Insight into the roles of selection in speciation from genomic patterns of divergence and introgression in secondary contact in venomous rattlesnakes

Investigating secondary contact of historically isolated lineages can provide insight into how selection and drift influence genomic divergence and admixture. Here, we studied the genomic landscape of divergence and introgression following secondary contact between lineages of the Western Diamondback Rattlesnake (Crotalus atrox) to determine whether genomic regions under selection in allopatry also contribute to reproductive isolation during introgression. We used thousands of nuclear loci to study genomic differentiation between two lineages that have experienced recent secondary contact following isolation, and incorporated sampling from a zone of secondary contact to identify loci that are resistant to gene flow in hybrids. Comparisons of patterns of divergence and introgression revealed a positive relationship between allelic differentiation and resistance to introgression across the genome, and greater‐than‐expected overlap between genes linked to lineage‐specific divergence and loci that resist introgression. Genes linked to putatively selected markers were related to prominent aspects of rattlesnake biology that differ between populations of Western Diamondback rattlesnakes (i.e., venom and reproductive phenotypes). We also found evidence for selection against introgression of genes that may contribute to cytonuclear incompatibility, consistent with previously observed biased patterns of nuclear and mitochondrial alleles suggestive of partial reproductive isolation due to cytonuclear incompatibilities. Our results provide a genome‐scale perspective on the relationships between divergence and introgression in secondary contact that is relevant for understanding the roles of selection in maintaining partial isolation of lineages, causing admixing lineages to not completely homogenize.     

A scalable, GFP-based pipeline for membrane protein overexpression screening and purification

We describe a generic, GFP-based pipeline for membrane protein overexpression and purification in Escherichia coli. We exemplify the use of the pipeline by the identification and characterization of E. coli YedZ, a new, membrane-integral flavocytochrome. The approach is scalable and suitable for high-throughput applications. The GFP-based pipeline will facilitate the characterization of the E. coli membrane proteome and serves as an important reference for the characterization of other membrane proteomes.     

Isolation of the mustard Napin protein Allergen Sin a 1 and characterisation of its antifungal activity

Proteins and peptides belonging to the plant immune system can possess natural antibacterial, antifungal and antiviral properties. Due to their broad range of activity and stability, they represent promising novel alternatives to commonly used antifungal agents to fight the emergence of resistant strains. An isolation protocol was optimised to target proteins found in plants’ defence system, and it was applied to white mustard (Brassica hirta) seeds. Firstly, a ～14 kDa protein with activity against S. cerevisiae was extracted and purified; secondly, the protein was identified as the mustard Napin protein named Allergen Sin a 1. Napin is the name given to seed storage (2S) albumin proteins belonging to the Brassicaceae family. While several Napins have been described for their antimicrobial potential, Sin a 1 has been mainly studied for its allergenic properties. The antimicrobial activity of Sin a 1 is described and characterised for the first time in this study; it possesses antifungal and antiyeast in vitro activity, but no antibacterial activity was recorded. The yeasts Zygosaccharomyces bailii Sa 1403 and Saccharomyces cerevisiae DSM 70449 along with the filamentous fungi Fusarium culmorum FST 4.05 were amongst the most senstitive strains to Sin a 1 (MICs range 3–6 μM). The antimicrobial mechanism of membrane permeabilisation was detected, and in general, the antifungal activity of Sin a 1 seemed to be expressed in a dose-dependent manner. Data collected confirmed Sin a 1 to be a stable and compact protein, as it displayed resistance to α-chymotrypsin digestion, heat denaturation and insensitivity to pH variations and the presence of salts. In addition, the protein did not show cytotoxicity towards mammalian cells.     

Iron overload down-regulates the expression of the HIV-1 Rev cofactor eIF5A in infected T lymphocytes

Background

Changes in iron metabolism frequently accompany HIV-1 infection. However, while many clinical and in vitro studies report iron overload exacerbates the development of infection, many others have found no correlation. Therefore, the multi-faceted role of iron in HIV-1 infection remains enigmatic.

Methods

RT-qPCR targeting the LTR region, gag, Tat and Rev were performed to measure the levels of viral RNAs in response to iron overload. Spike-in SILAC proteomics comparing i) iron-treated, ii) HIV-1-infected and iii) HIV-1-infected/iron treated T lymphocytes was performed to define modifications in the host cell proteome. Data from quantitative proteomics were integrated with the HIV-1 Human Interaction Database for assessing any viral cofactors modulated by iron overload in infected T lymphocytes.

Results

Here, we demonstrate that the iron overload down-regulates HIV-1 gene expression by decreasing the levels of viral RNAs. In addition, we found that iron overload modulates the expression of many viral cofactors. Among them, the downregulation of the REV cofactor eIF5A may correlate with the iron-induced inhibition of HIV-1 gene expression. Therefore, we demonstrated that eiF5A downregulation by shRNA resulted in a significant decrease of Nef levels, thus hampering HIV-1 replication.

Conclusions

Our study indicates that HIV-1 cofactors influenced by iron metabolism represent potential targets for antiretroviral therapy and suggests eIF5A as a selective target for drug development.

     

Karyotypic determinants of chromosome instability in aneuploid budding yeast

Recent studies in cancer cells and budding yeast demonstrated that aneuploidy, the state of having abnormal chromosome numbers, correlates with elevated chromosome instability (CIN), i.e. the propensity of gaining and losing chromosomes at a high frequency. Here we have investigated ploidy- and chromosome-specific determinants underlying aneuploidy-induced CIN by observing karyotype dynamics in fully isogenic aneuploid yeast strains with ploidies between 1N and 2N obtained through a random meiotic process. The aneuploid strains exhibited various levels of whole-chromosome instability (i.e. chromosome gains and losses). CIN correlates with cellular ploidy in an unexpected way: cells with a chromosomal content close to the haploid state are significantly more stable than cells displaying an apparent ploidy between 1.5 and 2N. We propose that the capacity for accurate chromosome segregation by the mitotic system does not scale continuously with an increasing number of chromosomes, but may occur via discrete steps each time a full set of chromosomes is added to the genome. On top of such general ploidy-related effect, CIN is also associated with the presence of specific aneuploid chromosomes as well as dosage imbalance between specific chromosome pairs. Our findings potentially help reconcile the divide between gene-centric versus genome-centric theories in cancer evolution.     

A two alternative forced choice method for assessing vibrotactile discrimination thresholds in the lower limb

The development of an easy to implement, quantitative measure to examine vibration perception would be useful for future application in clinical settings. Vibration sense in the lower limb of younger and older adults was examined using the method of constant stimuli (MCS) and the two-alternative forced choice paradigm. The focus of this experiment was to determine an appropriate stimulation site on the lower limb (tendon versus bone) to assess vibration threshold and to determine if the left and right legs have varying thresholds. Discrimination thresholds obtained at two stimulation sites in the left and right lower limbs showed differences in vibration threshold across the two ages groups, but not across sides of the body nor between stimulation sites within each limb. Overall, the MCS can be implemented simply, reliably, and with minimal time. It can also easily be implemented with low-cost technology. Therefore, it could be a good candidate method to assess the presence of specific deep sensitivity deficits in clinical practice, particularly in populations likely to show the onset of sensory deficits.     

Modeling Signal Propagation Mechanisms and Ligand-Based Conformational Dynamics of the Hsp90 Molecular Chaperone Full-Length Dimer

Hsp90 is a molecular chaperone essential for protein folding and activation in normal homeostasis and stress response. ATP binding and hydrolysis facilitate Hsp90 conformational changes required for client activation. Hsp90 plays an important role in disease states, particularly in cancer, where chaperoning of the mutated and overexpressed oncoproteins is important for function. Recent studies have illuminated mechanisms related to the chaperone function. However, an atomic resolution view of Hsp90 conformational dynamics, determined by the presence of different binding partners, is critical to define communication pathways between remote residues in different domains intimately affecting the chaperone cycle. Here, we present a computational analysis of signal propagation and long-range communication pathways in Hsp90. We carried out molecular dynamics simulations of the full-length Hsp90 dimer, combined with essential dynamics, correlation analysis, and a signal propagation model. All-atom MD simulations with timescales of 70 ns have been performed for complexes with the natural substrates ATP and ADP and for the unliganded dimer. We elucidate the mechanisms of signal propagation and determine “hot spots” involved in interdomain communication pathways from the nucleotide-binding site to the C-terminal domain interface. A comprehensive computational analysis of the Hsp90 communication pathways and dynamics at atomic resolution has revealed the role of the nucleotide in effecting conformational changes, elucidating the mechanisms of signal propagation. Functionally important residues and secondary structure elements emerge as effective mediators of communication between the nucleotide-binding site and the C-terminal interface. Furthermore, we show that specific interdomain signal propagation pathways may be activated as a function of the ligand. Our results support a “conformational selection model” of the Hsp90 mechanism, whereby the protein may exist in a dynamic equilibrium between different conformational states available on the energy landscape and binding of a specific partner can bias the equilibrium toward functionally relevant complexes.     

Reversal of the Myosin Power Stroke Induced by Fast Stretching of Intact Skeletal Muscle Fibers

Force generation and movement in skeletal muscle result from a cyclical interaction of overlapping myosin and actin filaments that permits the free energy of ATP hydrolysis to be converted into mechanical work. The rapid force recovery that occurs after a step release imposed on a muscle is thought to result from a synchronized tilting of myosin lever arms toward a position of lower free energy (the power stroke). We investigated the power stroke mechanism in intact muscle fibers of Rana esculenta using a fast stretch to detach forcibly cross-bridges. Stretches were applied either with or without a conditioning step release. Cross-bridge rupture tension was not significantly influenced by the release, whereas sarcomere elongation at the rupture point increased immediately after the release and returned to the prerelease condition within 15-20 ms, following a slower time course compared to the recovery of tension. These observations suggest that the rupture force of a bridge is unaltered by a conditioning release, but rupture must first be preceded by a power stroke reversal, which restores the prepower stroke state. The sarcomere extension at the rupture point indicates both the extent of this power stroke reversal and the time course of strained bridge replenishment.