Interglacial microrefugia and diversification of a cactus species complex: phylogeography and palaeodistributional reconstructions for Pilosocereus aurisetus and allies

The role of Pleistocene climate changes in promoting evolutionary diversification in global biota is well documented, but the great majority of data regarding this subject come from North America and Europe, which were greatly affected by glaciation. The effects of Pleistocene changes on cold‐ and/or dry‐adapted species in tropical areas where glaciers were not present remain sparsely investigated. Many such species are restricted to small areas surrounded by unfavourable habitats, which may represent potential interglacial microrefugia. Here, we analysed the phylogeographic structure and diversification history of seven cactus species in the Pilosocereus aurisetus complex that are restricted to rocky areas with high diversity and endemism within the Neotropical savannas of eastern South America. We combined palaeodistributional estimates with standard phylogeographic approaches based on two chloroplast DNA regions (trnT‐trnL and trnS‐trnG), exon 1 of the nuclear gene PhyC and 10 nuclear microsatellite loci. Our analyses revealed a phylogeographic history marked by multiple levels of distributional fragmentation, isolation leading to allopatric differentiation and secondary contact among divergent lineages within the complex. Diversification and demographic events appear to have been affected by the Quaternary climatic cycles as a result of isolation in multiple patches of xerophytic vegetation. These small patches presently harbouring P. aurisetus populations seem to operate as microrefugia, both at present and during Pleistocene interglacial periods; the role of such microrefugia should be explored and analysed in greater detail.     

Immunization of Chickens with Newcastle Disease Virus Expressing H5 Hemagglutinin Protects against Highly Pathogenic H5N1 Avian Influenza Viruses

Background

Highly-pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are the two most important poultry viruses in the world. Natural low-virulence NDV strains have been used as vaccines over the past 70 years with proven track records. We have previously developed a reverse genetics system to produce low-virulent NDV vaccine strain LaSota from cloned cDNA. This system allows us to use NDV as a vaccine vector for other avian pathogens.

Methodology/Principal Finding

Here, we constructed two recombinant NDVs (rNDVs) each of which expresses the hemagglutinin (HA) gene of HPAIV H5N1strain A/Vietnam/1203/2004 from an added gene. In one, rNDV (rNDV-HA), the open reading frame (ORF) of HA gene was expressed without modification. In the second, rNDV (rNDV-HAF), the ORF was modified so that the transmembrane and cytoplasmic domains of the encoded HA gene were replaced with those of the NDV F protein. The insertion of either version of the HA ORF did not increase the virulence of the rNDV vector. The HA protein was found to be incorporated into the envelopes of both rNDV-HA and rNDV-HAF. However, there was an enhanced incorporation of the HA protein in rNDV-HAF. Chickens immunized with a single dose of either rNDV-HA or rNDV-HAF induced a high titer of HPAIV H5-specific antibodies and were completely protected against challenge with NDV as well as lethal challenges of both homologous and heterologous HPAIV H5N1.

Conclusion and Significance

Our results suggest that these chimeric viruses have potential as safe and effective bivalent vaccines against NDV and. HPAIV. These vaccines will be convenient and affordable, which will be highly beneficial to the poultry industry. Furthermore, immunization with these vaccines will permit serological differentiation of vaccinated and avian influenza field virus infected animals.     

The CT20 peptide causes detachment and death of metastatic breast cancer cells by promoting mitochondrial aggregation and cytoskeletal disruption

Metastasis accounts for most deaths from breast cancer, driving the need for new therapeutics that can impede disease progression. Rationally designed peptides that take advantage of cancer-specific differences in cellular physiology are an emerging technology that offer promise as a treatment for metastatic breast cancer. We developed CT20p, a hydrophobic peptide based on the C terminus of Bax that exhibits similarities with antimicrobial peptides, and previously reported that CT20p has unique cytotoxic actions independent of full-length Bax. In this study, we identified the intracellular actions of CT20p which precede cancer cell-specific detachment and death. Previously, we found that CT20p migrated in the heavy membrane fractions of cancer cell lysates. Here, using MDA-MB-231 breast cancer cells, we demonstrated that CT20p localizes to the mitochondria, leading to fusion-like aggregation and mitochondrial membrane hyperpolarization. As a result, the distribution and movement of mitochondria in CT20p-treated MDA-MB-231 cells was markedly impaired, particularly in cell protrusions. In contrast, CT20p did not associate with the mitochondria of normal breast epithelial MCF-10A cells, causing little change in the mitochondrial membrane potential, morphology or localization. In MDA-MB-231 cells, CT20p triggered cell detachment that was preceded by decreased levels of α5β1 integrins and reduced F-actin polymerization. Using folate-targeted nanoparticles to encapsulate and deliver CT20p to murine tumors, we achieved significant tumor regression within days of peptide treatment. These results suggest that CT20p has application in the treatment of metastatic disease as a cancer-specific therapeutic peptide that perturbs mitochondrial morphology and movement ultimately culminating in disruption of the actin cytoskeleton, cell detachment, and loss of cell viability.     

Characterizing symbiont inheritance during host–microbiota evolution: Application to the great apes gut microbiota

Microbiota play a central role in the functioning of multicellular life, yet understanding their inheritance during host evolutionary history remains an important challenge. Symbiotic microorganisms are either acquired from the environment during the life of the host (i.e. environmental acquisition), transmitted across generations with a faithful association with their hosts (i.e. strict vertical transmission), or transmitted with occasional host switches (i.e. vertical transmission with horizontal switches). These different modes of inheritance affect microbes’ diversification, which at the two extremes can be independent from that of their associated host or follow host diversification. The few existing quantitative tools for investigating the inheritance of symbiotic organisms rely on cophylogenetic approaches, which require knowledge of both host and symbiont phylogenies, and are therefore often not well adapted to DNA metabarcoding microbial data. Here, we develop a model‐based framework for identifying vertically transmitted microbial taxa. We consider a model for the evolution of microbial sequences on a fixed host phylogeny that includes vertical transmission and horizontal host switches. This model allows estimating the number of host switches and testing for strict vertical transmission and independent evolution. We test our approach using simulations. Finally, we illustrate our framework on gut microbiota high‐throughput sequencing data of the family Hominidae and identify several microbial taxonomic units, including fibrolytic bacteria involved in carbohydrate digestion, that tend to be vertically transmitted.     

Starch metabolism in the leaf sheaths and culm of rice

The levels of starch and dextrin, free sugars, soluble protein, and enzymes involved in starch metabolism—α-amylase, β-amylase, phosphorylase, Q-enzyme, R-enzyme, and ADP-glucose starch synthetases—were assayed in the leaf sheaths and culm of the rice plant (Oryza sativa L., variety IR8) during growth.     

Role of arachidonic acid metabolism in Stat5 activation induced by oleic acid in MDA-MB-231 breast cancer cells

Epidemiological studies and animal models suggest an association between high levels of dietary fat intake and an increased risk of breast cancer. In breast cancer cells, the free fatty acid oleic acid (OLA) induces proliferation, migration, invasion and an increase of MMP-9 secretion. However, the role of OLA on Stat5 activation and the participation of COX-2 and LOXs activity in Stat5 activation induced by OLA remain to be investigated. We demonstrate here that stimulation of MDA-MB-231 breast cancer cells with 100 μM OLA induces Stat5 phosphorylation at Tyr-694 and an increase of Stat5–DNA complex formation. The Stat5 DNA-binding activity requires COX-2, LOXs, metalloproteinases and Src activities. In addition, OLA induces cell migration through a Stat5-dependent pathway. In summary, our findings establish that OLA induces cell migration through a Stat5-dependent pathway and that Stat5 activation requires AA metabolites in MDA-MB-231 breast cancer cells.     

A Spatially Explicit Model of Synchronization in Fiddler Crab Waving Displays

Fiddler crabs (Uca spp., Decapoda: Ocypodidae) are commonly found forming large aggregations in intertidal zones, where they perform rhythmic waving displays with their greatly enlarged claws. While performing these displays, fiddler crabs often synchronize their behavior with neighboring males, forming the only known synchronized visual courtship displays involving reflected light and moving body parts. Despite being one of the most conspicuous aspects of fiddler crab behavior, little is known about the mechanisms underlying synchronization of male displays. In this study we develop a spatially explicit model of fiddler crab waving displays using coupled logistic map equations. We explored two alternative models in which males either direct their attention at random angles or preferentially toward neighbors. Our results indicate that synchronization is possible over a fairly large region of parameter space. Moreover, our model was capable of generating local synchronization neighborhoods, as commonly observed in fiddler crabs under natural conditions.     

In vitro expression analysis of R68G and R68S mutations in phenylalanine hydroxylase gene

Phenylketonuria (PKU), an autosomal recessive disorder caused be a deficiency of hepatic phenylalanine hydroxylase (PAH), is clinically very heterogeneous. At the molecular level, more than 400 mutations in the PAH gene are known to date, which in different genotype combinations could account for biochemical and clinical variability of symptoms. In vitro expression studies on R68G and R68S mutations causing mild phenylketonuria are presented.     

Resequencing studies of nonmodel organisms using closely related reference genomes: optimal experimental designs and bioinformatics approaches for population genomics

Decreasing costs of next‐generation sequencing (NGS) experiments have made a wide range of genomic questions open for study with nonmodel organisms. However, experimental designs and analysis of NGS data from less well‐known species are challenging because of the lack of genomic resources. In this work, we investigate the performance of alternative experimental designs and bioinformatics approaches in estimating variability and neutrality tests based on the site‐frequency‐spectrum (SFS) from individual resequencing data. We pay particular attention to challenges faced in the study of nonmodel organisms, in particular the absence of a species‐specific reference genome, although phylogenetically close genomes are assumed to be available. We compare the performance of three alternative bioinformatics approaches – genotype calling, genotype–haplotype calling and direct estimation without calling genotypes. We find that relying on genotype calls provides biased estimates of population genetic statistics at low to moderate read depth (2–8×). Genotype–haplotype calling returns more accurate estimates irrespective of the divergence to the reference genome, but requires moderate depth (8–20×). Direct estimation without calling genotypes returns the most accurate estimates of variability and of most SFS tests investigated, including at low read depth (2–4×). Studies without species‐specific reference genome should thus aim for low read depth and avoid genotype calling whenever individual genotypes are not essential. Otherwise, aiming for moderate to high depth at the expense of number of individuals, and using genotype–haplotype calling, is recommended.     

Resolving structure and mechanical properties at the nanoscale of viruses with frequency modulation atomic force microscopy

Structural Biology (SB) techniques are particularly successful in solving virus structures. Taking advantage of the symmetries, a heavy averaging on the data of a large number of specimens, results in an accurate determination of the structure of the sample. However, these techniques do not provide true single molecule information of viruses in physiological conditions. To answer many fundamental questions about the quickly expanding physical virology it is important to develop techniques with the capability to reach nanometer scale resolution on both structure and physical properties of individual molecules in physiological conditions. Atomic force microscopy (AFM) fulfills these requirements providing images of individual virus particles under physiological conditions, along with the characterization of a variety of properties including local adhesion and elasticity. Using conventional AFM modes is easy to obtain molecular resolved images on flat samples, such as the purple membrane, or large viruses as the Giant Mimivirus. On the contrary, small virus particles (25-50 nm) cannot be easily imaged. In this work we present Frequency Modulation atomic force microscopy (FM-AFM) working in physiological conditions as an accurate and powerful technique to study virus particles. Our interpretation of the so called "dissipation channel" in terms of mechanical properties allows us to provide maps where the local stiffness of the virus particles are resolved with nanometer resolution. FM-AFM can be considered as a non invasive technique since, as we demonstrate in our experiments, we are able to sense forces down to 20 pN. The methodology reported here is of general interest since it can be applied to a large number of biological samples. In particular, the importance of mechanical interactions is a hot topic in different aspects of biotechnology ranging from protein folding to stem cells differentiation where conventional AFM modes are already being used.