Comparative Analysis of Techniques to Purify Plasma Membrane Proteins

The aim of this project was to identify the best method for the enrichment of plasma membrane (PM) proteins for proteomics experiments. Following tryptic digestion and extended liquid chromatography-tandem mass spectrometry acquisitions, data were processed using MaxQuant and Gene Ontology (GO) terms used to determine protein subcellular localization. The following techniques were examined for the total number and percentage purity of PM proteins identified: (a) whole cell lysate (total number, 84–112; percentage purity, 9–13%); (b) crude membrane preparation (104–111; 17–20%); (c) biotinylation of surface proteins with N-hydroxysulfosuccinimydyl-S,S-biotin and streptavidin pulldown (78–115; 27–31%); (d) biotinylation of surface glycoproteins with biocytin hydrazide and streptavidin pulldown (41–54; 59–85%); or (e) biotinylation of surface glycoproteins with amino-oxy-biotin (which labels the sialylated fraction of PM glycoproteins) and streptavidin pulldown (120; 65%). A two- to threefold increase in the overall number of proteins identified was achieved by using stop and go extraction tip (StageTip)-based anion exchange (SAX) fractionation. Combining technique (e) with SAX fractionation increased the number of proteins identified to 281 (54%). Analysis of GO terms describing these proteins identified a large subset of proteins integral to the membrane with no subcellular assignment. These are likely to be of PM location and bring the total PM protein identifications to 364 (68%). This study suggests that selective biotinylation of the cell surface using amino-oxy-biotin in combination with SAX fractionation is a useful method for identification of sialylated PM proteins.     

Exploiting new terrain: an advantage to sociality in the slime mold Dictyostelium discoideum

Understanding the ecological benefits of social actions is centralto explaining the evolution of social behavior. The social amoebaDictyostelium discoideum has been well studied and is a modelfor social evolution and development, but surprisingly littleis known about its ecology. When starving, thousands of thenormally solitary amoebae aggregate to form a differentiatedmulticellular organism known as a slug. The slug migrates towardthe soil surface where it metamorphoses into a fruiting bodyof hardy spores held up by a dead stalk comprising about one-fifthof the cells. Multicellularity in D. discoideum is thought tohave evolved to lift the spores above the hazards of the soilwhere spores can be picked up for long-distance dispersal. Here,we show that multicellularity has another advantage: local dispersalto new food sources. We find that cells shed by D. discoideumslugs during migration consume and remove bacteria in the pathof the slug, although slugs themselves do not breakup. We alsoshow that slugs are adept at local dispersal by comparing migrationof slugs with migration of individual cells of the mutant, CAP2,which cannot aggregate and so rely only on cellular movement.In particular, the solitary cells of the aggregation mutantare unable to cross a soil barrier, easily crossed by slugs.We propose that the exploitation of local food patches is animportant selective benefit favoring multicellular cooperationin D. discoideum.     

Influence of 5-N-carboxamide modifications on the thermodynamic stability of oligonucleotides

We have recently shown that the incorporation of modified nucleotides such as 5-N-carboxamide-deoxyuridines into random nucleic acid libraries improves success rates in SELEX experiments and facilitates the identification of ligands with slow off-rates. Here we report the impact of these modifications on the thermodynamic stability of both duplexes and intramolecular ‘single-stranded’ structures. Within duplexes, large, hydrophobic naphthyl groups were destabilizing relative to the all natural DNA duplex, while the hydrophilic groups exhibited somewhat improved duplex stability. All of the significant changes in stability were driven by opposing contributions from the enthalpic and entropic terms. In contrast, both benzyl and naphthyl modifications stabilized intramolecular single-stranded structures relative to their natural DNA analogs, consistent with the notion that intramolecular folding allows formation of novel, stabilizing hydrophobic interactions. Imino proton NMR data provided evidence that elements of the folded structure form at temperatures well below the T_m, with a melting transition that is distinctly less cooperative when compared to duplex DNA. Although there are no data to suggest that the unmodified DNA sequences fold into structures similar to their modified analogs, this still represents clear evidence that these modifications impart thermodynamic stability to the folded structure not achievable with unmodified DNA.     

Dog models of naturally occurring cancer

Studies using dogs provide an ideal solution to the gap in animal models for natural disease and translational medicine. This is evidenced by approximately 400 inherited disorders being characterized in domesticated dogs, most of which are relevant to humans. There are several hundred isolated populations of dogs (breeds) and each has a vastly reduced genetic variation compared with humans; this simplifies disease mapping and pharmacogenomics.?Dogs age five- to eight-fold faster than do humans, share environments with their owners, are usually kept until old age and receive a high level of health care. Farseeing investigators recognized this potential and, over the past decade, have developed the necessary tools and infrastructure to utilize this powerful model of human disease, including the sequencing of the dog genome in 2005. Here, we review the nascent convergence of genetic and translational canine models of spontaneous disease, focusing on cancer.     

Molecular Factors Affecting the Accumulation of Recombinant Proteins in the <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> Chloroplast

In an effort to develop microalgae as a robust system for the production of valuable proteins, we analyzed some of the factors affecting recombinant protein expression in the chloroplast of the green alga Chlamydomonas reinhardtii. We monitored mRNA accumulation, protein synthesis, and protein turnover for three codon-optimized transgenes including GFP, bacterial luciferase, and a large single chain antibody. GFP and luciferase proteins were quite stable, while the antibody was less so. Measurements of protein synthesis, in contrast, clearly showed that translation of the three chimeric mRNAs was greatly reduced when compared to endogenous mRNAs under control of the same atpA promoter/UTR. Only in a few conditions this could be explained by limited mRNA availability since, in most cases, recombinant mRNAs accumulated quite well when compared to the atpA mRNA. In vitro toeprint and in vivo polysome analyses suggest that reduced ribosome association might contribute to limited translational efficiency. However, when recombinant polysome levels and protein synthesis are analyzed as a whole, it becomes clear that other steps, such as inefficient protein elongation, are likely to have a considerable impact. Taken together, our results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast.     

Epigenetic regulation of flowering time in polyploids

Polyploidy plays a significant role in the evolution of flowering plants. Understanding the effects of polyploidy on the epigenetic regulation of adaptive traits may resolve questions about the success of polyploids. One such trait, flowering time, has been the subject of several gene expression studies because it has one of the best characterized genetic networks and because polyploidy has a significant impact on generating variation in flowering time. Future research on the epigenetic consequences of polyploidy on flowering time should begin to examine natural variation in an ecological context, while continuing to make use of resynthesized polyploids.     

The microalga Chlamydomonas reinhardtii as a platform for the production of human protein therapeutics

Microalgae are a diverse group of eukaryotic photosynthetic microorganisms. While microalgae play a crucial role in global carbon fixation and oxygen evolution, these organisms have recently gained much attention for their potential role in biotechnological and industrial applications, such as the production of biofuels. We investigated the potential of the microalga Chlamydomonas reinhardtii to be a platform for the production of human therapeutic proteins. C. reinhardtii is a unicellular freshwater green alga that has served as a popular model alga for physiological, molecular, biochemical and genetic studies. As such, the molecular toolkit for this microorganism is highly developed, including well-established methods for genetic transformation and recombinant gene expression. We transformed the chloroplast genome of C. reinhardtii with seven unrelated genes encoding for current or potential human therapeutic proteins and found that four of these genes supported protein accumulation to levels that are sufficient for commercial production. Furthermore, the algal-produced proteins were bioactive. Thus, the microalga C. reinhardtii has the potential to be a robust platform for human therapeutic protein production.     

Nonsterile immunity to cryptosporidiosis in infants is associated with mucosal IgA against the sporozoite and protection from malnutrition

We conducted a longitudinal study of cryptosporidiosis from birth to three years of age in an urban slum of Dhaka Bangladesh. Fecal DNA was extracted from monthly surveillance samples and diarrheal stool samples collected from 392 infants from birth to three years. A pan-Cryptosporidium qPCR assay was used to identify sub-clinical and symptomatic cryptosporidiosis. Anthropometric measurements were collected quarterly to assess child nutritional status. 31% (121/392) of children experienced a single and 57% (222/392) multiple infections with Cryptosporidium. Repeat infections had a lower burden of parasites in the stool (Cq slope = -1.85; p<0.0001) and were more likely to be sub-clinical (Chi square test for trend; p = 0.01). Repeat infections were associated with the development of growth faltering (Pearson correlation = -0.18; p = 0.0004). High levels of fecal IgA antibodies against the Cryptosporidium Cp23 sporozoite protein at one year of life were associated with a delay in reinfection and amelioration of growth faltering through three years of life (HAZ IgA high responders -1.323 ± 0.932 versus HAZ -1.731 ± 0.984 p = 0.0001). We concluded that nonsterile immunity to cryptosporidiosis in young children was associated with high levels of mucosal IgA anti-Cp23 and protection from diarrhea and growth faltering.Trial Registration: {"type":"clinical-trial","attrs":{"text":"NCT02764918","term_id":"NCT02764918"}}NCT02764918.