The Unseen World: Environmental Microbial Sequencing and Identification Methods for Ecologists

Microorganisms inhabit almost every environment, comprise the majority of diversity on Earth, are important in biogeochemical cycling, and may be vital to ecosystem responses to large-scale climatic change. In recent years, ecologists have begun to use rapidly advancing molecular techniques to address questions about microbial diversity, biogeography, and responses to environmental change. Studies of microbes in the environment generally focus on three broad objectives: determining which organisms are present, what their functional capabilities are, and which are active at any given time. However, comprehending the range of methodologies currently in use can be daunting. To provide an overview of environmental microbial sequence data collection and analysis approaches, we include case studies of microbiomes ranging from the human mouth to geothermal springs. We also suggest contexts in which each technique can be applied and highlight insights that result from their use

Gneiss-charnockite transformation at Kottavattam, Southern Kerala (India)

At Kottavattam, leucocratic granitic garnet-biotite gneisses (age less than 2 Ga) were partially transformed to coarse-grained charnockite along a system of conjugate fractures (N70E and N20W) and the foliation planes (N60 to 80W; dip 80 to 90 SW) about 550 m.y. ago. To examine and quantify changes in fabric, mineralogy, pore fluids and chemical composition associated with this process, large rock specimens showing gneiss-charnockite transition were studied in detail. The results of the present study corroborate the concept that charnockite formation at Kottavattam is an internally-generated phenomenon and was not triggered by the influx of carbonic fluids from a deep-seated source. It is suggested that charnockitization was caused by the following mechanism: (1) near-isothermal decompression during uplift of the gneiss complex led to an increase of the pore fluid pressure (P sub fluid greater than P sub lith) which - in a regime of anisotropic stress - triggered or at least promoted the development of conjugate fractures; (2) the simultaneous release of pore fluids from bursting fluid inclusions and their escape into the developing fracture system resulted in a drop of fluid pressure; and (3) the internal generation and buffering of the fluids and their, probably, limited migration in an entirely granitic rock system explains the absence of any significant metasomatic mass transfer

Differential Transgene Silencing of Myeloid-Specific Promoters in the AAVS1 Safe Harbor Locus of Induced Pluripotent Stem Cell-Derived Myeloid Cells

Targeted integration into a genomic safe harbor, such as the AAVS1 locus on chromosome 19, promises predictable transgene expression and reduces the risk of insertional mutagenesis in the host genome. The application of gamma-retroviral LTR-driven vectors, which semi-randomly integrate into the genome, has previously caused severe adverse events in some clinical studies due to transactivation of neighboring proto-oncogenes. Consequently, the site-specific integration of a therapeutic transgene into a genomic safe harbor locus would allow stable genetic correction with a reduced risk of insertional mutagenesis. However, recent studies revealed that transgene silencing, especially in case of weaker cell type-specific promoters, can occur in the AAVS1 locus of human pluripotent stem cells (PSC) and can impede transgene expression during differentiation. In this study, we aimed to correct p47phox-deficiency, which is the second most common cause of chronic granulomatous disease, by insertion of a therapeutic p47phox transgene into the AAVS1 locus of human induced PSC (iPSC) using CRISPR-Cas9. We analyzed transgene expression and functional correction from three different myeloid-specific promoters (miR223, CatG/cFes and MRP8). Upon myeloid differentiation of corrected iPSC clones, we observed that the miR223 and CatG/cFes promoter achieved therapeutic-relevant levels of p47phox expression and NADPH oxidase activity, whereas the MRP8 promoter was less efficient. Analysis of the different promoters revealed high CpG methylation of the MRP8 promoter in differentiated cells, which correlated with the transgene expression data. In summary, we identified the miR223 and CatG/cFes promoters as cell type-specific promoters that allow stable transgene expression in the AAVS1 locus of iPSC-derived myeloid cells. Our findings further indicate that promoter silencing can occur in the AAVS1 safe harbor locus in differentiated hematopoietic cells and that a comparison of different promoters is necessary to achieve optimal transgene expression for therapeutic application of iPSC-derived cells

Splenic pooling and loss of VCAM-1 causes an engraftment defect in patients with myelofibrosis after allogeneic hematopoietic stem cell transplantation

Myelofibrosis is a myeloproliferative neoplasm that results in cytopenia, bone marrow fibrosis and extramedullary hematopoiesis. Allogeneic hematopoietic stem cell transplantation is the only curative treatment but is associated with a risk of delayed engraftment and graft failure. In this study, patients with myelofibrosis (n=31) and acute myeloid leukemia (n=31) were analyzed for time to engraftment, graft failure and engraftment-related factors. Early and late neutrophil engraftment and late thrombocyte engraftment were significantly delayed in patients with myelofibrosis as compared to acute myeloid leukemia, and graft failure only occurred in myelofibrosis (6%). Only spleen size had a significant influence on engraftment efficiency in myelofibrosis patients. To analyze the cause for the engraftment defect, clearance of hematopoietic stem cells from peripheral blood was measured and immunohistological staining of bone marrow sections was performed. Numbers of circulating CD34+ were significantly reduced at early time points in myelofibrosis patients, whereas CD34+CD38- and colony-forming cells showed no significant difference in clearance. Staining of bone marrow sections for homing proteins revealed a loss of VCAM-1 in myelofibrosis with a corresponding significant increase in the level of soluble VCAM-1 within the peripheral blood. In conclusion, our data suggest that reduced engraftment and graft failure in myelofibrosis patients is caused by an early pooling of CD34+ hematopoietic stem cells in the spleen and a bone marrow homing defect caused by the loss of VCAM-1. Improved engraftment in myelofibrosis might be achieved by approaches that reduce spleen size and cleavage of VCAM-1 in these patients prior to hematopoietic stem cell transplantation

Minkowski Tensors of Anisotropic Spatial Structure

This article describes the theoretical foundation of and explicit algorithms for a novel approach to morphology and anisotropy analysis of complex spatial structure using tensor-valued Minkowski functionals, the so-called Minkowski tensors. Minkowski tensors are generalisations of the well-known scalar Minkowski functionals and are explicitly sensitive to anisotropic aspects of morphology, relevant for example for elastic moduli or permeability of microstructured materials. Here we derive explicit linear-time algorithms to compute these tensorial measures for three-dimensional shapes. These apply to representations of any object that can be represented by a triangulation of its bounding surface; their application is illustrated for the polyhedral Voronoi cellular complexes of jammed sphere configurations, and for triangulations of a biopolymer fibre network obtained by confocal microscopy. The article further bridges the substantial notational and conceptual gap between the different but equivalent approaches to scalar or tensorial Minkowski functionals in mathematics and in physics, hence making the mathematical measure theoretic method more readily accessible for future application in the physical sciences

Universal hidden order in amorphous cellular geometries

Partitioning space into cells with certain extreme geometrical properties is a central problem in many fields of science and technology. Here we investigate the Quantizer problem, defined as the optimisation of the moment of inertia of Voronoi cells, i.e., similarly-sized ‘sphere-like’ polyhedra that tile space are preferred. We employ Lloyd’s centroidal Voronoi diagram algorithm to solve this problem and find that it converges to disordered states associated with deep local minima. These states are universal in the sense that their structure factors are characterised by a complete independence of a wide class of initial conditions they evolved from. They moreover exhibit an anomalous suppression of long-wavelength density fluctuations and quickly become effectively hyperuniform. Our findings warrant the search for novel amorphous hyperuniform phases and cellular materials with unique physical properties

Universal hidden order in amorphous cellular geometries

Partitioning space into cells with certain extreme geometrical properties is a central problem in many fields of science and technology. Here we investigate the Quantizer problem, defined as the optimisation of the moment of inertia of Voronoi cells, i.e., similarly-sized ‘sphere-like’ polyhedra that tile space are preferred. We employ Lloyd’s centroidal Voronoi diagram algorithm to solve this problem and find that it converges to disordered states associated with deep local minima. These states are universal in the sense that their structure factors are characterised by a complete independence of a wide class of initial conditions they evolved from. They moreover exhibit an anomalous suppression of long-wavelength density fluctuations and quickly become effectively hyperuniform. Our findings warrant the search for novel amorphous hyperuniform phases and cellular materials with unique physical properties

Spatial variability of soil N₂O and CO₂ fluxes in different topographic positions in a tropical montane forest in Kenya

Quantifying and understanding the small-scale variability of nitrous oxide (N₂O) and carbon dioxide (CO₂) emission are essential for reporting accurate ecosystem greenhouse gas budgets. The objective of this study was to evaluate the spatial pattern of soil CO₂ and N₂O emissions and their relation to topography in a tropical montane forest. We measured fluxes of N₂O and CO₂ from 810 sampling locations across valley bottom, midslope, and ridgetop positions under controlled laboratory conditions. We further calculated the minimum number of samples necessary to provide best estimates of soil N₂O and CO₂ fluxes at the plot level. Topography exhibited a major influence on N₂O emissions, with soils at midslope position emitting significantly less than at ridgetops and valley bottoms, but no consistent effect of topography on soil CO₂ emissions was found. The high spatial variation of N₂O and CO₂ fluxes was further increased by changes in vegetation and soil properties resulting from human disturbance associated with charcoal production. Soil N₂O and CO₂ fluxes showed no spatial pattern at the plot level, with “hot spots” strongly contributing to the total emissions (10% of the soil cores represented 73 and 50% of the total N₂O and CO₂ emissions, respectively). Thus, a large number of samples are needed to obtain robust estimates of N₂O and CO₂ fluxes. Our results highlight the complex biogeochemical cycling in tropical montane forests, and the need to carefully address it in research experiments to robustly estimate soil CO₂ and N₂O fluxes at the ecosystem scale

Inferring Properties of Ancient Cyanobacteria from Biogeochemical Activity and Genomes of Siderophilic Cyanobacteria

Interrelationships between life and the planetary system could have simultaneously left landmarks in genomes of microbes and physicochemical signatures in the lithosphere. Verifying the links between genomic features in living organisms and the mineralized signatures generated by these organisms will help to reveal traces of life on Earth and beyond. Among contemporary environments, iron-depositing hot springs (IDHS) may represent one of the most appropriate natural models [1] for insights into ancient life since organisms may have originated on Earth and probably Mars in association with hydrothermal activity [2,3]. IDHS also seem to be appropriate models for studying certain biogeochemical processes that could have taken place in the late Archean and,-or early Paleoproterozoic eras [4, 5]. It has been suggested that inorganic polyphosphate (PPi), in chains of tens to hundreds of phosphate residues linked by high-energy bonds, is environmentally ubiquitous and abundant [6]. Cyanobacteria (CB) react to increased heavy metal concentrations and UV by enhanced generation of PPi bodies (PPB) [7], which are believed to be signatures of life [8]. However, the role of PPi in oxygenic prokaryotes for the suppression of oxidative stress induced by high Fe is poorly studied. Here we present preliminary results of a new mechanism of Fe mineralization in oxygenic prokaryotes, the effect of Fe on the generation of PPi bodies in CB, as well as preliminary analysis of the diversity and phylogeny of proteins involved in the prevention of oxidative stress in phototrophs inhabiting IDHS

Speech Communication

Contains research objectives and summary of research on four research projects.National Institutes of Health (Grant 5 RO1 NS04332-14)National Institutes of Health (Grant 5 T32 NS07040-02)National Institutes of Health (Fellowship 1 F22 NS00796-01)National Institutes of Health (Grant 1 ROI NS13028-01)National Institutes of Health (Grant 5 T3Z NS07040-02)National Institutes of Health (Fellowship 1 F22 MH58258-02)U. S. Army- Maryland Procurement Office (Contract MDA904-76-C-0331