Triplet repeat expansion generated by DNA slippage is suppressed by human flap endonuclease 1

Human flap endonuclease 1 (h-FEN1) mutations have dramatic effects on repeat instability. Current models for repeat expansion predict that h-FEN1 protein prevents mutations by removing 5'-flaps generated at ends of Okazaki fragments by strand displacement synthesis. The models propose that hairpin formations within flaps containing repeats enable them to escape h-FEN1 cleavage. Friedreich's ataxia is caused by expansion mutations in a d(GAA)n repeat tract. Single-stranded d(GAA)n repeat tracts, however, do not form stable hairpins until the repeat tracts are quite long. Therefore, to understand how d(GAA)n repeat expansions survive h-FEN1 activity, we determined the effects of h-FEN1 on d(GAA)n repeat expansion during replication of a d(TTC)n repeat template. Replication initiated within the repeat tract generated significant expansion that was suppressed by the addition of h-FEN1 at the start of replication. The ability of h-FEN1 to suppress expansion implies that DNA slippage generates a 5'-flap in the nascent strand independent of strand displacement synthesis by an upstream polymerase. Delaying the addition of h-FEN1 to the replication reaction abolished the ability of h-FEN1 ability to suppress d(GAA)n repeat expansion products of all sizes, including sizes unable to hairpin. Use of model substrates demonstrated that h-FEN1 cleaves d(GAA)n 5'-flaps joined to double-stranded nonrepeat sequences but not those joined to double-stranded repeat tracts. The results provide evidence that, given the opportunity, short d(GAA)n repeat expansion products rearrange from 5'-flaps to stable internal loops inside the repeat tract. Long expansion products are predicted to form hairpinned flaps and internal loops. Once formed, these DNA conformations resist h-FEN1. The biological implications of the results are discussed.     

Recombinant Human Collagen XV Regulates Cell Adhesion and Migration

The C-terminal end of collagen XV, restin, has been the focus of several studies, but the functions of full-length collagen XV have remained unknown. We describe here studies on the production, purification, and function of collagen XV and the production of a monoclonal N-terminal antibody to it. Full-length human collagen XV was produced in insect cells using baculoviruses and purified from the cell culture medium. The yield was 15 mg/liter of cell culture medium. The collagen XV was shown to be trimeric, with disulfide bonds in the collagenous region. Rotary shadowing electron microscopy revealed rod-like molecules with a mean length of 241.8 nm and with a globular domain at one end. The globular domain was verified to be the N-terminal end by N-terminal antibody binding. The molecules show flexibility in their conformation, presumably due to the many interruptions in their collagenous domains. The ability of collagen XV to serve as a substrate for cells was tested in cell adhesion assays, and it was shown that cells did not bind to collagen XV-coated surfaces. When added to the culture medium of fibroblasts and fibrosarcoma cells, however, collagen XV rapidly bound to their fibronectin network. Solid phase assays showed that collagen XV binds to fibronectin, laminin, and vitronectin and that it binds to the collagen/gelatin-binding domain of fibronectin. No binding was detected to fibrillar collagens, fibril-associated collagens, or decorin. Interestingly, collagen XV was found to inhibit the adhesion and migration of fibrosarcoma cells when present in fibronectin-containing matrices.     

Structure of the Epstein-Barr virus oncogene BARF1

The Epstein-Barr virus is a human gamma-herpesvirus that persistently infects more than 90% of the human population. It is associated with numerous epithelial cancers, principally undifferentiated nasopharyngeal carcinoma and gastric carcinoma. The BARF1 gene is expressed in a high proportion of these cancers. An oncogenic, mitogenic and immortalizing activity of the BARF1 protein has been shown. We solved the structure of the secreted BARF1 glycoprotein expressed in a human cell line by X-ray crystallography at a resolution of 2.3A. The BARF1 protein consists of two immunoglobulin (Ig)-like domains. The N-terminal domain belongs to the subfamily of variable domains whereas the C-terminal one is related to a constant Ig-domain. BARF1 shows an unusual hexamerisation involving two principal contacts, one between the C-terminal domains and one between the N-terminal domains. The C-terminal contact with an uncommonly large contact surface extends the beta-sandwich of the Ig-domain through the second molecule. The N-terminal contact involves Ig-domains with an unusual relative orientation but with a more classical contact surface with a size in the range of dimer interactions of Ig-domains. The structure of BARF1 is most closely related to CD80 or B7-1, a co-stimulatory molecule present on antigen presenting cells, from which BARF1 must have been derived during evolution. Still, domain orientation and oligomerization differ between BARF1 and CD80. It had been shown that BARF1 binds to hCSF-1, the human colony-stimulating factor 1, but this interaction has to be principally different from the one between CSF-1 and CSF-1 receptor.     

Predictors of belief that genetic test information about hemochromatosis should be shared with family members

We queried 101,951 white, Hispanic, black, Asian, American Indian (i.e., American Indian or Alaska Native in the United States and North American Indian, Metis, or Inuit in Canada) and Pacific Islander (including Native Hawaiian) adults who agreed to be genotypically and phenotypically screened for hemochromatosis as part of the Hemochromatosis and Iron Overload Screening (HEIRS) study about their views on sharing genetic test information with family members. Multiple logistic regression (adjusting for study site, age group, race/ethnicity, preferred language, gender, education group, income group, SF-36 General Health and Mental Health subscales, perceived benefits and limitations of genetic testing, and belief that genetic testing is a good idea) evaluated independent predictors of responding "Strongly Agree" or "Agree" versus "Disagree" or "Strongly Disagree" to the statement "Information about a person's genetic risk should be shared with family members". Agreement that genetic risk information should be shared with family members was high (93% in the overall sample of 78,952 who answered this question), but differed among racial/ethnic groups. Hispanics were significantly less likely to agree that genetic test information should be shared with family members (i.e., 88% versus 92% or more among all other ethnicities). The relationship of perceived limitations and benefits of testing, gender, and age group to the belief that information should be shared differed among racial/ethnic groups, with Spanish-preferring Hispanics being the most different from other subgroups.     

DHEA supplementation improves follicular microenviroment in poor responder patients

Objective: To analyze the effect of dehydroepiandrosterone (DHEA) supplementation on follicular microenvironment and on in vitro fertilization (IVF) outcomes among poor responder patients. Study design: We enrolled 24 patients diagnosed as poor responders based on ESHRE consensus criteria. One group received 25?mg/die three times daily of DHEA supplementation for 3 months previous to IVF cycle, while the other did not receive any treatment. COH was performed with rFSH and hMG, and a GnRH antagonist was administered according to a flexible protocol. We evaluated perifollicular vascularization of recruited follicles through power Doppler blood flow analysis and follicles were graded as described by Chui et al. Follicular fluids (FF) from F3-F4 follicles were collected, and FF levels of vascular endothelial growth factor (VEGF) and hypoxic inducible factor1 (HIF1) were measured. Results: FF levels of HIF1 were statistically significant lower in women treated with DHEA (14.76 ± 51.13 vs. 270.03 ± 262.18 pg/ml; p = 0.002). On the contrary, VEGF levels did not differ between the two groups. Concerning COH, in the DHEA-group the mean duration of treatment was significantly shorter (9.83 ± 1.85 vs. 12.09?±?2.81; p = 0.023). Total numbers of oocytes retrieved, fertilized oocytes, good quality embryos, number of transferred embryos and clinical pregnancies tended to be higher in study group, but the results were not significant. On the other hand, considering the oocytes retrieved in selected F3-F4 follicles, there was a relation between HIF1 levels and oocytes quality. In fact, mature oocytes retrieved in selected follicles were significantly more numerous in DHEA-group (0.50 ± 0.52 vs. 0.08?±?0.29; p = 0.018). Conclusions: The improvement of reproductive parameters after DHEA supplementation in poor responders may be explained through the effect that this pro-hormone exerts on follicular microenvironment.     

Microbiome dynamics of human epidermis following skin barrier disruption

Background

Recent advances in sequencing technologies have enabled metagenomic analyses of many human body sites. Several studies have catalogued the composition of bacterial communities of the surface of human skin, mostly under static conditions in healthy volunteers. Skin injury will disturb the cutaneous homeostasis of the host tissue and its commensal microbiota, but the dynamics of this process have not been studied before. Here we analyzed the microbiota of the surface layer and the deeper layers of the stratum corneum of normal skin, and we investigated the dynamics of recolonization of skin microbiota following skin barrier disruption by tape stripping as a model of superficial injury.

Results

We observed gender differences in microbiota composition and showed that bacteria are not uniformly distributed in the stratum corneum. Phylogenetic distance analysis was employed to follow microbiota development during recolonization of injured skin. Surprisingly, the developing neo-microbiome at day 14 was more similar to that of the deeper stratum corneum layers than to the initial surface microbiome. In addition, we also observed variation in the host response towards superficial injury as assessed by the induction of antimicrobial protein expression in epidermal keratinocytes.

Conclusions

We suggest that the microbiome of the deeper layers, rather than that of the superficial skin layer, may be regarded as the host indigenous microbiome. Characterization of the skin microbiome under dynamic conditions, and the ensuing response of the microbial community and host tissue, will shed further light on the complex interaction between resident bacteria and epidermis.     

Virological consequences of early events following cell-cell contact between human immunodeficiency virus type 1-infected and uninfected CD4+ cells

Human immunodeficiency virus type 1 (HIV-1)-infected cells transmit viral products to uninfected CD4⁺ cells very rapidly. However, the natures of the transmitted viral products and the mechanism of transmission, as well as the relative virological consequences, have not yet been fully clarified. We studied the virological events occurring a few hours after contact between HIV-1-infected and uninfected CD4⁺ cells using a coculture cell system in which the virus expression in target cells could be monitored through the induction of a green fluorescent protein reporter gene driven by HIV-1 long terminal repeats. Within 16 h of coculture, we observed two phenomena not related to the cell-free virus infection, i.e., the formation of donor-target cell fusions and a fusion-independent internalization of viral particles likely occurring at least in part through intercellular connections. Both events depended on the expression of Env and CD4 in donor and target cells, respectively, whereas the HIV-1 internalization required clathrin activity in target cells. Importantly, both phenomena were also observed in cocultures of primary CD4⁺ lymphocytes, while primary macrophages supported only HIV-1 endocytosis. By investigating the virological consequences of these events, we noticed that while fused cells released infectious HIV-1 particles, albeit with reduced efficiency compared with donor cells, no virus expression was detectable upon HIV-1 endocytosis in target cells. In sum, the HIV-1 transmission following contact between an HIV-1-infected and an uninfected CD4⁺ cell can occur through different mechanisms, leading to distinguishable virological outcomes.     

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.