High resolution footprinting of the hepatitis C virus polymerase NS5B in complex with RNA

The nucleic acid binding channel of the hepatitis C virus RNA polymerase remains to be defined. Here we employed complementary footprinting techniques and show that the enzyme binds to a newly synthesized duplex of approximately seven to eight base pairs. Comparative analysis of surface topologies of free enzyme versus the nucleoprotein complex revealed certain lysines and arginines that are protected from chemical modification upon RNA binding. The protection pattern helps to define the trajectory of the nucleic acid substrate. Lys(81), Lys(98), Lys(100), Lys(106), Arg(158), Arg(386), and Arg(394) probably interact with the bound RNA. The selective protection of amino acids of the arginine-rich region in helix T points to RNA-induced conformational rearrangements. Together, these findings suggest that RNA-protein interaction through the entire substrate binding channel can modulate intradomain contacts at the C terminus.     

A single amino acid mutation in zebrafish (Danio rerio) liver bile acid-binding protein can change the stoichiometry of ligand binding

In all of the liver bile acid-binding proteins (L-BABPs) studied so far, it has been found that the stoichiometry of binding is of two cholate molecules per internal binding site. In this paper, we describe the expression, purification, crystallization, and three-dimensional structure determination of zebrafish (Danio rerio) L-BABP to 1.5A resolution, which is currently the highest available for a protein of this family. Since we have found that in zebrafish, the stoichiometry of binding in the protein cavity is of only one cholate molecule per wild type L-BABP, we examined the role of two crucial amino acids present in the binding site. Using site-directed mutagenesis, we have prepared, crystallized, and determined the three-dimensional structure of co-crystals of two mutants. The mutant G55R has the same stoichiometry of binding as the wild type protein, whereas the C91T mutant changes the stoichiometry of binding from one to two ligand molecules in the cavity and therefore appears to be more similar to the other members of the L-BABP family. Based on the presence or absence of a single disulfide bridge, it can be postulated that fish should bind a single cholate molecule, whereas amphibians and higher vertebrates should bind two. Isothermal titration calorimetry has also revealed the presence in the wild type protein and the G55R mutant of an additional binding site, different from the first and probably located on the surface of the molecule.     

Mitosis persists in the absence of Cdk1 activity when proteolysis or protein phosphatase activity is suppressed

Cellular transition to anaphase and mitotic exit has been linked to the loss of cyclin-dependent kinase 1 (Cdk1) kinase activity as a result of anaphase-promoting complex/cyclosome (APC/C)–dependent specific degradation of its cyclin B1 subunit. Cdk1 inhibition by roscovitine is known to induce premature mitotic exit, whereas inhibition of the APC/C-dependent degradation of cyclin B1 by MG132 induces mitotic arrest. In this study, we find that combining both drugs causes prolonged mitotic arrest in the absence of Cdk1 activity. Different Cdk1 and proteasome inhibitors produce similar results, indicating that the effect is not drug specific. We verify mitotic status by the retention of mitosis-specific markers and Cdk1 phosphorylation substrates, although cells can undergo late mitotic furrowing while still in mitosis. Overall, we conclude that continuous Cdk1 activity is not essential to maintain the mitotic state and that phosphatase activity directed at Cdk1 substrates is largely quiescent during mitosis. Furthermore, the degradation of a protein other than cyclin B1 is essential to activate a phosphatase that, in turn, enables mitotic exit.     

Lessons from a Beetle and an Ant: Coping with Taxon-Dependent Differences in Microsatellite Development Success

Microsatellites are powerful markers often isolated de novo for species yet to be investigated. Enriched genomic libraries are usually used for isolation purposes. We critically evaluate the outcome of an enrichment-based protocol applied to two insect species (the ant Lasius austriacus and the beetle Pityogenes chalcographus) which yielded contrasting numbers of suitable loci. Our findings of differences in microsatellite isolation are consistent with the available data on differences in genomic characteristics across these taxa. In the beetle repeated isolation of identical motifs, difficulties in primer development, and multibanded products caused loss of most candidate clones. We identified critical steps during marker development. Reviewing Editor: Dr. John Oakeshott     

The current situation and potential effects of climate change on the microbial load of marine bivalves of the Greek coastlines: an integrative review

Global warming affects the aquatic ecosystems, accelerating pathogenic microorganisms' and toxic microalgae's growth and spread in marine habitats, and in bivalve molluscs. New parasite invasions are directly linked to oceanic warming. Consumption of pathogen-infected molluscs impacts human health at different rates, depending, inter alia, on the bacteria taxa. It is therefore necessary to monitor microbiological and chemical contamination of food. Many global cases of poisoning from bivalve consumption can be traced back to Mediterranean regions. This article aims to examine the marine bivalve's infestation rate within the scope of climate change, as well as to evaluate the risk posed by climate change to bivalve welfare and public health. Biological and climatic data literature review was performed from international scientific sources, Greek authorities and State organizations. Focusing on Greek aquaculture and bivalve fisheries, high-risk index pathogenic parasites and microalgae were observed during summer months, particularly in Thermaikos Gulf. Considering the climate models that predict further temperature increases, it seems that marine organisms will be subjected in the long term to higher temperatures. Due to the positive linkage between temperature and microbial load, the marine areas most affected by this phenomenon are characterized as ‘high risk’ for consumer health.     

Infection and Inflammation in Schizophrenia and Bipolar Disorder: A Genome Wide Study for Interactions with Genetic Variation

Inflammation and maternal or fetal infections have been suggested as risk factors for schizophrenia (SZ) and bipolar disorder (BP). It is likely that such environmental effects are contingent on genetic background. Here, in a genome-wide approach, we test the hypothesis that such exposures increase the risk for SZ and BP and that the increase is dependent on genetic variants. We use genome-wide genotype data, plasma IgG antibody measurements against Toxoplasma gondii, Herpes simplex virus type 1, Cytomegalovirus, Human Herpes Virus 6 and the food antigen gliadin as well as measurements of C-reactive protein (CRP), a peripheral marker of inflammation. The subjects are SZ cases, BP cases, parents of cases and screened controls. We look for higher levels of our immunity/infection variables and interactions between them and common genetic variation genome-wide. We find many of the antibody measurements higher in both disorders. While individual tests do not withstand correction for multiple comparisons, the number of nominally significant tests and the comparisons showing the expected direction are in significant excess (permutation p=0.019 and 0.004 respectively). We also find CRP levels highly elevated in SZ, BP and the mothers of BP cases, in agreement with existing literature, but possibly confounded by our inability to correct for smoking or body mass index. In our genome-wide interaction analysis no signal reached genome-wide significance, yet many plausible candidate genes emerged. In a hypothesis driven test, we found multiple interactions among SZ-associated SNPs in the HLA region on chromosome 6 and replicated an interaction between CMV infection and genotypes near the CTNNA3 gene reported by a recent GWAS. Our results support that inflammatory processes and infection may modify the risk for psychosis and suggest that the genotype at SZ-associated HLA loci modifies the effect of these variables on the risk to develop SZ.     

Implementation of a Pan-Genomic Approach to Investigate Holobiont-Infecting Microbe Interaction: A Case Report of a Leukemic Patient with Invasive Mucormycosis

Disease can be conceptualized as the result of interactions between infecting microbe and holobiont, the combination of a host and its microbial communities. It is likely that genomic variation in the host, infecting microbe, and commensal microbiota are key determinants of infectious disease clinical outcomes. However, until recently, simultaneous, multiomic investigation of infecting microbe and holobiont components has rarely been explored. Herein, we characterized the infecting microbe, host, micro- and mycobiomes leading up to infection onset in a leukemia patient that developed invasive mucormycosis. We discovered that the patient was infected with a strain of the recently described Mucor velutinosus species which we determined was hypervirulent in a Drosophila challenge model and has a predisposition for skin dissemination. After completing the infecting M. velutinosus genome and genomes from four other Mucor species, comparative pathogenomics was performed and assisted in identifying 66 M. velutinosus-specific putatively secreted proteins, including multiple novel secreted aspartyl proteinases which may contribute to the unique clinical presentation of skin dissemination. Whole exome sequencing of the patient revealed multiple non-synonymous polymorphisms in genes critical to control of fungal proliferation, such as TLR6 and PTX3. Moreover, the patient had a non-synonymous polymorphism in the NOD2 gene and a missense mutation in FUT2, which have been linked to microbial dysbiosis and microbiome diversity maintenance during physiologic stress, respectively. In concert with host genetic polymorphism data, the micro- and mycobiome analyses revealed that the infection developed amid a dysbiotic microbiome with low α-diversity, dominated by staphylococci. Additionally, longitudinal mycobiome data showed that M. velutinosus DNA was detectable in oral samples preceding disease onset. Our genome-level study of the host-infecting microbe-commensal triad extends the concept of personalized genomic medicine to the holobiont-infecting microbe interface thereby offering novel opportunities for using synergistic genetic methods to increase understanding of infectious diseases pathogenesis and clinical outcomes.