based on direct sequencing. An example of transfer of a CRISPR cluster including repeats and spacers was accidentally found supporting the dynamic nature and frequent transfer of this peculiar prokaryotic mechanism of cell protection.This work was supported by projects MAGYK (BIO2008-02444), MICROGEN (Programa CONSOLIDERINGENIO 2010 CDS2009-00006), CGL2'09-12651-C02-01 from the Spanish Ministerio de Ciencia e Innovación, DIMEGEN (PROMETEO/2010/089) and ACOMP/2009/155 from the Generalitat Valenciana. FEDER funds supported this project. IG-H was supported by MAGYK from Ministerio de Ciencia e Innovación. A-BM-C was supported by CONSOLIDER-INGENIO 2010

Ferredoxin C2 is required for chlorophyll biosynthesis and accumulation of photosynthetic antennae in Arabidopsis

Ferredoxins (Fd) are small iron-sulphur proteins, with sub-types that have evolved for specific redox functions. Ferredoxin C2 (FdC2) proteins are essential Fd homologues conserved in all photosynthetic organisms and a number of different FdC2 functions have been proposed in angiosperms. Here we use RNAi silencing in Arabidopsis thaliana to generate a viable fdC2 mutant line with near-depleted FdC2 protein levels. Mutant leaves have ~50% less chlorophyll a and b, and chloroplasts have poorly developed thylakoid membrane structure. Transcriptomics indicates upregulation of genes involved in stress responses. Although fdC2 antisense plants show increased damage at photosystem II (PSII) when exposed to high light, PSII recovers at the same rate as wild type in the dark. This contradicts literature proposing that FdC2 regulates translation of the D1 subunit of PSII, by binding to psbA transcript. Measurement of chlorophyll biosynthesis intermediates revealed a build-up of Mg-protoporphyrin IX, the substrate of the aerobic cyclase. We localise FdC2 to the inner chloroplast envelope and show that the FdC2 RNAi line has a disproportionately lower protein abundance of antennae proteins, which are nuclear-encoded and must be refolded at the envelope after import

Dual ifgMosaic: A Versatile Method for Multispectral and Combinatorial Mosaic Gene-Function Analysis

Improved methods for manipulating and analyzing gene function have provided a better understanding of how genes work during organ development and disease. Inducible functional genetic mosaics can be extraordinarily useful in the study of biological systems; however, this experimental approach is still rarely used in vertebrates. This is mainly due to technical difficulties in the assembly of large DNA constructs carrying multiple genes and regulatory elements and their targeting to the genome. In addition, mosaic phenotypic analysis, unlike classical single gene-function analysis, requires clear labeling and detection of multiple cell clones in the same tissue. Here, we describe several methods for the rapid generation of transgenic or gene-targeted mice and embryonic stem (ES) cell lines containing all the necessary elements for inducible, fluorescent, and functional genetic mosaic (ifgMosaic) analysis. This technology enables the interrogation of multiple and combinatorial gene function with high temporal and cellular resolution.This work was supported by grants to the PI R.B. from the Spanish Ministry of Economy, Industry and Competitiveness (SAF2013-44329-P, SAF2013-42359-ERC, and RYC-2013-13209) and European Research Council (ERC-2014-StG - 638028). S.P.-Q., M.F.-C., and I.G.-G. were supported by PhD fellowships from Fundacion La Caixa (CX-SO-2013-02, CX\_E-2015-01, and CX-SO-16-1, respectively). W.L. by a FP7-PEOPLE-2012-COFUND GA600396 postdoctoral contract. We thank Simon Bartlett for English editing, Ralf H. Adams for sharing the Cdh5(PAC)-CreERT2 mice, Jose Luis de La Pompa for comments throughout the project and for sharing the Tie2-Cre mice, Gonzalo Gancedo for the help with the mouse colony, Valeria Caiolfa for the help with the microscopy, and all the members of the CNIC gene targeting, transgenesis, cellomics, and microscopy units. The CNIC is supported by MEIC/MINECO and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Pilot Study to Assess the Effectiveness of the Sustainable Culturally adaptive Nutrition Program (Scan) to Improve adherence to the National Diabetes Prevention Program

PURPOSE: The Sustainable Culturally Adapted Nutrition Program (SCAN) is a novel adaptation to the National Diabetes Prevention Program (NDPP) that aims to improve attendance and effectiveness. This paper presents its feasibility and impact through the initial 6-month outcomes. DESIGN: A pragmatic quasi-experimental pilot study with intervention (DPP plus SCAN) and control (DPP only) groups. SAMPLES AND INCLUSION CRITERIA: Sustainable Culturally Adapted Nutrition Program participants were recruited from federally qualified health center (FQHC) clinic patients enrolled in a NDPP in Houston, Texas. Participants needed to be (1) ≥18 years old, (2) body mass index \u3e25, (3) no prior diagnosis of diabetes, and (4) not pregnant. INTERVENTION: Sustainable Culturally Adapted Nutrition Program cooking classes were designed to teach skills to prepare fresh produce, and utilized Motivational Interviewing (MI) techniques to encourage participants to adapt these skills for foods that were culturally important to them. OUTCOME MEASURES: (1) National Diabetes Prevention Program attendance, (2) BMI and (3) percent weight loss. ANALYSIS: We used linear mixed models to test the association between weights and NDPP attendance. RESULTS: 22 intervention and 15 control participants completed the program to the 6-month point. Intervention participants had increased DPP attendance over controls (7.14 vs 6.87 session). Intervention participants also demonstrated on average, 1.5% weight loss for each additional SCAN class attended ( CONCLUSIONS: The SCAN adaptation shows promising results for effectively increasing both NDPP attendance and weight loss

Stent tunnel technique to save thrombosed native hemodialysis fistula with extensive venous aneurysm

Introduction and purpose: The increasing number of patients undergoing hemodialysis and the limited number of access sites have resulted in an increasing number of techniques to maintain vascular access for hemodialysis. Thrombosed arteriovenous (AV) fistulas with large venous aneurysms have poor treatment results, with both endovascular and surgical techniques, leading to a high rate of definitive AV access loss. The purpose of this study was to review the feasibility and initial results of this novel endovascular treatment of thrombosed AV fistulas with large venous aneurysms. Materials and methods: A novel endovascular treatment technique of inserting nitinol auto-expandable uncovered stents stretching through the whole puncture site area, thus creating a tunnel inside the thrombus, was retrospectively analyzed and described. Results: A total of 17 stents were placed in 10 hemodialysis fistulas, with a mean venous coverage length of 17.8 cm. In all the cases, 100% technical success was achieved, with complete restoration of blood flow in all patients. There were no procedure-related complications. The mean follow-up was 167 days (range 60–420 days), with a primary and assisted patency of 80% and 100%, respectively. No multiple trans-stent struts-related complications were observed. Three stent fractures were diagnosed with plain films at the site of puncture without consequence in the venous access permeability. Conclusion: The “stent tunnel technique” is a feasible, safe and effective alternative to salvage native hemodialysis access, thus extending the function of the venous access with no signs of stent-related complications and a respectable midterm patency

The role of probiotics on the microbiota: effect on obesity

The microbiota and the human host maintain a symbiotic association. Nowadays, metagenomic analyses are providing valuable knowledge on the diversity and functionality of the gut microbiota. However, with regard to the definition of a “healthy microbiota” and the characterization of the dysbiosis linked to obesity, there is still not a clear answer. Despite this fact, attempts have been made to counteract obesity through probiotic supplementation. A literature search of experimental studies relevant to the topic was performed in PubMed database with the keywords “probiotic” and “obesity” and restricted to those with “Lactobacillus” or “Bifidobacterium” in the title. So far, evidence of an antiobesity effect of different lactobacilli and bifidobacteria has been mainly obtained from animal models of dietary-induced obesity. Using these experimental models, a substantial number of studies have reported reductions in weight gain and, in particular, fat tissue mass at different locations following administration of bacteria, as compared with controls. Antiatherogenic and anti-inflammatory effects—including regulation of expression of lipogenic and lipolytic genes in the liver, reduction in liver steatosis, improvement of blood lipid profile and glucose tolerance, decreased endotoxemia, and regulation of inflammatory pathways—are also reported in many of them. The number of human studies focused on probiotic administration for obesity management is still very scarce, and it is too soon to judge their potential efficacy, especially when considering the fact that the actions of probiotics are always strain specific and the individual response varies according to intrinsic factors, the overall composition of diet, and their interactions

Dyskeratosis congenita: natural history of the disease through the study of a cohort of patients diagnosed in childhood

BackgroundDyskeratosis congenita (DC) is a multisystem and ultra-rare hereditary disease characterized by somatic involvement, bone marrow failure, and predisposition to cancer. The main objective of this study is to describe the natural history of DC through a cohort of patients diagnosed in childhood and followed up for a long period of time.Material and methodsMulticenter, retrospective, longitudinal study conducted in patients followed up to 24 years since being diagnosed in childhood (between 1998 and 2020).ResultsFourteen patients were diagnosed with DC between the ages of 3 and 17 years (median, 8.5 years). They all had hematologic manifestations at diagnosis, and nine developed mucocutaneous manifestations during the first decade of life. Seven presented severe DC variants. All developed non-hematologic manifestations during follow-up. Mutations were identified in 12 patients. Thirteen progressed to bone marrow failure at a median age of 8 years [range, 3–18 years], and eight received a hematopoietic stem cell transplant. Median follow-up time was 9 years [range, 2–24 years]. Six patients died, the median age was 13 years [range, 6–24 years]. As of November 2022, eight patients were still alive, with a median age of 18 years [range, 6–32 years]. None of them have developed myeloblastic syndrome or cancer.ConclusionsDC was associated with high morbidity and mortality in our series. Hematologic manifestations appeared early and consistently. Non-hematologic manifestations developed progressively. No patient developed cancer possibly due to their young age. Due to the complexity of the disease multidisciplinary follow-up and adequate transition to adult care are essential

Pan-Atlantic analysis of the overlap of a highly migratory species, the leatherback turtle, with pelagic longline fisheries

Large oceanic migrants play important roles in ecosystems, yet many species are of conservation concern as a result of anthropogenic threats, of which incidental capture by fisheries is frequently identified. The last large populations of the leatherback turtle, Dermochelys coriacea, occur in the Atlantic Ocean, but interactions with industrial fisheries could jeopardize recent positive population trends, making bycatch mitigation a priority. Here, we perform the first pan-Atlantic analysis of spatio-temporal distribution of the leatherback turtle and ascertain overlap with longline fishing effort. Data suggest that the Atlantic probably consists of two regional management units: northern and southern (the latter including turtles breeding in South Africa). Although turtles and fisheries show highly diverse distributions, we highlight nine areas of high susceptibility to potential bycatch (four in the northern Atlantic and five in the southern/equatorial Atlantic) that are worthy of further targeted investigation and mitigation. These are reinforced by reports of leatherback bycatch at eight of these sites. International collaborative efforts are needed, especially from nations hosting regions where susceptibility to bycatch is likely to be high within their exclusive economic zone (northern Atlantic: Cape Verde, Gambia, Guinea Bissau, Mauritania, Senegal, Spain, USA and Western Sahara; southern Atlantic: Angola, Brazil, Namibia and UK) and from nations fishing in these high-susceptibility areas, including those located in international waters

Pan-Atlantic analysis of the overlap of a highly migratory species, the leatherback turtle, with pelagic longline fisheries

This is the final version of the article. Available from the publisher via the DOI in this record.Large oceanic migrants play important roles in ecosystems, yet many species are of conservation concern as a result of anthropogenic threats, of which incidental capture by fisheries is frequently identified. The last large populations of the leatherback turtle, Dermochelys coriacea, occur in the Atlantic Ocean, but interactions with industrial fisheries could jeopardize recent positive population trends, making bycatch mitigation a priority. Here, we perform the first pan-Atlantic analysis of spatio-temporal distribution of the leatherback turtle and ascertain overlap with longline fishing effort. Data suggest that the Atlantic probably consists of two regional management units: northern and southern (the latter including turtles breeding in South Africa). Although turtles and fisheries show highly diverse distributions, we highlight nine areas of high susceptibility to potential bycatch (four in the northern Atlantic and five in the southern/equatorial Atlantic) that are worthy of further targeted investigation and mitigation. These are reinforced by reports of leatherback bycatch at eight of these sites. International collaborative efforts are needed, especially from nations hosting regions where susceptibility to bycatch is likely to be high within their exclusive economic zone (northern Atlantic: Cape Verde, Gambia, Guinea Bissau, Mauritania, Senegal, Spain, USA and Western Sahara; southern Atlantic: Angola, Brazil, Namibia and UK) and from nations fishing in these high-susceptibility areas, including those located in international waters.Work in Gabon was financially supported by the Large Pelagics Research Center through National Oceanographic and Atmospheric Agency award no. NA04NMF4550391, the UK Defra Darwin Initiative, the Shellshock Campaign (European Association for Zoos and Aquaria) and the UK Natural Environment Research Council. Sea turtle monitoring programmes in Gabon were financially supported by the Wildlife Conservation Society and by the Gabon Sea Turtle Partnership with funding from the Marine Turtle Conservation Fund (United States Fish and Wildlife Service, US Department of the Interior). Four of the satellite tags were deployed in Canadian waters by M. James (Dalhousie University) and the Canadian Sea Turtle Network, with the funding support of Canadian Sea Turtle Network leatherback field research provided by R. A. Myers, the Canadian Wildlife Federation, Environment Canada and WWF-Canada. Work in French Guiana was financially supported by CNES, DEAL and the European Union.This study results from the collaborative effort of 10 data providers, which have satellite-tracked leatherback turtles in the Atlantic Ocean since 1995, through their voluntary participation in the Trans-Atlantic Leatherback Conservation Initiative (TALCIN), a WWF-led initiative. We thank C. Drews (WWF-International) and Jean-Yves Georges (IPHC-CNRS) for having initiated this project. Significant contributions were made by A. Fonseca and M. L. Felix and the WWF Guianas office in fostering this project to secure its continuation. We thank those involved in the sea turtle restoration plan in French Guiana (DEAL, ONCFS, Kulalasi NGO, Kwata, the Reserve Naturelle de l'Amana, Chiefs of Awala and Yalimapo), Yvon Le Maho (IPHC-CNRS) for having initiated the leatherback tracking programme in French Guiana, colleagues from the Regional Program for Sea Turtles Research and Conservation of Argentina–PRICTMA, Aquamarina and Fundación Mundo Marino, the onboard scientific observers from PNOFA-DINARA, the crew and owner of the F/V Torres del Paine, the artisanal fishermen from Kiyú, San José, Uruguay, D. del Bene (PROFAUMA), Z. Di Rienzo and colleagues from Karumbé, the University of Pisa for initiating the satellite tagging programmes in South Africa, and the South African Department of Environmental Affairs for continuing the work in cooperation with Dr Nel from the Nelson Mandela Metropolitan University, Port Elizabeth and Ezemvelo KZN Wildlife. We thank M. L. Felix for her efforts in the deployment of satellite tags in Suriname and the Nature Conservation Division Suriname for facilitating these research efforts. P.M. thanks C. Palma for his help in dealing with ICCAT's database, C. Ere, as well as the GIS training and support received from SCGIS and the ESRI Conservation Program, which allowed processing of fishing-effort data. We thank J. Parezo for her careful reading of the manuscript. All authors designed the study and contributed data; S.F, M.S.C., P.M. and M.J.W. compiled the data; S.F., M.A.N. and A.L. coordinated and supervised the project; S.F., M.J.W., P.M. and B.J.G. led the data analysis and interpretation with contributions from all authors; the manuscript was developed by S.F. and M.J.W. as lead authors, with contributions from all authors

Local piezoresponse and polarization switching in nucleobase thymine microcrystals

Thymine (2-oxy-4-oxy-5 methyl pyrimidine) is one of the four nucleobases of deoxyribonucleic acid (DNA). In the DNA molecule, thymine binds to adenine via two hydrogen bonds, thus stabilizing the nucleic acid structure and is involved in pairing and replication. Here, we show that synthetic thymine microcrystals grown from the solution exhibit local piezoelectricity and apparent ferroelectricity, as evidenced by nanoscale electromechanical measurements via Piezoresponse Force Microscopy. Our experimental results demonstrate significant electromechanical activity and polarization switchability of thymine, thus opening a pathway for piezoelectric and ferroelectric-based applications of thymine and, perhaps, of other DNA nucleobase materials. The results are supported by molecular modeling of polarization switching under an external electric field. (C) 2015 AIP Publishing LLC