Separating Instability from Aggregation Propensity in γS-Crystallin Variants

Molecular dynamics (MD) simulations, circular dichroism (CD), and dynamic light scattering (DLS) measurements were used to investigate the aggregation propensity of the eye-lens protein γS-crystallin. The wild-type protein was investigated along with the cataract-related G18V variant and the symmetry-related G106V variant. The MD simulations suggest that local sequence differences result in dramatic differences in dynamics and hydration between these two apparently similar point mutations. This finding is supported by the experimental measurements, which show that although both variants appear to be mostly folded at room temperature, both display increased aggregation propensity. Although the disease-related G18V variant is not the most strongly destabilized, it aggregates more readily than either the wild-type or the G106V variant. These results indicate that γS-crystallin provides an excellent model system for investigating the role of dynamics and hydration in aggregation by locally unfolded proteins.     

Controlling tick-borne diseases through domestic animal management: a theoretical approach

Vector-borne diseases are of global importance to human and animal health. Empirical trials of effective methods to control vectors and their pathogens can be difficult for practical, financial and ethical reasons. Here, therefore, we use a mathematical model to predict the effectiveness of a vector-borne disease control method. As a case study, we use the tick-louping ill virus system, where sheep are treated with acaricide in an attempt to control ticks and disease in red grouse , an economically important game bird. We ran the model under different scenarios of sheep flock sizes, alternative host (deer) densities, acaricide efficacies and tick burdens. The model predicted that, with very low deer densities, using sheep as tick mops can reduce the tick population and virus prevalence. However, treatment is ineffective above a certain threshold deer density, dependent on the comparative tick burden on sheep and deer. The model also predicted that high efficacy levels of acaricide must be maintained for effective tick control. This study suggests that benignly managing one host species to protect another host species from a vector and pathogen can be effective under certain conditions. It also highlights the importance of understanding the ecological complexity of a system, in order to target control methods only under certain circumstances for maximum effectiveness.     

Expanding the Symbiodinium (Dinophyceae,Suessiales) Toolkit Through Protoplast Technology

Dinoflagellates within the genus Symbiodinium are photosymbionts of many tropical reef invertebrates, including corals, making them central to the health of coral reefs. Symbiodinium have therefore gained significant research attention, though studies have been constrained by technical limitations. In particular, the generation of viable cells with their cell walls removed (termed protoplasts) has enabled a wide range of experimental techniques for bacteria, fungi, plants, and algae such as ultrastructure studies, virus infection studies, patch clamping, genetic transformation, and protoplast fusion. However, previous studies have struggled to remove the cell walls from armored dinoflagellates, potentially due to the internal placement of their cell walls. Here, we produce the first Symbiodinium protoplasts from three genetically and physiologically distinct strains via incubation with cellulase and osmotic agents. Digestion of the cell walls was verified by a lack of Calcofluor White fluorescence signal and by cell swelling in hypotonic culture medium. Fused protoplasts were also observed, motivating future investigation into intra‐ and inter‐specific somatic hybridization of Symbiodinium. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Generation of Symbiodinium protoplasts opens exciting, new avenues for researching these crucial symbiotic dinoflagellates, including genetic modification.     

The choline transporter resurfaces: new roles for synaptic vesicles?

Presynaptic choline uptake is vital to sustained neuronal acetylcholine (ACh) release; however, only with the recent cloning of choline transporters (CHTs) (i.e., SLC5A7), has a picture emerged of the regulatory pathways supporting CHT modulation. Studies arising from the development of CHT-specific antibodies reveal a large, intracellular reserve of CHT proteins, localized to ACh-containing, synaptic vesicles. The intersection of mechanisms supporting vesicular ACh release and choline uptake demonstrates an elegant mechanism for linking regulation of CHT membrane density to rates of ACh release. Furthermore, these studies point to control of the CHT endocytic process as an important target for novel therapeutics that could offset functional deficits in disorders bearing diminished cholinergic tone, including myasthenias and dementias.     

Synthesis of β-Peptide Standards for Use in Model Prebiotic Reactions

A one-pot method was developed for the preparation of a series of β-alanine standards of moderate size (2 to ≥12 residues) for studies concerning the prebiotic origins of peptides. The one-pot synthesis involved two sequential reactions: (1) dry-down self-condensation of β-alanine methyl ester, yielding β-alanine peptide methyl ester oligomers, and (2) subsequent hydrolysis of β-alanine peptide methyl ester oligomers, producing a series of β-alanine peptide standards. These standards were then spiked into a model prebiotic product mixture to confirm by HPLC the formation of β-alanine peptides under plausible reaction conditions. The simplicity of this approach suggests it can be used to prepare a variety of β-peptide standards for investigating differences between α- and β-peptides in the context of prebiotic chemistry.     

High affinity inhibitors of the dopamine transporter (DAT): novel biotinylated ligands for conjugation to quantum dots

Compounds capable of inhibiting the dopamine transporter protein (DAT) that can be conjugated to cadmium selenide/zinc sulfide/core shell nanocrystals may be used to image the location and distribution of the DAT in neuronal cell membranes. This letter describes the synthesis of biotinylated analogs of the DAT antagonists GBR 12909 and GBR 12935 that can be attached to streptavidin coated cadmium selenide/zinc sulfide/core shell nanocrystals.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Recent amplification of the kangaroo endogenous retrovirus, KERV, limited to the centromere

Mammalian retrotransposons, transposable elements that are processed through an RNA intermediate, are categorized as short interspersed elements (SINEs), long interspersed elements (LINEs), and long terminal repeat (LTR) retroelements, which include endogenous retroviruses. The ability of transposable elements to autonomously amplify led to their initial characterization as selfish or junk DNA; however, it is now known that they may acquire specific cellular functions in a genome and are implicated in host defense mechanisms as well as in genome evolution. Interactions between classes of transposable elements may exert a markedly different and potentially more significant effect on a genome than interactions between members of a single class of transposable elements. We examined the genomic structure and evolution of the kangaroo endogenous retrovirus (KERV) in the marsupial genus Macropus. The complete proviral structure of the kangaroo endogenous retrovirus, phylogenetic relationship among relative retroviruses, and expression of this virus in both Macropus rufogriseus and M. eugenii are presented for the first time. In addition, we show the relative copy number and distribution of the kangaroo endogenous retrovirus in the Macropus genus. Our data indicate that amplification of the kangaroo endogenous retrovirus occurred in a lineage-specific fashion, is restricted to the centromeres, and is not correlated with LINE depletion. Finally, analysis of KERV long terminal repeat sequences using massively parallel sequencing indicates that the recent amplification in M. rufogriseus is likely due to duplications and concerted evolution rather than a high number of independent insertion events.