Two decades of describing the unseen majority of aquatic microbial diversity

Aquatic environments harbour large and diverse microbial populations that ensure their functioning and sustainability. In the current context of global change, characterizing microbial diversity has become crucial, and new tools have been developed to overcome the methodological challenges posed by working with microbes in nature. The advent of Sanger sequencing and now next-generation sequencing technologies has enabled the resolution of microbial communities to an unprecedented degree of precision. However, to correctly interpret microbial diversity and its patterns this revolution must also consider conceptual and methodological matters. This review presents advances, gaps and caveats of these recent approaches when considering microorganisms in aquatic ecosystems. We also discuss potentials and limitations of the available methodologies, from water sampling to sequence analysis, and suggest alternative ways to incorporate results in a conceptual and methodological framework. Together, these methods will allow us to gain an unprecedented understanding of microbial diversity in aquatic ecosystems.     

Low 2-dimensional CD4 T cell receptor affinity for myelin sets in motion delayed response kinetics

T cells recognizing self-peptides that mediate autoimmune disease and those that are responsible for efficacious immunity against pathogens may differ in affinity for antigen due to central and peripheral tolerance mechanisms. Here we utilize prototypical self-reactive (myelin) and viral-specific (LCMV) T cells from T cell receptor (TCR) transgenic mice (2D2 and SMARTA, respectively) to explore affinity differences. The T cells responsive to virus possessed >10,000 fold higher 2D affinity as compared to the self-reactive T cells. Despite their dramatically lower affinity for their cognate ligand, 2D2 T cells respond with complete, albeit delayed, activation (proliferation and cytokine production). SMARTA activation occurs rapidly, achieving peak phosphorylation of p38 (1 minute), Erk (30 minutes), and Jun (3 hours) as well as CD69 and CD25 upregulation (3 and 6 hours, respectively), with a corresponding early initiation of proliferation. 2D2 stimulation with MOG results in altered signaling--no phospho-Erk or phospho-p38 accumulation, significantly delayed activation kinetics of Jun (12 hours), and delayed but sustained SHP-1 activity--as well as delayed CD69 and CD25 expression (12-24 hours), and slow initiation of proliferation. This delay was not intrinsic to the 2D2 T cells, as a more potent antigen with >100-fold increased 2D affinity restored rapid response kinetics in line with those identified for the viral antigen. Taken together, these data demonstrate that time can offset low TCR affinity to attain full activation and suggest a mechanism by which low affinity T cells participate in autoimmune disease.     

Interbasin water transfer, riverine connectivity, and spatial controls on fish biodiversity

Background

Large-scale inter-basin water transfer (IBWT) projects are commonly proposed as solutions to water distribution and supply problems. These problems are likely to intensify under future population growth and climate change scenarios. Scarce data on the distribution of freshwater fishes frequently limits the ability to assess the potential implications of an IBWT project on freshwater fish communities. Because connectivity in habitat networks is expected to be critical to species'' biogeography, consideration of changes in the relative isolation of riverine networks may provide a strategy for controlling impacts of IBWTs on freshwater fish communities.

Methods/Principal Findings

Using empirical data on the current patterns of freshwater fish biodiversity for rivers of peninsular India, we show here how the spatial changes alone under an archetypal IBWT project will (1) reduce freshwater fish biodiversity system-wide, (2) alter patterns of local species richness, (3) expand distributions of widespread species throughout peninsular rivers, and (4) decrease community richness by increasing inter-basin similarity (a mechanism for the observed decrease in biodiversity). Given the complexity of the IBWT, many paths to partial or full completion of the project are possible. We evaluate two strategies for step-wise implementation of the 11 canals, based on economic or ecological considerations. We find that for each step in the project, the impacts on freshwater fish communities are sensitive to which canal is added to the network.

Conclusions/Significance

Importantly, ecological impacts can be reduced by associating the sequence in which canals are added to characteristics of the links, except for the case when all 11 canals are implemented simultaneously (at which point the sequence of canal addition is inconsequential). By identifying the fundamental relationship between the geometry of riverine networks and freshwater fish biodiversity, our results will aid in assessing impacts of IBWT projects and balancing ecosystem and societal demands for freshwater, even in cases where biodiversity data are limited.     

A method for quantifying pulmonary Legionella pneumophila infection in mouse lungs by flow cytometry

ABSTRACT: BACKGROUND: Pulmonary load of Legionella pneumophila in mice is normally determined by counting serial dilutions of bacterial colony forming units (CFU) on agar plates. This process is often tedious and time consuming. We describe a novel, rapid and versatile flow cytometric method that detects bacteria phagocytosed by neutrophils. FINDINGS: Mice were infected with L. pneumophila via intratracheal or intranasal administration. At various times after bacteria inoculation, mouse lungs were harvested and analysed concurrently for bacterial load by colony counting and flow cytometry analysis. The number of L. pneumophila-containing neutrophils correlated strongly with CFU obtained by bacteriological culture. CONCLUSIONS: This technique can be utilised to determine pulmonary bacterial load and may be used in conjunction with other flow cytometric based analyses of the resulting immune response.     

Banded cucumber beetle (Coleoptera: Chrysomelidae) resistance in romaine lettuce: understanding latex chemistry

Many plants subjected to herbivore damage exude latex, a rich source of biochemicals, which plays important roles in host plant resistance. Our previous studies showed that fresh latex from Valmaine, a resistant cultivar of romaine lettuce Lactuca sativa L., applied to artificial diet is highly deterrent to feeding by banded cucumber beetle, Diabrotica balteata LeConte, compared to the latex of a closely related susceptible cultivar Tall Guzmaine. The deterrent factor(s) could be extracted from Valmaine latex with water–methanol (20:80). In this study, further fractionation of the methanolic crude extract of Valmaine latex was performed using reverse-phase and ion-exchange solid-phase extraction to isolate the deterrent compounds. Retention of deterrent compounds on anion and cation exchange resin suggested the presence of highly polar compounds with both carboxylic and amine groups in Valmaine latex. Further bioassay-directed fractionation of cation exchange extract using LC/MS indicated the presence of at least 3 major and an unknown number of minor compounds in the bioactive fraction between 3 and 4 min. The m/z 210 out of the 3 major compounds showed strong amino acid characteristics (glutamine and/or glutamic acid) when subjected to further MS ⁿ degradation. Our studies suggest that nitrogenous ingredients of latex play a key role in Valmaine resistance to D. balteata, and latex may be a source of bioactive compounds with a potential use in pest management.     

Age-associated elevation in TLR5 leads to increased inflammatory responses in the elderly

Aging is accompanied by a progressive decline in immune function. Studies have shown age-related decreases in the expression and signaling efficiency of Toll-like receptors (TLRs) in monocytes and dendritic cells and dysregulation of macrophage TLR3. Using a multivariable mixed effect model, we report a highly significant increase in TLR5-induced production of IL-8 from monocytes of older individuals (P < 0.0001). Elevated IL-8 is accompanied by increased expression of TLR5, both protein and mRNA, and by increased levels of TLR5-mediated phosphorylation of MAPK p38 and ERK. We noted incomplete activation of NF-κB in response to TLR5 signaling in monocytes of elderly donors, as reflected by the absence of an associated increase in the production of TNF-α. Elevated TLR5 may provide a critical mechanism to enhance immune responsiveness in older individuals.     

Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells

To assess the genetic consequences of induced pluripotent stem cell (iPSC) reprogramming, we sequenced the genomes of ten murine iPSC clones derived from three independent reprogramming experiments, and compared them to their parental cell genomes. We detected hundreds of single nucleotide variants (SNVs) in every clone, with an average of 11 in coding regions. In two experiments, all SNVs were unique for each clone and did not cluster in pathways, but in the third, all four iPSC clones contained 157 shared genetic variants, which could also be detected in rare cells (<1 in 500) within the parental MEF pool. These data suggest that most of the genetic variation in iPSC clones is not caused by reprogramming per se, but is rather a consequence of cloning individual cells, which "captures" their mutational history. These findings have implications for the development and therapeutic use of cells that are reprogrammed by any method.     

Clear genetic distinctiveness between human- and pig-derived Trichuris based on analyses of mitochondrial datasets

The whipworm, Trichuris trichiura, causes trichuriasis in ～600 million people worldwide, mainly in developing countries. Whipworms also infect other animal hosts, including pigs (T. suis), dogs (T. vulpis) and non-human primates, and cause disease in these hosts, which is similar to trichuriasis of humans. Although Trichuris species are considered to be host specific, there has been considerable controversy, over the years, as to whether T. trichiura and T. suis are the same or distinct species. Here, we characterised the entire mitochondrial genomes of human-derived Trichuris and pig-derived Trichuris, compared them and then tested the hypothesis that the parasites from these two host species are genetically distinct in a phylogenetic analysis of the sequence data. Taken together, the findings support the proposal that T. trichiura and T. suis are separate species, consistent with previous data for nuclear ribosomal DNA. Using molecular analytical tools, employing genetic markers defined herein, future work should conduct large-scale studies to establish whether T. trichiura is found in pigs and T. suis in humans in endemic regions.