Polymorphisms of two loci at the oxytocin receptor gene in populations of Africa, Asia and South Europe.

BACKGROUND: The oxytocin (OT) system is known to be implicated in the regulation of complex social behavior, particularly empathy and parenting. The goal of this study was to estimate the gender and population differences in polymorphisms of two oxytocin receptor gene SNPs, rs53576 and rs2254298, in four populations. RESULTS: These data were compared with each other and with 14 samples from the corresponding regions retrieved from the 1000 Genomes database. Low level of heterozygosity was observed for both SNPs in all populations in this study (rs53576: Catalonian, Hobs = 0.413; Hadza, Hobs = 0.556; sr2254698: Khanty-Mansi, Hobs = 0.250; Datoga, Hobs = 0.550). The amount of variance due to regional variability was almost equal for both SNPs (rs53576: FRT = 0.086, rs2554298: FRT = 0.072), whereas variance for the population level of variability was twice bigger for rs2554298 (rs53576: FST = 0.127, rs2554298: FST = 0.162). Pairwise coefficients of fixation demonstrate that the Hadza were well differentiated from other African populations except of Datoga, the Datoga were weakly differentiated from other African origin populations, the Ob Ugric people were extremely differentiated from all other populations. Catalans were extremely differentiated of Asian populations. CONCLUSIONS: It is hypothesized on the base of spatial distribution of the evolutionary novel A alleles of the both OXTR gene loci, that the spread of alleles of rs22542298 and rs53376 SNPs may be associated to some extant with manipulation of parental investment in humans.

Intravenous infusion of phage-displayed antibody library in human cancer patients: enrichment and cancer-specificity of tumor-homing phage-antibodies.

Phage display is a powerful method for target discovery and selection of ligands for cancer treatment and diagnosis. Our goal was to select tumor-binding antibodies in cancer patients. Eligibility criteria included absence of preexisting anti-phage-antibodies and a Stage IV cancer status. All patients were intravenously administered 1 × 10(11) TUs/kg of an scFv library 1 to 4 h before surgical resection of their tumors. No significant adverse events related to the phage library infusion were observed. Phage were successfully recovered from all tumors. Individual clones from each patient were assessed for binding to the tumor from which clones were recovered. Multiple tumor-binding phage-antibodies were identified. Soluble scFv antibodies were produced from the phage clones showing higher tumor binding. The tumor-homing phage-antibodies and derived soluble scFvs were found to bind varying numbers (0-5) of 8 tested normal human tissues (breast, cervix, colon, kidney, liver, spleen, skin, and uterus). The clones that showed high tumor-specificity were found to bind corresponding tumors from other patients also. Clone enrichment was observed based on tumor binding and DNA sequence data. Clone sequences of multiple variable regions showed significant matches to certain cancer-related antibodies. One of the clones (07-2,355) that was found to share a 12-amino-acid-long motif with a reported IL-17A antibody was further studied for competitive binding for possible antigen target identification. We conclude that these outcomes support the safety and utility of phage display library panning in cancer patients for ligand selection and target discovery for cancer treatment and diagnosis.

Private Spaceflight: A New Landscape for Dealing with Medical Risk.

As NASA and other space agencies make plans to proceed with human exploration missions beyond low earth orbit (LEO), the private sector, including Space X, Virgin Galactic, Blue Origin, Space Adventures and others, echo these plans with initiatives of their own to send humans further into space. Development of more sub-orbital flight opportunities, orbital flight opportunities to LEO and even higher risk endeavors will certainly result in exposure to medical risks for an expanding and heterogeneous population of civilians. To date, a handful of "space tourists" have flown to the International Space Station (ISS), at their own expense, ushering in a new era in which anyone with reasonably good health and even those with physical disability may consider becoming space travelers. Indeed, medical and behavioral issues of healthy, professional astronauts, have not been problematic on short orbital flights. However, recent attempts to test the potential limitations in astronauts on extended duration orbital flights in preparation for future missions beyond LEO raise concern about individual differences in ability to tolerate the hazardous spaceflight environment. Given the rapid development of opportunities for non-professionals and the employees of private companies to travel into space, this is an appropriate time to consider the development of selection strategies for non-government space travelers, including the development of genomic and other modern tools to assess susceptibility to spaceflight risk.

Localization of ASV integrase-DNA contacts by site-directed crosslinking and their structural analysis.

BACKGROUND: We applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins. METHODOLOGY/RESULTS: Photoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and amino-modified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate. CONCLUSION: Analysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories shows good agreement with the structure of PFV IN and derived ASV, HIV, and MuLV models for the core domain, but only partial agreement for the N- and C-terminal domains. These differences might be explained by structural variations and evolutionary selection for residues at alternate positions to perform analogous functions, and by methodological differences: i.e., a static picture of a particular assembly from crystallography vs. a variety of interactions that might occur during formation of functional IN complexes in solution.

Structural basis of binding by cyclic nonphosphorylated peptide antagonists of Grb7 implicated in breast cancer progression.

Growth-receptor-bound protein (Grb)7 is an adapter protein aberrantly overexpressed, along with the erbB-2 receptor in breast cancer and in other cancers. Normally recruited to focal adhesions with a role in cell migration, it is associated with erbB-2 in cancer cells and is found to exacerbate cancer progression via stimulation of cell migration and proliferation. The G7-18NATE peptide (sequence: WFEGYDNTFPC cyclized via a thioether bond) is a nonphosphorylated peptide that was developed for the specific inhibition of Grb7 by blocking its SH2 domain. Cell-permeable versions of G7-18NATE are effective in the reduction of migration and proliferation in Grb7-overexpressing cells. It thus represents a promising starting point for the development of a therapeutic against Grb7. Here, we report the crystal structure of the G7-18NATE peptide in complex with the Grb7-SH2 domain, revealing the structural basis for its interaction. We also report further rounds of phage display that have identified G7-18NATE analogues with micromolar affinity for Grb7-SH2. These peptides retained amino acids F2, G4, and F9, as well as the YDN motif that the structural biology study showed to be the main residues in contact with the Grb7-SH2 domain. Isothermal titration calorimetry measurements reveal similar and better binding affinity of these peptides compared with G7-18NATE. Together, this study facilitates the optimization of second-generation inhibitors of Grb7.

Nonnucleoside inhibitor binding affects the interactions of the fingers subdomain of human immunodeficiency virus type 1 reverse transcriptase with DNA.

Site-directed photoaffinity cross-linking experiments were performed by using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (i.e., positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Since neither the introduction of the unique cysteine residues into the fingers nor the modification of the SH groups of these residues with photoaffinity cross-linking reagents caused a significant decrease in the enzymatic activities of RT, we were able to use this system to measure distances between specific positions in the fingers domain of RT and double-stranded DNA. HIV-1 RT is quite flexible. There are conformational changes associated with binding of the normal substrates and nonnucleoside RT inhibitors (NNRTIs). Cross-linking was used to monitor intramolecular movements associated with binding of an NNRTI either in the presence or in the absence of an incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreased the efficiency of cross-linking but caused only modest changes in the preferred positions of cross-linking. This finding suggests that the fingers of p66 are closer to an extended template in the "open" configuration of the enzyme with the fingers away from the active site than in the closed configuration with the fingers in direct contact with the incoming dNTP. NNRTI binding caused increased cross-linking in experiments with diazirine reagents (especially with a diazirine reagent with a longer linker) and a moderate shift in the preferred sites of interaction with the template. Cross-linking occurred closer to the polymerase active site for RTs modified at positions 70 and 74. The effects of NNRTI binding were more pronounced in the absence of a bound dNTP; pretreatment of HIV-1 RT with an NNRTI reduced the effect of dNTP binding. These observations can be explained if the binding of NNRTI causes a decrease in the flexibility in the fingers subdomain of RT-NNRTI complex and a decrease in the distance from the fingers to the template extension.

Combinatorial evolution of high-affinity peptides that bind to the Thomsen-Friedenreich carcinoma antigen.

Thomsen-Friedenreich (TF) antigen occurs on approximately 90% of human carcinomas, is likely involved in carcinoma cell homotypic aggregation, and has clinical value as a prognostic indicator and marker of metastasized cells. Previously, we isolated anti-TF antigen peptides from bacteriophage display libraries. These bound to TF antigen on carcinoma cells but were of low affinity and solubility. We hypothesized that peptide amino acid sequence changes would result in increased affinity and solubility, which would translate into improved carcinoma cell binding and increased inhibition of aggregation. The new peptides were more soluble and exhibited up to fivefold increase in affinity (Kd approximately equal to 60 nM). They bound cultured human breast and prostate carcinoma cells at low concentrations, whereas the earlier peptides did not. Moreover, the new peptides were potent inhibitors of homotypic aggregation. The maturated peptides will have expanded applications in basic studies of the TF antigen and particular utility as clinical carcinoma-targeting agents.