The scale of population structure in Arabidopsis thaliana

The population structure of an organism reflects its evolutionary history and influences its evolutionary trajectory. It constrains the combination of genetic diversity and reveals patterns of past gene flow. Understanding it is a prerequisite for detecting genomic regions under selection, predicting the effect of population disturbances, or modeling gene flow. This paper examines the detailed global population structure of Arabidopsis thaliana. Using a set of 5,707 plants collected from around the globe and genotyped at 149 SNPs, we show that while A. thaliana as a species self-fertilizes 97% of the time, there is considerable variation among local groups. This level of outcrossing greatly limits observed heterozygosity but is sufficient to generate considerable local haplotypic diversity. We also find that in its native Eurasian range A. thaliana exhibits continuous isolation by distance at every geographic scale without natural breaks corresponding to classical notions of populations. By contrast, in North America, where it exists as an exotic species, A. thaliana exhibits little or no population structure at a continental scale but local isolation by distance that extends hundreds of km. This suggests a pattern for the development of isolation by distance that can establish itself shortly after an organism fills a new habitat range. It also raises questions about the general applicability of many standard population genetics models. Any model based on discrete clusters of interchangeable individuals will be an uneasy fit to organisms like A. thaliana which exhibit continuous isolation by distance on many scales

Patterns of Polymorphism and Demographic History in Natural Populations of Arabidopsis lyrata

Many of the processes affecting genetic diversity act on local populations. However, studies of plant nucleotide diversity have largely ignored local sampling, making it difficult to infer the demographic history of populations and to assess the importance of local adaptation. Arabidopsis lyrata, a self-incompatible, perennial species with a circumpolar distribution, is an excellent model system in which to study the roles of demographic history and local adaptation in patterning genetic variation.We studied nucleotide diversity in six natural populations of Arabidopsis lyrata, using 77 loci sampled from 140 chromosomes. The six populations were highly differentiated, with a median FST of 0.52, and structure analysis revealed no evidence of admixed individuals. Average within-population diversity varied among populations, with the highest diversity found in a German population; this population harbors 3-fold higher levels of silent diversity than worldwide samples of A. thaliana. All A. lyrata populations also yielded positive values of Tajima's D. We estimated a demographic model for these populations, finding evidence of population divergence over the past 19,000 to 47,000 years involving non-equilibrium demographic events that reduced the effective size of most populations. Finally, we used the inferred demographic model to perform an initial test for local adaptation and identified several genes, including the flowering time gene FCA and a disease resistance locus, as candidates for local adaptation events.Our results underscore the importance of population-specific, non-equilibrium demographic processes in patterning diversity within A. lyrata. Moreover, our extensive dataset provides an important resource for future molecular population genetic studies of local adaptation in A. lyrata

Lipid-Lowering Effects of Ethyl 2-Phenacyl-3-aryl-1H-pyrrole- 4-carboxylates in Rodents

A series of substituted 2-phenacyl-3-phenyl-1H-pyrrole-4-carboxylates were prepared from substituted acetophenones in 6 steps. The final condensations between a chloroenal and an aminoketone were carried out under neutral conditions in parallel to yield the series listed below. Selected pyrrole derivatives proved to be potent hypolipidemic agents lowering serum triglyceride concentrations in CF-1 male mice after 14 days of I.P. administration. One agent orally lowered serum cholesterol in Sprague-Dawley male rats at 2mg/kg/day after 14 days. The agents demonstrated a lowering of mouse serum LDL- cholesterol levels and selected compounds showed an elevation of serum HDL-cholesterol levels. The cholesterol concentrations in the liver were raised while the cholesterol and triglyceride contents of the aorta were significantly lowered by the selected trisubstituted pyrrole

Bioconjugation to functionalize peptidomimetics: Peptoid-based selective estrogen receptor modulators

Oligo-N-substituted glycines (peptoids) are a class of sequence specific peptidomimetics that are notable for their facile synthesis, sequence specificity, and peptide-like architectures. Additionally, due to their proteolytic resistance and enhanced cell permeability, peptoids are considered attractive candidates for therapeutics. In an effort to expand the chemical diversity and biomedical relevance of peptoids, we report here a novel technique to site-specifically functionalize linear peptoid oligomers utilizing the copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) reaction. Peptoid sequences including azide or alkyne-functionalized side chains were synthesized on solid-phase support and used as substrates for CuAAC. We were successful at conjugating such diverse pendant groups as fluorophores, nucleobases, metallocenes, and steroid ligands at up to six positions along the oligomer scaffold. We have also demonstrated that the triazole linkages formed from the CuAAC reaction are compatible with standard peptoid submonomer addition chemistry. This reactive orthogonality allows for multiple cycles of conjugation and oligomer extension to be performed, facilitating the generation of highly functionalized peptidomimetic oligomers outfitted with multiple heterogeneous pendant groups. This CuAAC-mediated ligation technique was used to generate novel multivalent bioactive constructs. Estradiol-peptidomimetic conjugates (EPCs) were synthesized via site-specific conjugation of 17α-ethynylestradiol to multiple sites on azide-functionalized peptoid scaffolds. Multivalent EPCs were generated in high yield and their efficacy on binding the human estrogen receptor α (hERα) was evaluated in vitro. It was determined that EPC-hERα binding avidities are enhanced when the valency of hormone ligand is increased. To monitor EPC-mediated activation of the hERα in vivo, HEK293 cells were stably transfected with plasmids coding for hERα and subsequently treated with EPCs. EPC length and valency influenced both cell uptake and receptor modulation in this system, with highest levels of hERα activation coming from divalent 18-mer and trivalent 9-mer EPCs. Non-steroidal peptidomimetic conjugates did not activate the hERα when compared to their EPC analogues, indicating that the estradiol pendant groups are primarily responsible for the bioactivity of these constructs. Given intrinsic length and valence "tunability", and the ability to modulate the hERα in living systems, EPCs may prove to be beneficial chemical tools in activating specific nuclear, and perhaps extranuclear effects of estrogens

Improved Assays for Determining the Cytosolic Access of Peptides, Proteins, and Their Mimetics

Proteins and other macromolecules that cross biological membranes have great potential as tools for research and next-generation therapeutics. Here, we describe two assays that effectively quantify the cytosolic localization of a number of previously reported peptides and protein domains. One assay, which we call GIGI (glucocorticoid-induced eGFP induction), is an amplified assay that informs on relative cytosolic access without the need for sophisticated imaging equipment or adherent cells. The second, GIGT (glucocorticoid-induced eGFP translocation), is a nonamplified assay that informs on relative cytosolic access and exploits sophisticated imaging equipment to facilitate high-content screens in live cells. Each assay was employed to quantify the cytosolic delivery of several canonical "cell permeable peptides," as well as more recently reported minimally cationic miniature proteins and zinc finger nuclease domains. Our results show definitively that both overall charge as well as charge distribution influence cytosolic access and that small protein domains containing a discrete, helical, penta-Arg motif can dramatically improve the cytosolic delivery of small folded proteins such as zinc finger domains. We anticipate that the assays described herein will prove useful to explore and discover the fundamental physicochemical and genetic properties that influence both the uptake and endosomal release of peptidic molecules and their mimetics

Improved Assays for Determining the Cytosolic Access of Peptides, Proteins, and Their Mimetics

Proteins and other macromolecules that cross biological membranes have great potential as tools for research and next-generation therapeutics. Here, we describe two assays that effectively quantify the cytosolic localization of a number of previously reported peptides and protein domains. One assay, which we call GIGI (glucocorticoid-induced eGFP induction), is an amplified assay that informs on relative cytosolic access without the need for sophisticated imaging equipment or adherent cells. The second, GIGT (glucocorticoid-induced eGFP translocation), is a nonamplified assay that informs on relative cytosolic access and exploits sophisticated imaging equipment to facilitate high-content screens in live cells. Each assay was employed to quantify the cytosolic delivery of several canonical “cell permeable peptides,” as well as more recently reported minimally cationic miniature proteins and zinc finger nuclease domains. Our results show definitively that both overall charge as well as charge distribution influence cytosolic access and that small protein domains containing a discrete, helical, penta-Arg motif can dramatically improve the cytosolic delivery of small folded proteins such as zinc finger domains. We anticipate that the assays described herein will prove useful to explore and discover the fundamental physicochemical and genetic properties that influence both the uptake and endosomal release of peptidic molecules and their mimetics

A novel peptide antagonist of the human growth hormone receptor

Excess circulating human growth hormone (hGH) in vivo is linked to metabolic and growth disorders such as cancer, diabetes, and acromegaly. Consequently, there is considerable interest in developing antagonists of hGH action. Here, we present the design, synthesis, and characterization of a 16- residue peptide (site 1-binding helix [S1H]) that inhibits hGH-mediated STAT5 phosphorylation in cultured cells. S1H was designed as a direct sequence mimetic of the site 1 minihelix (residues 36-51) of wild-type hGH and acts by inhibiting the interaction of hGH with the human growth hormone receptor (hGHR). In vitro studies indicated that S1H is stable in human serum and can adopt an α-helix in solution. Our results also show that S1H mitigates phosphorylation of STAT5 in cells co-treated with hGH, reducing intracellular STAT5 phosphorylation levels to those observed in untreated controls. Furthermore, S1H was found to attenuate the activity of the hGHR and the human prolactin receptor, suggesting that this peptide acts as an antagonist of both lactogenic and somatotrophic hGH actions. Finally, we used alanine scanning to determine how discrete amino acids within the S1H sequence contribute to its structural organization and biological activity. We observed a strong correlation between helical propensity and inhibitory effect, indicating that S1H-mediated antagonism of the hGHR is largely dependent on the ability for S1H to adopt an α-helix. Taken together, these results show that S1H not only acts as a novel peptide-based antagonist of the hGHR but can also be applied as a chemical tool to study the molecular nature of hGH-hGHR interactions

Do Men Produce Higher Quality Ejaculates When Primed With Thoughts of Partner Infidelity?

Sperm competition theory can be used to generate the hypothesis that men alter the quality of their ejaculates as a function of sperm competition risk. Using a repeated measures experimental design, we investigated whether men produce a higher quality ejaculate when primed with cues to sperm competition (i.e., imagined partner infidelity) relative to a control prime. Men ( n = 45) submitted two masturbatory ejaculates—one ejaculate sample for each condition (i.e., sperm competition and control conditions). Ejaculates were assessed on 17 clinical parameters. The results did not support the hypothesis: Men did not produce higher quality ejaculates in the sperm competition condition relative to the control condition. Despite the null results of the current research, there is evidence for psychological and physiological adaptations to sperm competition in humans. We discuss methodological limitations that may have produced the null results and present methodological suggestions for research on human sperm competition