Development of proteomic tools to study protein adsorption on a biomaterial, titanium grafted with poly(sodium styrene sulfonate)

It is known that protein adsorption is the initial interaction between implanted biomaterials and biological environment. Generally, a complex protein layer will be formed on material surfaces within a few minutes and the composition of this layer at the interface determines the biological response to the implanted material, and therefore the long-term compatibility of the biomaterial. Despite different techniques exist to observe protein adsorption on biomaterials, none of them led to the identification of adsorbed proteins. In this paper, we report a chromatographic technique coupled to proteomics to analyse and identify proteins from complex biological samples adsorbed on biomaterial surfaces. This approach is based on (1) elaboration of the chromatographic support containing the biomaterial (2) a chromatography step involving adsorption of proteins on the biomaterial (3) the high-resolution separation of eluted proteins by 2-DE gel and (4) the identification of proteins by mass spectrometry. Experiments were performed with proteins from platelets rich plasma (PRP) adsorbed on a biomaterial which consist in titanium bioactivated with PolyNaSS. Our results show that chromatographic approach combined to 2-DE gels and mass spectrometry provides a powerful tool for the analysis and identification of proteins adsorbed on various surfaces.     

Long-lasting inhibitory effects of fetal liver mesenchymal stem cells on T-lymphocyte proliferation

Human bone marrow mesenchymal stem cells (BM-MSC) are multipotent progenitor cells that have transient immunomodulatory properties on Natural Killer (NK) cells, Dendritic Cells (DC), and T cells. This study compared the use of MSC isolated from bone marrow and fetal liver (FL-MSC) to determine which displayed the most efficient immunosuppressive effects on T cell activation. Although both types of MSC exhibit similar phenotype profile, FL-MSC displays a much more extended in vitro life-span and immunomodulatory properties. When co-cultured with CD3/CD28-stimulated T cells, both BM-MSC and FL-MSC affected T cell proliferation by inhibiting their entry into the cell cycle, by inducing the down-regulation of phospho-retinoblastoma (pRb), cyclins A and D1, as well as up-regulating p27(kip1) expression. The T cell inhibition by MSC was not due to the soluble HLA-G5 isoform, but to the surface expression of HLA-G1, as shown by the need of cell-cell contact and by the use of neutralizing anti-HLA-G antibodies. To note, in a HLA-G-mediated fashion, MSC facilitated the expansion of a CD4(low)/CD8(low) T subset that had decreased secretion of IFN-γ, and an induced secretion of the immunomodulatory cytokine IL-10. Because of their longer lasting in vitro immunosuppressive properties, mainly mediated by HLA-G, and their more efficient induction of IL-10 production and T cell apoptosis, fetal liver MSC could be considered a new tool for MSC therapy to prevent allograft rejection.     

HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase

The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨ_m) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor.     

Characterization of the innate immune response in goats after intrauterine infusion of E. coli using histopathological, cytologic and molecular analyses

The objective was to characterize the innate immune response in dairy goats after intrauterine infusion of E. coli. A suspension of Escherichia coli (E. coli; 4 × 10⁹ cfu (cfu)/mL; experimental group, n = 6) or 5 mL PBS (control group, n = 6) were infused once into each uterine horn in goats at 25 days postpartum. Blood and endometrial biopsy samples were collected preinoculation (0 h) and at 3, 6, 12, 24, 72, 120, and 168 h post inoculation (pi). Relative gene expression analyses of Toll-like receptor4 (TLR4), tumor necrosis factorα (TNF-α), β-defensin2, and interleukins (IL-1β, IL-6, IL-8) were performed on RNA extracted from endometrial tissue and peripheral white blood cells (WBCs) using quantitative real-time PCR. Endometrial tissue was also used for histopathology and cytology. In experimental goats, the mRNA expression of TLR4 and proinflammatory cytokines were increased within 24 h pi (P < 0.01) in endometrium and WBCs. Similarly, expression of β-defensin2 was higher at 72 h pi in endometrium (P < 0.001), and at 120 h pi in WBCs (P < 0.05). The %PMNs in the experimental group increased up to 92.16 ± 3.95% at 3 h pi (P < 0.001). Endometrial histopathology revealed a severe inflammatory response at 3 to 12 h pi, whereas no changes were detected in the control group. In conclusion, intrauterine infusion of E. coli in goats resulted in a rapid activation of the local innate immune response, characterized by infiltration of PMNs into the endometrium and up-regulation of gene expression for TLR4, cytokines and β-defensin2.     

Virologic failures on initial boosted-PI regimen infrequently possess low-level variants with major PI resistance mutations by ultra-deep sequencing

Background

It is unknown whether HIV-positive patients experiencing virologic failure (VF) on boosted-PI (PI/r) regimens without drug resistant mutations (DRM) by standard genotyping harbor low-level PI resistant variants. CASTLE compared the efficacy of atazanavir/ritonavir (ATV/r) with lopinavir/ritonavir (LPV/r), each in combination with TVD in ARV-naïve subjects.

Objective

To determine if VF on an initial PI/r-based regimen possess low-level resistant variants that may affect a subsequent PI-containing regimen.

Methods/Results

Patients experiencing VF on a Tenofovir/Emtricitabine+PI/r regimen were evaluated by ultra deep sequencing (UDS) for mutations classified/weighted by Stanford HIVdb. Samples were evaluated for variants to 0.4% levels. 36 VF subjects were evaluated by UDS; 24 had UDS for PI and RT DRMs. Of these 24, 19 (79.2%) had any DRM by UDS. The most common UDS-detected DRM were NRTI in 18 subjects: M184V/I (11), TAMs(7) & K65R(4); PI DRMs were detected in 9 subjects: M46I/V(5), F53L(2), I50V(1), D30N(1), and N88S(1). The remaining 12 subjects, all with VLs<10,000, had protease gene UDS, and 4 had low-level PI DRMs: F53L(2), L76V(1), I54S(1), G73S(1). Overall, 3/36(8.3%) subjects had DRMs identified with Stanford-HIVdb weights >12 for ATV or LPV: N88S (at 0.43% level-mutational load 1,828) in 1 subject on ATV; I50V (0.44%-mutational load 110) and L76V (0.52%-mutational load 20) in 1 subject each, both on LPV. All VF samples remained phenotypically susceptible to the treatment PI/r.

Conclusion

Among persons experiencing VF without PI DRMs with standard genotyping on an initial PI/r regimen, low-level variants possessing major PI DRMs were present in a minority of cases, occurred in isolation, and did not result in phenotypic resistance. NRTI DRMs were detected in a high proportion of subjects. These data suggest that PIs may remain effective in subjects experiencing VF on a PI/r-based regimen when PI DRMs are not detected by standard or UDS genotyping.     

Molecular characteristics of the porcine DLK1 and MEG3 genes

Imprinted genes play important roles in embryo survival and postnatal growth regulation. The DLK1 and MEG3 (previously GTL2) genes are linked and reciprocally imprinted in several mammals, but their imprinting status is still unknown in pigs. In this study, we report polymorphisms, imprinting status and QTL analyses of the porcine DLK1 and MEG3 genes. Muscle and adipose DNA and RNA samples from 30-day-old animals generated with reciprocal crosses between the Korean native pig (KNP) and Yorkshire breeds were used to analyse DLK1 and MEG3 variation and expression. The samples exhibited paternal expression of DLK1 and maternal expression of MEG3 in pigs. These results indicated that the imprinting status of the DLK1 and MEG3 genes is conserved across mammalian species. By linkage analyses, we assigned the DLK1 and MEG3 genes to the telomeric region of SSC7. By QTL analyses, we confirmed a significant polar overdominance (POD) effect in DLK1 , which was previously detected for several growth traits in pigs. However, no significant POD effect was found with the MEG3 locus.     

Substantial hydraulic signals are triggered by leaf-biting insects in tomato

In various plants, defence responses can be induced throughoutthe shoot by localized damage or insect attack. Activation ofsuch systemic defence responses must involve a rapid long-distancesignal of wounding. There is firm evidence that, in the caseof localized heat wounds, systemic signalling occurs by hydraulicdispersal of chemical elicitors. However, more natural wounds(such as those imposed by leaf-biting insects) may trigger onlysmall hydraulic events, and it is not clear whether hydraulicdispersal could account for wound signalling in these cases. It is shown here that partial defoliation offers a method foramplifying wound-induced hydraulic events in tomato. Using thisamplification, it is demonstrated that brief feeding by individualleaf-eating insects triggers substantial hydraulic events. Themass flows associated with these events are shown to be sufficientto drive hydraulic dispersal of elicitors through the tomatoplant. It is concluded that hydraulic dispersal could be ofmajor importance for wound signalling in plants in the naturalenvironment. Key words: Lycopersicon esculentum, Spodoptera lit-toralis, wound signalling, systemic defence responses, hydraulic signals