Pleistocene origin and population history of a neoendemic alpine butterfly

Alpine environments underwent dramatic transformation during glacial–interglacial cycles, with the consequence that geographical, ecological and demographic changes of alpine populations provided the opportunity for formation of neoendemic species. Several biogeographical models have been proposed to account for the unique history of alpine populations, with different expectations of genetic divergence and speciation. The expanding alpine archipelago model proposes that alpine populations expand spatially and demographically during glacial events, dispersing between mountain ranges. Under this model, alpine populations are unlikely to diverge in isolation due to substantial interpopulation gene flow. In contrast, the alpine archipelago refuge model proposes that gene flow during glacial phases is limited and populations expand demographically during interglacial phases, increasing genetic isolation and the likelihood of speciation. We assess these models by reconstructing the evolutionary history of Colias behrii, a morphologically and ecologically distinct alpine butterfly restricted to the California Sierra Nevada. C. behrii exhibits very low genetic diversity at mitochondrial and nuclear loci, limited population structure and evidence of population expansion. C. behrii and Rocky Mountain C. meadii share identical mitochondrial haplotypes, while in contrast, nuclear data indicate common ancestry between C. behrii and Cascades Range Colias pelidne. The conflict in gene genealogies may be a result of recent expansion in North American Colias, but an isolation with migration analysis indicates that genetic patterns in C. behrii might result from differential introgression following hybridization. Based on the timing of population expansion and gene flow between mountain ranges, the expanding alpine archipelago model is supported in C. behrii.     

Enediol mimics as inhibitors of the D-arabinose 5-phosphate isomerase (KdsD) from Francisella tularensis

We explored the d-arabinose 5-phosphate isomerase (KdsD, E.C. 5.3.1.13) from Francisella tularensis, a highly infectious Gram-negative pathogen that has raised concern as a potential bioweapon, as a target for the development of novel chemotherapeutics. F. tularensis KdsD was expressed in Escherichia coli from a synthetic gene, purified, and characterized. A group of hydroxamates designed to be mimics of the putative enediol intermediate in the enzyme’s catalytic mechanism were prepared and tested as inhibitors of F. tularensis KdsD. The best inhibitor, which has an IC₅₀ of 7 μM, is the most potent KdsD inhibitor reported to date.     

Climate signals in the ring widths and stable carbon, hydrogen and oxygen isotopic composition of Larix decidua growing at the forest limit in the southeastern European Alps

The southeastern border of the European Alps is not well resourced with high-resolution climate proxies and experiences a distinct climatic regime from the northern and western Alpine zones. Here, we present new high-resolution climatic proxies (AD 1907–2006) from ring widths and stable carbon (δ¹³C), non-exchangeable hydrogen (δ²H) and oxygen (δ¹⁸O) isotope ratios of cellulose extracted from Larix decidua tree rings, growing at the forest limit in the southeastern European Alps (Slovenia). δ¹³C, δ²H and δ¹⁸O are strongly (p < 0.001) and positively correlated with each other. June temperature has the strongest control on tree ring width (TRW), while later summer conditions (July–August) influence the stable isotope composition. All four proxies are strongly correlated (r > 0.4; p < 0.001) with summer temperature and also sunshine hours, while precipitation has less impact. A combination of TRW and δ¹³C provides the greatest potential for reconstructing past temperatures (June–August) with significant (p < 0.001) correlations with gridded temperatures extending across a very large part of southern and western Europe west of the Carpathian Mountains. The water isotopes (oxygen and hydrogen) record conditions in the Adriatic and Mediterranean, which are the source area for the air masses that bring precipitation to this region giving strong correlations with temperatures in southern Italy and the western part of the Balkan Peninsula. Combining proxies with different spatial and temporal signals allows the strength and spatial footprint of climate signals to be enhanced. These findings open new perspectives for climate reconstruction in the southeastern European Alps and Western Balkans.     

A Novel Respiratory Syncytial Virus (RSV) F Subunit Vaccine Adjuvanted with GLA-SE Elicits Robust Protective TH1-Type Humoral and Cellular Immunity In Rodent Models

Background

Illness associated with Respiratory Syncytial Virus (RSV) remains an unmet medical need in both full-term infants and older adults. The fusion glycoprotein (F) of RSV, which plays a key role in RSV infection and is a target of neutralizing antibodies, is an attractive vaccine target for inducing RSV-specific immunity.

Methodology and Principal Findings

BALB/c mice and cotton rats, two well-characterized rodent models of RSV infection, were used to evaluate the immunogenicity of intramuscularly administered RSV vaccine candidates consisting of purified soluble F (sF) protein formulated with TLR4 agonist glucopyranosyl lipid A (GLA), stable emulsion (SE), GLA-SE, or alum adjuvants. Protection from RSV challenge, serum RSV neutralizing responses, and anti-F IgG responses were induced by all of the tested adjuvanted RSV sF vaccine formulations. However, only RSV sF + GLA-SE induced robust F-specific T_H1-biased humoral and cellular responses. In mice, these F-specific cellular responses include both CD4 and CD8 T cells, with F-specific polyfunctional CD8 T cells that traffic to the mouse lung following RSV challenge. This RSV sF + GLA-SE vaccine formulation can also induce robust RSV neutralizing titers and prime IFNγ-producing T cell responses in Sprague Dawley rats.

Conclusions/Significance

These studies indicate that a protein subunit vaccine consisting of RSV sF + GLA-SE can induce robust neutralizing antibody and T cell responses to RSV, enhancing viral clearance via a T_H1 immune-mediated mechanism. This vaccine may benefit older populations at risk for RSV disease.     

An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum

Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn²⁺ bound (PDB_ID:4PCA), and SAM- Mn²⁺ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations.     

Most of the Dominant Members of Amphibian Skin Bacterial Communities Can Be Readily Cultured

Currently, it is estimated that only 0.001% to 15% of bacteria in any given system can be cultured by use of commonly used techniques and media, yet culturing is critically important for investigations of bacterial function. Despite this situation, few studies have attempted to link culture-dependent and culture-independent data for a single system to better understand which members of the microbial community are readily cultured. In amphibians, some cutaneous bacterial symbionts can inhibit establishment and growth of the fungal pathogen Batrachochytrium dendrobatidis, and thus there is great interest in using these symbionts as probiotics for the conservation of amphibians threatened by B. dendrobatidis. The present study examined the portion of the culture-independent bacterial community (based on Illumina amplicon sequencing of the 16S rRNA gene) that was cultured with R2A low-nutrient agar and whether the cultured bacteria represented rare or dominant members of the community in the following four amphibian species: bullfrogs (Lithobates catesbeianus), eastern newts (Notophthalmus viridescens), spring peepers (Pseudacris crucifer), and American toads (Anaxyrus americanus). To determine which percentage of the community was cultured, we clustered Illumina sequences at 97% similarity, using the culture sequences as a reference database. For each amphibian species, we cultured, on average, 0.59% to 1.12% of each individual''s bacterial community. However, the average percentage of bacteria that were culturable for each amphibian species was higher, with averages ranging from 2.81% to 7.47%. Furthermore, most of the dominant operational taxonomic units (OTUs), families, and phyla were represented in our cultures. These results open up new research avenues for understanding the functional roles of these dominant bacteria in host health.     

Promiscuous Binding of Karyopherinβ1 Modulates FG Nucleoporin Barrier Function and Expedites NTF2 Transport Kinetics

The transport channel of nuclear pore complexes (NPCs) contains a high density of intrinsically disordered proteins that are rich in phenylalanine-glycine (FG)-repeat motifs (FG Nups). The FG Nups interact promiscuously with various nuclear transport receptors (NTRs), such as karyopherins (Kaps), that mediate the trafficking of nucleocytoplasmic cargoes while also generating a selectively permeable barrier against other macromolecules. Although the binding of NTRs to FG Nups increases molecular crowding in the NPC transport channel, it is unclear how this impacts FG Nup barrier function or the movement of other molecules, such as the Ran importer NTF2. Here, we use surface plasmon resonance to evaluate FG Nup conformation, binding equilibria, and interaction kinetics associated with the multivalent binding of NTF2 and karyopherinβ1 (Kapβ1) to Nsp1p molecular brushes. NTF2 and Kapβ1 show different long- and short-lived binding characteristics that emerge from varying degrees of molecular retention and FG repeat binding avidity within the Nsp1p brush. Physiological concentrations of NTF2 produce a collapse of Nsp1p brushes, whereas Kapβ1 binding generates brush extension. However, the presence of prebound Kapβ1 inhibits Nsp1p brush collapse during NTF2 binding, which is dominated by weak, short-lived interactions that derive from steric hindrance and diminished avidity with Nsp1p. This suggests that binding promiscuity confers kinetic advantages to NTF2 by expediting its facilitated diffusion and reinforces the proposal that Kapβ1 contributes to the integral barrier function of the NPC.     

Compromised CDK1 activity sensitizes BRCA-proficient cancers to PARP inhibition

Cells that are deficient in homologous recombination, such as those that lack functional breast cancer-associated 1 (BRCA1) or BRCA2, are hypersensitive to inhibition of poly(ADP-ribose) polymerase (PARP). However, BRCA-deficient tumors represent only a small fraction of adult cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. Cyclin-dependent kinase 1 (Cdk1) phosphorylates BRCA1, and this is essential for efficient formation of BRCA1 foci. Here we show that depletion or inhibition of Cdk1 compromises the ability of cells to repair DNA by homologous recombination. Combined inhibition of Cdk1 and PARP in BRCA-wild-type cancer cells resulted in reduced colony formation, delayed growth of human tumor xenografts and tumor regression with prolonged survival in a mouse model of lung adenocarcinoma. Inhibition of Cdk1 did not sensitize nontransformed cells or tissues to inhibition of PARP. Because reduced Cdk1 activity impaired BRCA1 function and consequently, repair by homologous recombination, inhibition of Cdk1 represents a plausible strategy for expanding the utility of PARP inhibitors to BRCA-proficient cancers.     

Recent emergence and worldwide spread of the red tomato spider mite, <Emphasis Type="Italic">Tetranychus evansi</Emphasis>: genetic variation and multiple cryptic invasions

Plant biosecurity is increasingly challenged by emerging crop pests. The spider mite Tetranychus evansi has recently emerged as a new threat to solanaceous crops in Africa and the Mediterranean basin, with invasions characterized by a high reproductive output and an ability to withstand a wide range of temperatures. Mitochondrial (868 bp of COI) and nuclear (1,137 bp of ITS) loci were analyzed in T. evansi samples spanning the current geographical distribution to study the earliest stages of the invasive process. The two sets of markers separate the samples into two main clades that are only present together in South America and Southern Europe. The highest COI diversity was found in South America, consistent with the hypothesis of a South American origin of T. evansi. Among the invaded areas, the Mediterranean region displayed a high level of genetic diversity similar to that present in South America, that is likely the result of multiple colonization events. The invasions of Africa and Asia by T. evansi are characterized by a low genetic variation associated with distinct introductions. Genetic data demonstrate two different patterns of invasions: (1) populations in the Mediterranean basin that are a result of multiple cryptic introductions and (2) emerging invasions of Africa and Asia, each likely the result of propagules from one or limited sources. The recent invasions of T. evansi illustrate not only the importance of human activities in the spread of agricultural pests, but also the limits of international quarantine procedures, particularly for cryptic invasions.     

Structural chemistry of dihalogenopalladium(II) and platinum(II) complexes of heteroleptic N,S- and N,Se-donor ligands based on the 2-organochalcogenomethylpyridine motif

The structural chemistry of dihalogenopalladium(II) and platinum(II) complexes of 2-organochalcogenomethylpyridine ligands is described. Complexes with a methyl group in the 6-position of the pyridyl ring, 6-MepyCH₂ER, form dimeric complexes [trans-PdX₂(μ-6-MepyCH₂SePh-N,Se)]₂ (X = Br (1), I (2)) or mononuclear complexes trans-PdI₂(6-MepyCH₂SR-N)₂ (R = Me (5), Ph (6)). Absence of a 6-methyl substituent results in the bidentate configuration observed for PdI₂(pyCH₂SePh-N,Se) (3) and PdI₂(4-MepyCH₂SMe-N,S) (4). Related platinum(II) complexes are mononuclear including PtCl₂(6-MepyCH₂SPh-N,S) (8) as an analogue of trimeric [trans-PdCl₂(μ-6-MepyCH₂SPh-N,S)]₃. Differences between palladium and platinum appear to result from a combination of steric and electronic factors.