Comparative hydrogen-deuterium exchange for a mesophilic vs thermophilic dihydrofolate reductase at 25 °C: identification of a single active site region with enhanced flexibility in the mesophilic protein

The technique of hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has been applied to a mesophilic (E. coli) dihydrofolate reductase under conditions that allow direct comparison to a thermophilic (B. stearothermophilus) ortholog, Ec-DHFR and Bs-DHFR, respectively. The analysis of hydrogen-deuterium exchange patterns within proteolytically derived peptides allows spatial resolution, while requiring a series of controls to compare orthologous proteins with only ca. 40% sequence identity. These controls include the determination of primary structure effects on intrinsic rate constants for HDX as well as the use of existing 3-dimensional structures to evaluate the distance of each backbone amide hydrogen to the protein surface. Only a single peptide from the Ec-DHFR is found to be substantially more flexible than the Bs-DHFR at 25 °C in a region located within the protein interior at the intersection of the cofactor and substrate-binding sites. The surrounding regions of the enzyme are either unchanged or more flexible in the thermophilic DHFR from B. stearothermophilus. The region with increased flexibility in Ec-DHFR corresponds to one of two regions previously proposed to control the enthalpic barrier for hydride transfer in Bs-DHFR [Oyeyemi et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 10074].     

Regulation of Na(+)-coupled glucose carrier SGLT1 by human papillomavirus 18 E6 protein

Tumor cells utilize preferably glucose for energy production. They accomplish cellular glucose uptake in part through Na⁺-coupled glucose transport mediated by SGLT1 (SLC5A1). This study explored the possibility that the human papillomavirus 18 E6 protein HPV18 E6 (E6) participates in the stimulation of SGLT1 activity. E6 is one of the two major oncoproteins of high-risk human papillomaviruses, which are the causative agent for cervical carcinoma. According to Western blotting, SGLT1 is expressed in the HPV18-positive cervical carcinoma cell line HeLa. To explore whether E6 affects SGLT1 activity, SGLT1 was expressed in Xenopus oocytes with and without E6 and electrogenic glucose transport determined by dual electrode voltage clamp. In SGLT1-expressing oocytes, but not in oocytes injected with water or expressing E6 alone, glucose triggered a current (I_g). I_g was significantly increased by coexpression of E6 but not by coexpression of E2. According to chemiluminescence and confocal microscopy, coexpression of E6 significantly increased the SGLT1 protein abundance in the cell membrane. The decay of I_g following inhibition of carrier insertion by Brefeldine A (5 μM) was not significantly affected E6 coexpression. Accrodingly, E6 was not effective by increasing carrier protein stability in the membrane. In conclusion, HPV18 E6 oncoprotein participates in the upregulation of SGLT1.     

Genetic structure among Charadrius plovers on the African mainland and islands of Madagascar and St Helena

Colonization of islands by long-distance dispersers has great impact on genetic diversification among populations and may spearhead speciation events. We investigated intra- and interspecific divergence in Charadrius plovers with populations on mainland Africa, Madagascar and St Helena. We analysed microsatellite loci and sequence data from four nuclear and two mitochondrial gene regions. Charadrius plovers are shorebirds with high dispersal and mobility. Our results confirmed genetic differentiation between Madagascar and mainland populations of three plover species (White-fronted Plover Charadrius marginatus, Kittlitz's Plover Charadrius pecuarius and, based on sequence data only, Three-banded Plover Charadrius tricollaris) but highlight substantial variation in levels of intraspecific divergence among the three species. Namely, the Kittlitz's Plover, a dispersive habitat generalist with a polygamous mating system, exhibited lower island–mainland differentiation (0.05% COI sequence divergence) compared with the two monogamous species, the White-fronted Plover (0.6% COI divergence) and Three-banded Plover (1.6% COI divergence). In addition, past colonization of the islands of St Helena and Madagascar by ancestors of today's Kittlitz's Plover has led to the evolution of two endemic island species, the Madagascar Plover Charadrius thoracicus and the more closely-related St Helena Plover Charadrius sanctaehelenae. We discuss the factors driving species differences in island–mainland divergence and highlight the importance of conserving genetically unique island populations and island habitats to safeguard future evolutionary potential.     

Developmental trap or demographic bonanza? Opposing consequences of earlier phenology in a changing climate for a multivoltine butterfly

A rapidly changing climate has the potential to interfere with the timing of environmental cues that ectothermic organisms rely on to initiate and regulate life history events. Short‐lived ectotherms that exhibit plasticity in their life history could increase the number of generations per year under warming climate. If many individuals successfully complete an additional generation, the population experiences an additional opportunity to grow, and a warming climate could lead to a demographic bonanza. However, these plastic responses could become maladaptive in temperate regions, where a warmer climate could trigger a developmental pathway that cannot be completed within the growing season, referred to as a developmental trap. Here we incorporated detailed demography into commonly used photothermal models to evaluate these demographic consequences of phenological shifts due to a warming climate on the formerly widespread, multivoltine butterfly (Pieris oleracea). Using species‐specific temperature‐ and photoperiod‐sensitive vital rates, we estimated the number of generations per year and population growth rate over the set of climate conditions experienced during the past 38 years. We predicted that populations in the southern portion of its range have added a fourth generation in recent years, resulting in higher annual population growth rates (demographic bonanzas). We predicted that populations in the Northeast United States have experienced developmental traps, where increases in the thermal window initially caused mortality of the final generation and reduced growth rates. These populations may recover if more growing degree days are added to the year. Our framework for incorporating detailed demography into commonly used photothermal models demonstrates the importance of using both demography and phenology to predict consequences of phenological shifts.     

Comparative Genomics Reveal That Host-Innate Immune Responses Influence the Clinical Prevalence of Legionella pneumophila Serogroups

Legionella pneumophila is the primary etiologic agent of legionellosis, a potentially fatal respiratory illness. Amongst the sixteen described L. pneumophila serogroups, a majority of the clinical infections diagnosed using standard methods are serogroup 1 (Sg1). This high clinical prevalence of Sg1 is hypothesized to be linked to environmental specific advantages and/or to increased virulence of strains belonging to Sg1. The genetic determinants for this prevalence remain unknown primarily due to the limited genomic information available for non-Sg1 clinical strains. Through a systematic attempt to culture Legionella from patient respiratory samples, we have previously reported that 34% of all culture confirmed legionellosis cases in Ontario (n = 351) are caused by non-Sg1 Legionella. Phylogenetic analysis combining multiple-locus variable number tandem repeat analysis and sequence based typing profiles of all non-Sg1 identified that L. pneumophila clinical strains (n = 73) belonging to the two most prevalent molecular types were Sg6. We conducted whole genome sequencing of two strains representative of these sequence types and one distant neighbour. Comparative genomics of the three L. pneumophila Sg6 genomes reported here with published L. pneumophila serogroup 1 genomes identified genetic differences in the O-antigen biosynthetic cluster. Comparative optical mapping analysis between Sg6 and Sg1 further corroborated this finding. We confirmed an altered O-antigen profile of Sg6, and tested its possible effects on growth and replication in in vitro biological models and experimental murine infections. Our data indicates that while clinical Sg1 might not be better suited than Sg6 in colonizing environmental niches, increased bloodstream dissemination through resistance to the alternative pathway of complement mediated killing in the human host may explain its higher prevalence.     

Bone Marrow Cell Recruitment to the Brain in the Absence of Irradiation or Parabiosis Bias

The engraftment of bone marrow-derived cells has been described not only during diseases of the central nervous system (CNS) but also under healthy conditions. However, previous studies pointing to an ample bone marrow cell engraftment used irradiation-induced bone marrow chimeras that evoked severe alterations of the CNS micromilieu including disturbances of the blood brain barrier (BBB), damage of endothelial cells and local induction of proinflammatory cytokines. On the other hand, parabiosis experiments using temporarily joined circulatory systems generally yielded low levels of myeloid cell chimerism thereby potentially underestimating bone marrow cell turnover with the CNS. To avoid these drawbacks we established a protocol using the alkylating agent busulfan prior to allogenic bone marrow transplantation from CX₃CR1^GFP/+ donors. This regimen resulted in a stable and high peripheral myeloid chimerism, significantly reduced cytokine induction and preserved BBB integrity. Importantly, bone marrow cell recruitment to the CNS was strongly diminished under these conditions and only weakly enhanced during local neurodegeneration induced by facial nerve axotomy. These results underscore the requirement of local CNS conditioning for efficient recruitment of bone marrow cells, establish busulfan as an alternative treatment for studying bone marrow chimeras and suggest a critical re-evaluation of earlier chimeric studies involving irradiation or parabiosis regimens.     

Toll-Like Receptor 4 Engagement Drives Differentiation of Human and Murine Dendritic Cells from a Pro- into an Anti-Inflammatory Mode

The dendritic cell (DC) coordinates innate and adaptive immunity to fight infections and cancer. Our observations reveal that DCs exposed to the microbial danger signal lipopolysaccharide (LPS) in the presence of interferon-γ (IFN-γ) acquire a continuously changing activation/maturation phenotype. The DCs’ initial mode of action is pro-inflammatory via up-regulation among others of the signaling molecule interleukin (IL) 12, which polarizes IFN-γ secreting type 1 helper T-cells (Th1). Within 24 hours the same DC switches from the pro- into an anti-inflammatory phenotype. This is mediated by autocrine IL-10 release and secretion of soluble IL-2 receptor alpha (sIL-2RA) molecules. T-cells, when contacted with DCs during their anti-inflammatory phase loose their proliferative capacity and develop regulatory T-cell (Treg) -like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels. Studying the kinetics of IL-12 and IL-10 expression from LPS/IFN-γ activated myeloid DCs on a single cell level confirmed these observations. When T-cells are separated from DCs within 24 hours, they are spared from the anti-inflammatory DC activity. We conclude that, in addition to differentiation of DCs into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response. A better understanding of time-kinetic DC features is required for optimally exploiting the therapeutic capacity of DCs in cancer immune therapy.     

Coverage Bias and Sensitivity of Variant Calling for Four Whole-genome Sequencing Technologies

The emergence of high-throughput, next-generation sequencing technologies has dramatically altered the way we assess genomes in population genetics and in cancer genomics. Currently, there are four commonly used whole-genome sequencing platforms on the market: Illumina’s HiSeq2000, Life Technologies’ SOLiD 4 and its completely redesigned 5500xl SOLiD, and Complete Genomics’ technology. A number of earlier studies have compared a subset of those sequencing platforms or compared those platforms with Sanger sequencing, which is prohibitively expensive for whole genome studies. Here we present a detailed comparison of the performance of all currently available whole genome sequencing platforms, especially regarding their ability to call SNVs and to evenly cover the genome and specific genomic regions. Unlike earlier studies, we base our comparison on four different samples, allowing us to assess the between-sample variation of the platforms. We find a pronounced GC bias in GC-rich regions for Life Technologies’ platforms, with Complete Genomics performing best here, while we see the least bias in GC-poor regions for HiSeq2000 and 5500xl. HiSeq2000 gives the most uniform coverage and displays the least sample-to-sample variation. In contrast, Complete Genomics exhibits by far the smallest fraction of bases not covered, while the SOLiD platforms reveal remarkable shortcomings, especially in covering CpG islands. When comparing the performance of the four platforms for calling SNPs, HiSeq2000 and Complete Genomics achieve the highest sensitivity, while the SOLiD platforms show the lowest false positive rate. Finally, we find that integrating sequencing data from different platforms offers the potential to combine the strengths of different technologies. In summary, our results detail the strengths and weaknesses of all four whole-genome sequencing platforms. It indicates application areas that call for a specific sequencing platform and disallow other platforms. This helps to identify the proper sequencing platform for whole genome studies with different application scopes.