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1.  The Timing of Pigmentation Lightening in Europeans 
The inverse correlation between skin pigmentation and latitude observed in human populations is thought to have been shaped by selective pressures favoring lighter skin to facilitate vitamin D synthesis in regions far from the equator. Several candidate genes for skin pigmentation have been shown to exhibit patterns of polymorphism that overlap the geospatial variation in skin color. However, little work has focused on estimating the time frame over which skin pigmentation has changed and on the intensity of selection acting on different pigmentation genes. To provide a temporal framework for the evolution of lighter pigmentation, we used forward Monte Carlo simulations coupled with a rejection sampling algorithm to estimate the time of onset of selective sweeps and selection coefficients at four genes associated with this trait in Europeans: KITLG, TYRP1, SLC24A5, and SLC45A2. Using compound haplotype systems consisting of rapidly evolving microsatellites linked to one single-nucleotide polymorphism in each gene, we estimate that the onset of the sweep shared by Europeans and East Asians at KITLG occurred approximately 30,000 years ago, after the out-of-Africa migration, whereas the selective sweeps for the European-specific alleles at TYRP1, SLC24A5, and SLC45A2 started much later, within the last 11,000–19,000 years, well after the first migrations of modern humans into Europe. We suggest that these patterns were influenced by recent increases in size of human populations, which favored the accumulation of advantageous variants at different loci.
doi:10.1093/molbev/mss207
PMCID: PMC3525146  PMID: 22923467
pigmentation genes; age of selection; selection coefficient; European populations
2.  Genomic Data Reveal a Complex Making of Humans 
PLoS Genetics  2012;8(7):e1002837.
In the last few years, two paradigms underlying human evolution have crumbled. Modern humans have not totally replaced previous hominins without any admixture, and the expected signatures of adaptations to new environments are surprisingly lacking at the genomic level. Here we review current evidence about archaic admixture and lack of strong selective sweeps in humans. We underline the need to properly model differential admixture in various populations to correctly reconstruct past demography. We also stress the importance of taking into account the spatial dimension of human evolution, which proceeded by a series of range expansions that could have promoted both the introgression of archaic genes and background selection.
doi:10.1371/journal.pgen.1002837
PMCID: PMC3400556  PMID: 22829785
3.  Relationships between Membrane Binding, Affinity and Cell Internalization Efficacy of a Cell-Penetrating Peptide: Penetratin as a Case Study 
PLoS ONE  2011;6(9):e24096.
Background
Penetratin is a positively charged cell-penetrating peptide (CPP) that has the ability to bind negatively charged membrane components, such as glycosaminoglycans and anionic lipids. Whether this primary interaction of penetratin with these cell surface components implies that the peptide will be further internalized is not clear.
Methodology
Using mass spectrometry, the amount of internalized and membrane bound penetratin remaining after washings, were quantified in three different cell lines: wild type (WT), glycosaminoglycans- (GAGneg) and sialic acid-deficient (SAneg) cells. Additionally, the affinity and kinetics of the interaction of penetratin to membrane models composed of pure lipids and membrane fragments from the referred cell lines was investigated, as well as the thermodynamics of such interactions using plasmon resonance and calorimetry.
Principal Findings
Penetratin internalized with the same efficacy in the three cell lines at 1 µM, but was better internalized at 10 µM in SAneg>WT>GAGneg. The heat released by the interaction of penetratin with these cells followed the ranking order of internalization efficiency. Penetratin had an affinity of 10 nM for WT cells and µM for SAneg and GAGneg cells and model membrane of phospholipids. The remaining membrane-bound penetratin after cells washings was similar in WT and GAGneg cells, which suggested that these binding sites relied on membrane phospholipids. The interaction of penetratin with carbohydrates was more superficial and reversible while it was stronger with phospholipids, likely because the peptide can intercalate between the fatty acid chains.
Conclusion/Significance
These results show that accumulation and high-affinity binding of penetratin at the cell-surface do not reflect the internalization efficacy of the peptide. Altogether, these data further support translocation (membrane phospholipids interaction) as being the internalization pathway used by penetratin at low micromolecular concentration, while endocytosis is activated at higher concentration and requires accumulation of the peptide on GAG and GAG clustering.
doi:10.1371/journal.pone.0024096
PMCID: PMC3167814  PMID: 21915283
4.  Distinct Behaviour of the Homeodomain Derived Cell Penetrating Peptide Penetratin in Interaction with Different Phospholipids 
PLoS ONE  2010;5(12):e15819.
Background
Penetratin is a protein transduction domain derived from the homeoprotein Antennapedia. Thereby it is currently used as a cell penetrating peptide to introduce diverse molecules into eukaryotic cells, and it could also be involved in the cellular export of transcription factors. Moreover, it has been shown that it is able to act as an antimicrobial agent. The mechanisms involved in all these processes are quite controversial.
Methodology/Principal Findings
In this article, we report spectroscopic, calorimetric and biochemical data on the penetratin interaction with three different phospholipids: phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to mimic respectively the outer and the inner leaflets of the eukaryotic plasma membrane and phosphatidylglycerol (PG) to mimic the bacterial membrane. We demonstrate that with PC, penetratin is able to form vesicle aggregates with no major change in membrane fluidity and presents no well defined secondary structure organization. With PE, penetratin aggregates vesicles, increases membrane rigidity and acquires an α-helical structure. With PG membranes, penetratin does not aggregate vesicles but decreases membrane fluidity and acquires a structure with both α-helical and β–sheet contributions.
Conclusions/Significance
These data from membrane models suggest that the different penetratin actions in eukaryotic cells (membrane translocation during export and import) and on prokaryotes may result from different peptide and lipid structural arrangements. The data suggest that, for eukaryotic cell penetration, penetratin does not acquire classical secondary structure but requires a different conformation compared to that in solution.
doi:10.1371/journal.pone.0015819
PMCID: PMC3012702  PMID: 21209890
6.  Quantitative Evaluation of Human δ Opioid Receptor Desensitization Using the Operational Model of Drug Action 
Molecular pharmacology  2007;71(5):1416-1426.
Agonist-mediated desensitization of the opioid receptors is thought to function as a protective mechanism against sustained opioid signaling and therefore may prevent the development of opioid tolerance. However, the exact molecular mechanism of opioid receptor desensitization remains unresolved because of difficulties in measuring and interpreting receptor desensitization. In the present study, we investigated deltorphin II-mediated rapid desensitization of the human δ opioid receptors (hDOR) by measuring guanosine 5′-O-(3-[35S]thio)-triphosphate binding and inhibition of cAMP accumulation. We developed a mathematical analysis based on the operational model of agonist action (Black et al., 1985) to calculate the proportion of desensitized receptors. This approach permits a correct analysis of the complex process of functional desensitization by taking into account receptor-effector coupling and the time dependence of agonist pretreatment. Finally, we compared hDOR desensitization with receptor phosphorylation at Ser363, the translocation of β-arrestin2, and hDOR internalization. We found that in Chinese hamster ovary cells expressing the hDOR, deltorphin II treatment leads to phosphorylation of Ser363, translocation of β-arrestin2 to the plasma membrane, receptor internalization, and uncoupling from G proteins. It is noteworthy that mutation of the primary phosphorylation site Ser363 to alanine had virtually no effect on agonist-induced β-arrestin2 translocation and receptor internalization yet significantly attenuated receptor desensitization. These results strongly indicate that phosphorylation of Ser363 is the primary mechanism of hDOR desensitization.
doi:10.1124/mol.106.030023
PMCID: PMC2694736  PMID: 17322005
7.  The Two NK-1 Binding Sites Correspond to Distinct, Independent, and Non-Interconvertible Receptor Conformational States As Confirmed by Plasmon-Waveguide Resonance Spectroscopy 
Biochemistry  2006;45(16):5309-5318.
Two nonstoichiometric ligand binding sites have been previously reported for the NK-1 receptor, with the use of classical methods (radioligand binding and second messenger assays). The most populated (major, NK-1M) binding site binds substance P (SP) and is related to the adenylyl cyclase pathway. The less populated (minor, NK-1m) binding site binds substance P, C-terminal hexa- and heptapeptide analogues of SP, and the NK-2 endogenous ligand, neurokinin A, and is coupled to the phospholipase C pathway. Here, we have examined these two binding sites with plasmon-waveguide resonance (PWR) spectroscopy that allows the thermodynamics and kinetics of ligand–receptor binding processes and the accompanying structural changes of the receptor to be monitored, through measurements of the anisotropic optical properties of lipid bilayers into which the receptor is incorporated. The binding of the three peptides, substance P, neurokinin A, and propionyl[Met(O2)11]SP(7-11), to the partially purified NK-1 receptor has been analyzed by this method. Substance P and neurokinin A bind to the reconstituted receptor in a biphasic manner with two affinities (Kd1 = 0.14 ± 0.02 nM and Kd2 = 1.4 ± 0.18 nM, and Kd1 = 5.5 ± 0.7 nM and Kd2 = 620 ± 117 nM, respectively), whereas only one binding affinity (Kd = 5.5 ± 0.4 nM) could be observed for propionyl[Met(O2)11]SP(7-11). Moreover, binding experiments in which one ligand was added after another one has been bound to the receptor have shown that the binding of these ligands to each binding site was unaffected by the fact that the other site was already occupied. These data strongly suggest that these two binding sites are independent and non-interconvertible on the time scale of these experiments (1-2 h).
doi:10.1021/bi052586d
PMCID: PMC1865500  PMID: 16618119
8.  New Paradigms and Tools in Drug Design for Pain and Addiction 
The AAPS journal  2006;8(3):E450-E460.
New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at μ and δ opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in G-protein coupled receptors (GPCRs) as illustrated by the δ opioid receptor.
doi:10.1208/aapsj080353
PMCID: PMC1764851  PMID: 17025262
drug design; neuropathic pain; bifunctional ligands; plasmon waveguide resonance spectroscopy; GPCRs; opioid receptors; cholecystokinin receptors
9.  New paradigms and tools in drug design for pain and addiction 
The AAPS Journal  2006;8(3):E450-E460.
New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at μ and σ opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in g-protein coupled receptors (GPCRs) as illustrated by the δ opioid receptor.
doi:10.1208/aapsj080353
PMCID: PMC1764851  PMID: 17025262
drug design; neuropathic pain; bifunctional ligands; plasmon waveguide resonance spectroscopy; GPCRs; opioid receptors; cholecystokinin receptors
10.  LIGAND MODULATION OF GPCR LATERAL SEGREGATION INTO LIPID MICRODOMAINS IN SPHINGOMYELIN/PHOSPHATIDYLCHOLINE SOLID-SUPPORTED BILAYERS 
Biochemistry  2005;44(25):9168-9178.
Increasing evidence supports the idea that the plasma membrane bilayer is characterized by a laterally inhomogeneous mixture of lipids, having an organized structure in which lipid molecules segregate into small domains or patches. Such microdomains are characterized by high contents of sphingolipids that form thicker liquid-ordered regions having resistance to extraction with nonionic detergents. The existence of lipid lateral segregation has been demonstrated in both model and biological membranes, although its role in protein sorting and membrane function still remains unclear. In the present studies, plasmon-waveguide resonance (PWR) spectroscopy was employed to investigate the properties of microdomains in a model system consisting of a solid-supported lipid bilayer composed of a 1:1 mixture of palmitoyloleoylphosphatidylcholine (POPC) and brain sphingomyelin (SM), and their influence on the partitioning and functioning of the human delta opioid receptor (hDOR; a G-protein coupled receptor, GPCR). Resonance signals corresponding to two microdomains (POPC-rich and SM-rich) were observed in such bilayers, and the sorting of the receptor into each domain was highly dependent on the type of ligand that was bound. When no ligand was bound, the receptor incorporated preferentially into the POPC-rich domain; when an agonist or an antagonist was bound, the receptor incorporated preferentially into the SM-rich component, although with a two-fold greater propensity for this microdomain in the case of the agonist. G-protein binding to the agonist-bound receptor in the SM-rich domain occurred with a 30-fold higher affinity than to the receptor in the PC-rich domain. The binding of agonist to an unliganded receptor in the bilayer produced receptor trafficking from the PC-rich into the SM-rich component. Since the SM-rich domain is thicker than the PC-rich domain, and previous studies with the hDOR have shown that the receptor elongates upon agonist-activation, we propose that hydrophobic matching between the receptor and the lipid is a driving force for receptor trafficking to the SM-rich component.
doi:10.1021/bi050207a
PMCID: PMC1440485  PMID: 15966741
microenvironmental effects; G-protein binding and activation; phospholipids; sphingolipids; protein sorting

Results 1-10 (10)