Biogeography of Rhizobium radiobacter and distribution of associated temperate phages in deep subseafloor sediments

Bacteriophages might be the main ‘predators'' in the marine deep subsurface and probably have a major impact on indigenous microbial communities. To identify their function within this habitat, we have determined their abundance and distribution along the sediment columns of two continental margin and two open ocean sites that were recovered during Leg 201 of the Ocean Drilling Program. For all investigated sites, viral abundance followed the total cell numbers with a virus-to-cell ratio between 1 and 10 in the upper 100 mbsf (meters below seafloor). An increasing ratio of about 20 in deeper layers indicated an ongoing viral production in up to 11 Ma old sediments. We have used Rhizobium radiobacter as the most frequently isolated organism from the deep subsurface with a high in situ abundance to identify the frequency of associated rhizobiophages. In this study, 16S rRNA gene copies of R. radiobacter accounted for up to 5.6% of total bacterial 16S rRNA genes (average: 0.75%) as detected by quantitative PCR. A distinctive distribution was identified for R. radiobacter as indicated by a site-specific arrangement of genetically similar populations. Whole genome information of rhizobiophage RR1-A was used to generate a primer system for quantitative PCR specifically targeting the prophage antirepressor gene, indicative for temperate phages. The quantification of this gene within various sediment horizons showed a contribution of temperate phages of up to 14.3% to the total viral abundance. Thus, the high amount of temperate phages within the sediments and among all investigated isolates indicates that lysogeny is the main viral proliferation mode in deep subsurface populations.     

Blood Parasite Infection Intensity Covaries with Risk‐Taking Personality in Male Carpetan Rock Lizards (Iberolacerta cyreni)

Identifying evolutionary and developmental mechanisms underlying consistent between‐individual differences in behaviour is the main goal in ‘animal personality studies’. Here, we explored whether activity and risk‐taking varied consistently between individuals and correlated to various – potentially fitness linked – male traits in Carpetan rock lizards (Iberolacerta cyreni). Lizards showed significant consistency within both behaviours, implying the presence of activity and risk‐taking personalities. However, there were no correlation between activity and risk‐taking, neither on the between‐ nor on the within‐individual levels, implying the absence of a behavioural syndrome. We found a strong link between the intensity of blood parasite (Haemogregarinidae) infection and risk‐taking: lizards with higher infection intensity took more risk. While we cannot distinguish cause from causative in the parasite intensity – risk‐taking correlation – our results are in line with the asset protection hypothesis predicting that individuals with lower future reproductive value should focus on the current reproductive event and take higher risk.     

A multiscale computational fluid dynamics approach to simulate the micro-fluidic environment within a tissue engineering scaffold with highly irregular pore geometry

Mechanical stimulation can regulate cellular behavior, e.g., differentiation, proliferation, matrix production and mineralization. To apply fluid-induced wall shear stress (WSS) on cells, perfusion bioreactors have been commonly used in tissue engineering experiments. The WSS on cells depends on the nature of the micro-fluidic environment within scaffolds under medium perfusion. Simulating the fluidic environment within scaffolds will be important for gaining a better insight into the actual mechanical stimulation on cells in a tissue engineering experiment. However, biomaterial scaffolds used in tissue engineering experiments typically have highly irregular pore geometries. This complexity in scaffold geometry implies high computational costs for simulating the precise fluidic environment within the scaffolds. In this study, we propose a low-computational cost and feasible technique for quantifying the micro-fluidic environment within the scaffolds, which have highly irregular pore geometries. This technique is based on a multiscale computational fluid dynamics approach. It is demonstrated that this approach can capture the WSS distribution in most regions within the scaffold. Importantly, the central process unit time needed to run the model is considerably low.

     

Genetic knockouts and knockins in human somatic cells

Gene targeting by homologous recombination with exogenous DNA constructs is the most powerful technique available for analysis of mammalian gene function. Over the past several years, the methods used to generate knockout and knockin mice have been modified for use in cultured human cells. The most significant innovation has been the adaptation of recombinant adeno-associated viruses (rAAVs) for such targeting. The stages of rAAV-mediated gene targeting include (i) the design and construction of a DNA targeting vector, (ii) the production of an infectious rAAV stock, (iii) the generation of cell clones that harbor rAAV transgenes, (iv) screening for homologous recombinants and (v) the iterative targeting of multiple alleles. The protocol described herein allows the generation of a cell line with a single altered allele in 3 months. A second allele of the same gene can be targeted in an additional 3 months.     

Metabolic syndrome in Yup'ik Eskimos: the Center for Alaska Native Health Research (CANHR) Study

Objective: This study investigated the prevalence of metabolic syndrome and its defining components among Yup'ik Eskimos. Research Methods and Procedures: A cross‐sectional study design that included 710 adult Yup'ik Eskimos ≥18 years of age residing in 8 communities in Southwest Alaska. The prevalence of metabolic syndrome was determined using the recently updated Adult Treatment Panel III criteria. Results: The prevalence of metabolic syndrome in this study cohort was 14.7%, and varied by sex with 8.6% of the men and 19.8% of the women having metabolic syndrome. This is lower than the prevalence of 23.9% in the general U.S. adult population. The most common metabolic syndrome components/risk factors were increased waist circumference and elevated blood glucose. High‐density lipoprotein (HDL) cholesterol levels in Yup'ik Eskimos were significantly higher, and triglycerides lower than levels reported in National Health and Nutritional Examination III. Discussion: Compared with other populations, metabolic syndrome is relatively uncommon in Yup'ik Eskimos. The higher prevalence among Yup'ik women is primarily explained by their large waist circumference, suggesting central body fat accumulation. Further increases in metabolic syndrome risk factors among Yup'ik Eskimos could lead to increases in the prevalence of type 2 diabetes and cardiovascular disease, once rare in this population.     

Elimination of affinity reagent interference for the mass spectrometric detection of low-abundance proteins following immunoprecipitation

The presence of affinity reagents such as immunoglobulin in preparations for sensitive mass spectrometry analyses can preclude the identification of low-abundance proteins of interest. We report a method whereby antisera are purified and biotinylated prior to use in immunoprecipitation that allows for its efficient removal from proteomic samples via streptavidin capture. This method can similarly be extended to other affinity reagents such as recombinant fusion proteins for enhanced identification of interacting proteins.     

GROmaρs: A GROMACS-Based Toolset to Analyze Density Maps Derived from Molecular Dynamics Simulations

We introduce a computational toolset, named GROmaρs, to obtain and compare time-averaged density maps from molecular dynamics simulations. GROmaρs efficiently computes density maps by fast multi-Gaussian spreading of atomic densities onto a three-dimensional grid. It complements existing map-based tools by enabling spatial inspection of atomic average localization during the simulations. Most importantly, it allows the comparison between computed and reference maps (e.g., experimental) through calculation of difference maps and local and time-resolved global correlation. These comparison operations proved useful to quantitatively contrast perturbed and control simulation data sets and to examine how much biomolecular systems resemble both synthetic and experimental density maps. This was especially advantageous for multimolecule systems in which standard comparisons like RMSDs are difficult to compute. In addition, GROmaρs incorporates absolute and relative spatial free-energy estimates to provide an energetic picture of atomistic localization. This is an open-source GROMACS-based toolset, thus allowing for static or dynamic selection of atoms or even coarse-grained beads for the density calculation. Furthermore, masking of regions was implemented to speed up calculations and to facilitate the comparison with experimental maps. Beyond map comparison, GROmaρs provides a straightforward method to detect solvent cavities and average charge distribution in biomolecular systems. We employed all these functionalities to inspect the localization of lipid and water molecules in aquaporin systems, the binding of cholesterol to the G protein coupled chemokine receptor type 4, and the identification of permeation pathways through the dermicidin antimicrobial channel. Based on these examples, we anticipate a high applicability of GROmaρs for the analysis of molecular dynamics simulations and their comparison with experimentally determined densities.     

Zinc tolerance and hyperaccumulation are genetically independent characters

The hyperaccumulation of metals by a rare class of plants is a fascinating and little understood phenomenon. No genetic analysis has been possible since no intraspecific variation is known for this character. Here, we report on crosses between the zinc-hyperaccumulating and -tolerant species Arabidopsis halleri and the non-hyperaccumulating, non-tolerant species Arabidopsis petraea. The F2 segregates for both characters and it appears that the two characters are genetically independent. The data for tolerance are consistent with a single major gene for this character (although the number of genes for hyperaccumulation cannot be determined), and is probably not very large.