Impacts of 3 years of elevated atmospheric CO2 on rhizosphere carbon flow and microbial community dynamics

Carbon (C) uptake by terrestrial ecosystems represents an important option for partially mitigating anthropogenic CO₂ emissions. Short‐term atmospheric elevated CO₂ exposure has been shown to create major shifts in C flow routes and diversity of the active soil‐borne microbial community. Long‐term increases in CO₂ have been hypothesized to have subtle effects due to the potential adaptation of soil microorganism to the increased flow of organic C. Here, we studied the effects of prolonged elevated atmospheric CO₂ exposure on microbial C flow and microbial communities in the rhizosphere. Carex arenaria (a nonmycorrhizal plant species) and Festuca rubra (a mycorrhizal plant species) were grown at defined atmospheric conditions differing in CO₂ concentration (350 and 700 ppm) for 3 years. During this period, C flow was assessed repeatedly (after 6 months, 1, 2, and 3 years) by ¹³C pulse‐chase experiments, and label was tracked through the rhizosphere bacterial, general fungal, and arbuscular mycorrhizal fungal (AMF) communities. Fatty acid biomarker analyses and RNA‐stable isotope probing (RNA‐SIP), in combination with real‐time PCR and PCR‐DGGE, were used to examine microbial community dynamics and abundance. Throughout the experiment the influence of elevated CO₂ was highly plant dependent, with the mycorrhizal plant exerting a greater influence on both bacterial and fungal communities. Biomarker data confirmed that rhizodeposited C was first processed by AMF and subsequently transferred to bacterial and fungal communities in the rhizosphere soil. Over the course of 3 years, elevated CO₂ caused a continuous increase in the ¹³C enrichment retained in AMF and an increasing delay in the transfer of C to the bacterial community. These results show that, not only do elevated atmospheric CO₂ conditions induce changes in rhizosphere C flow and dynamics but also continue to develop over multiple seasons, thereby affecting terrestrial ecosystems C utilization processes.     

Lysosomal Membrane Proteins and Their Central Role in Physiology

The lysosomal membrane was thought for a long time to primarily act as a physical barrier separating the luminal acidic milieu from the cytoplasmic environment. Meanwhile, it has been realized that unique lysosomal membranes play essential roles in a number of cellular events ranging from phagocytosis, autophagy, cell death, virus infection to membrane repair. This review provides an overview about the most interesting emerging functions of lysosomal membrane proteins and how they contribute to health and disease. Their importance is exemplified by their role in acidification, transport of metabolites and ions across the membrane, intracellular transport of hydrolases and the regulation of membrane fusion events. Studies in patient cells, non‐mammalian model organisms and knockout mice contributed to our understanding of how the different lysosomal membrane proteins affect cellular homeostasis, developmental processes as well as tissue functions. Because these proteins are central for the biogenesis of this compartment they are also considered as attractive targets to modulate the lysosomal machinery in cases where impaired lysosomal degradation leads to cellular pathologies. We are only beginning to understand the complex composition and function of these proteins which are tightly linked to processes occurring throughout the endocytic and biosynthetic pathways.      

Fetal Fibroblasts and Keratinocytes with Immunosuppressive Properties for Allogeneic Cell-Based Wound Therapy

Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.     

Phylogenetic and population genetic analyses suggest a potential species boundary between Mountain (Gazella gazella) and Arabian Gazelles (G. arabica) in the Levant

Two cryptic lineages of ‘Mountain Gazelles’ have been reported based on molecular phylogenetic analyses using maternally inherited (mitochondrial) sequence markers, namely Gazella gazella in the Levant and G. arabica south of the Arava Valley into the Arabian Peninsula. Here, we provide a rigorous test for the existence of two distinct lineages based on bi-parentally inherited (nuclear microsatellite) markers. Our study confirms two genetically distinct clusters in the Levant and detected no gene-flow between them. Divergence time (inferred from a cytochrome b-based phylogeny) was approximately one MYA. Treating and breeding both lineages separately in future conservation and captive breeding programmes is highly recommended.     

Molecular cross-talk between sponge host and associated microbes

Marine organisms especially those that live sessile, as sponges, are well known to have specific relationships with a great variety of microorganisms including bacteria and fungi. As most simple metazoan phylum, the Porifera, which emerged first during the transition from the non-Metazoa to the Metazoa from the common ancestor, comprise wide arrays of recognition molecules, both for Gram-negative bacteria and for Gram-positive bacteria as well as for fungi. They react specifically with effector molecules to inhibit or kill the invading microorganisms. The elicitation and the subsequent effector reactions of the sponges towards these microbes are outlined. However, besides of the elimination of bacteria and fungi, some of those taxa are kept as symbionts of the sponges, allowing them, for example, to accumulate the essential element manganese or to synthesize carotinoids. The sponges produce low-molecular-weight bioactive compounds, secondary metabolites, to eliminate the microorganisms. In addition, they are armed with cationic antimicrobial peptides allowing them to defend against invasive microorganisms and, in parallel, to kill or repel also metazoan invaders. The broad range of chemically and functionally different compounds qualifies the Porifera as the most important animal phylum to be exploited as a source for the isolation of new potential drugs. First molecular biological strategies have been outlined to obtain those compounds in a sustainable way, by producing them recombinantly.     

A DNA Metabarcoding Study of a Primate Dietary Diversity and Plasticity across Its Entire Fragmented Range

In tropical regions, most primary ecosystems have been replaced by mosaic landscapes in which species must cope with a large shift in the distribution of their habitat and associated food resources. Primates are particularly vulnerable to habitat modifications. Most species persist in small fragments surrounded by complex human-mediated matrices whose structure and connectivity may strongly influence their dispersal and feeding behavior. Behavioral plasticity appears to be a crucial parameter governing the ability of organisms to exploit the resources offered by new matrix habitats and thus to persist in fragmented habitats. In this study, we were interested in the dietary plasticity of the golden-crowned sifaka (Propithecus tattersalli), an endangered species of lemur, found only in the Daraina region in north-eastern Madagascar. We used a DNA-based approach combining the barcoding concept and Illumina next-generation sequencing to (i) describe the species diet across its entire range and (ii) evaluate the influence of landscape heterogeneity on diet diversity and composition. Faeces from 96 individuals were sampled across the entire species range and their contents were analyzed using the trnL metabarcoding approach. In parallel, we built a large DNA reference database based on a checklist of the plant species of the Daraina region. Our results suggest that golden-crowned sifakas exhibit remarkable dietary diversity with at least 130 plant species belonging to 80 genera and 49 different families. We highlighted an influence of both habitat type and openness on diet composition suggesting a high flexibility of foraging strategies. Moreover, we observed the presence of numerous cultivated and naturalized plants in the faeces of groups living in forest edge areas. Overall, our findings support our initial expectation that P. tattersalli is able to cope with the current level of alteration of the landscape and confirm our previous results on the distribution and the dispersal ability of this species.     

Porcine E. coli: Virulence-Associated Genes,Resistance Genes and Adhesion and Probiotic Activity Tested by a New Screening Method

We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars.Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation.In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars.     

Hepatocyte Growth Factor Activator Inhibitor-1 Is Induced by Bone Morphogenetic Proteins and Regulates Proliferation and Cell Fate of Neural Progenitor Cells

Background

Neural progenitor cells (NPCs) in the developing neuroepithelium are regulated by intrinsic and extrinsic factors. There is evidence that NPCs form a self-supporting niche for cell maintenance and proliferation. However, molecular interactions and cell-cell contacts and the microenvironment within the neuroepithelium are largely unknown. We hypothesized that cellular proteases especially those associated with the cell surface of NPCs play a role in regulation of progenitor cells in the brain.

Methodology/Principal Findings

In this work, we show that NPCs, isolated from striatal anlage of developing rat brain, express hepatocyte growth factor activator inhibitor-1 and -2 (HAI-1 and HAI-2) that are cell surface-linked serine protease inhibitors. In addition, radial glia cells derived from mouse embryonic stem cells also express HAI-1 and HAI-2. To study the functional significance of HAI-1 and HAI-2 in progenitor cells, we modulated their levels using expression plasmids or silencing RNA (siRNA) transfected into the NPCs. Data showed that overexpression of HAI-1 or HAI-2 decreased cell proliferation of cultured NPCs, whilst their siRNAs had opposite effects. HAI-1 also influenced NPC differentiation by increasing the number of glial fibrillary acidic protein (GFAP) expressing cells in the culture. Expression of HAI-1 in vivo decreased cell proliferation in developing neuroepithelium in E15 old animals and promoted astrocyte cell differentiation in neonatal animals. Studying the regulation of HAI-1, we observed that Bone morphogenetic protein-2 (BMP-2) and BMP-4 increased HAI-1 levels in the NPCs. Experiments using HAI-1-siRNA showed that these BMPs act on the NPCs partly in a HAI-1-dependent manner.

Conclusions

This study shows that the cell-surface serine protease inhibitors, HAI-1 and HAI-2 influence proliferation and cell fate of NPCs and their expression levels are linked to BMP signaling. Modulation of the levels and actions of HAI-1 in NPCs may be of a potential value in stem cell therapies in various brain diseases.     

MultiPSQ: A Software Solution for the Analysis of Diagnostic n-Plexed Pyrosequencing Reactions

Background

Pyrosequencing can be applied for Single-Nucleotide-Polymorphism (SNP)-based pathogen typing or for providing sequence information of short DNA stretches. However, for some pathogens molecular typing cannot be performed relying on a single SNP or short sequence stretch, necessitating the consideration of several genomic regions. A promising rapid approach is the simultaneous application of multiple sequencing primers, called multiplex pyrosequencing. These primers generate a fingerprint-pyrogram which is constituted by the sum of all individual pyrograms originating from each primer used.

Methods

To improve pyrosequencing-based pathogen typing, we have developed the software tool MultiPSQ that expedites the analysis and evaluation of multiplex-pyrograms. As a proof of concept, a multiplex pyrosequencing assay for the typing of orthopoxviruses was developed to analyse clinical samples diagnosed in the German Consultant Laboratory for Poxviruses.

Results

The software tool MultiPSQ enabled the analysis of multiplex-pyrograms originating from various pyrosequencing primers. Thus several target regions can be used for pathogen typing based on pyrosequencing. As shown with a proof of concept assay, SNPs present in different orthopoxvirus strains could be identified correctly with two primers by MultiPSQ.

Conclusions

Software currently available is restricted to a fixed number of SNPs and sequencing primers, severely limiting the usefulness of this technique. In contrast, our novel software MultiPSQ allows analysis of data from multiplex pyrosequencing assays that contain any number of sequencing primers covering any number of polymorphisms.