Identification and immunogenicity of immunodominant mimotopes of outer membrane protein U (OmpU) of Vibrio mimicus from phage display peptide library

Vibrio mimicus (V. mimicus) is the causative agent of ascites disease in aquatic animals. Outer membrane protein U (OmpU) is an important antigen of V. mimicus, but its protective epitopes are still unclear. A random 12-mer phage-displayed peptide library was used to screen and identify immunodominant mimotopes of the OmpU protein in V. mimicus by panning against purified OmpU-specific polyclonal antibody. Then the immunogenicity and immunoprotection in fish of these mimotopes was evaluated. Nine positive phage clones presented seven different 12- peptide sequences and more than 50% of them carried a consensus core motif of DSSK–P. These positive clones reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by OmpU protein. Intraperitoneal injection of seven positive phage clones into fish induced a speci?c antibody response to OmpU protein. The fish immunized respectively with the positive phage clones C17, C24, C60 and C66 obtained 100% immunoprotective effect against experimental V. mimicus challenge. Taken together, these mimotopes presented by clone C17, C24, C60 and C66 were immunodominant mimotopes of the OmpU protein and exhibited a more appropriate candidate as epitope-based vaccine against V. mimicus infection in aquatic animals.     

An Anti-CD34 Antibody-Functionalized Clinical-Grade POSS-PCU Nanocomposite Polymer for Cardiovascular Stent Coating Applications: A Preliminary Assessment of Endothelial Progenitor Cell Capture and Hemocompatibility

In situ endothelialization of cardiovascular implants has emerged in recent years as an attractive means of targeting the persistent problems of thrombosis and intimal hyperplasia. This study aimed to investigate the efficacy of immobilizing anti-CD34 antibodies onto a POSS-PCU nanocomposite polymer surface to sequester endothelial progenitor cells (EPCs) from human blood, and to characterize the surface properties and hemocompatibility of this surface. Amine-functionalized fumed silica was used to covalently conjugate anti-CD34 to the polymer surface. Water contact angle, fluorescence microscopy, and scanning electron microscopy were used for surface characterization. Peripheral blood mononuclear cells (PBMCs) were seeded on modified and pristine POSS-PCU polymer films. After 7 days, adhered cells were immunostained for the expression of EPC and endothelial cell markers, and assessed for the formation of EPC colonies. Hemocompatibility was assessed by thromboelastography, and platelet activation and adhesion assays. The number of EPC colonies formed on anti-CD34-coated POSS-PCU surfaces was not significantly higher than that of POSS-PCU (5.0±1.0 vs. 1.7±0.6, p>0.05). However, antibody conjugation significantly improved hemocompatibility, as seen from the prolonged reaction and clotting times, decreased angle and maximum amplitude (p<0.05), as well as decreased platelet adhesion (76.8±7.8 vs. 8.4±0.7, p<0.05) and activation. Here, we demonstrate that POSS-PCU surface immobilized anti-CD34 antibodies selectively captured CD34⁺ cells from peripheral blood, although only a minority of these were EPCs. Nevertheless, antibody conjugation significantly improves the hemocompatibility of POSS-PCU, and should therefore continue to be explored in combination with other strategies to improve the specificity of EPC capture to promote in situ endothelialization.     

Novel Cell-Free Strategy for Therapeutic Angiogenesis: In Vitro Generated Conditioned Medium Can Replace Progenitor Cell Transplantation

Background

Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy.

Methodology/Principal Findings

EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2±2.9% and 83.7±3.0% vs. 53.5±2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62±0.03 and 1.68±0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6±0.3 and 8.1±0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7±44.1 vs. 340.0±29.1 CD34⁺/CD45⁻ cells/1×10⁵ mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9±0.7 vs. 2.6±0.4 CD34⁺ cells/HPF, P<0.001) 3 days after the last injection.

Conclusions/Significance

Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.     

Characterization of a Cross-Reactive,Immunodominant and HLA-Promiscuous Epitope of Mycobacterium tuberculosis-Specific Major Antigenic Protein PPE68

PPE68 (Rv3873), a major antignic protein encoded by Mycobacteriun tuberculosis-specific genomic region of difference (RD)1, is a strong stimulator of peripheral blood mononuclear cells (PBMCs) obtained from tuberculosis patients and Mycobacterium bovis bacillus Calmette Guerin (BCG)-vaccianted healthy subjects in T helper (Th)1 cell assays, i.e. antigen-induced proliferation and interferon-gamma (IFN-γ) secretion. To confirm the antigen-specific recognition of PPE68 by T cells in IFN-γ assays, antigen-induced human T-cell lines were established from PBMCs of M. Bovis BCG-vaccinated and HLA-heterogeneous healthy subjects and tested with peptide pools of RD1 proteins. The results showed that PPE68 was recognized by antigen-specific T-cell lines from HLA-heteregeneous subjects. To further identify the immunodominant and HLA-promiscuous Th1-1 cell epitopes present in PPE68, 24 synthetic peptides covering the sequence of PPE68 were indivdually analyzed for HLA-DR binding prediction analysis and tested with PBMCs from M. bovis BCG-vaccinated and HLA-heterogeuous healthy subjects in IFN-γ assays. The results identified the peptide P9, i.e. aa 121-VLTATNFFGINTIPIALTEMDYFIR-145, as an immunodominant and HLA-DR promiscuous peptide of PPE68. Furthermore, by using deletion peptides, the immunodominant and HLA-DR promiscuous core sequence was mapped to aa 127-FFGINTIPIA-136. Interestingly, the core sequence is present in several PPE proteins of M. tuberculosis, and conserved in all sequenced strains/species of M. tuberculosis and M. tuberculosis complex, and several other pathogenic mycobacterial species, including M. leprae and M. avium-intracellulalae complex. These results suggest that the peptide aa 121–145 may be exploited as a peptide-based vaccine candidate against tuberculosis and other mycobacterial diseases.     

Erythroid Progenitor Cells Expanded from Peripheral Blood without Mobilization or Preselection: Molecular Characteristics and Functional Competence

Background

Continued development of in-vitro procedures for expansion and differentiation of erythroid progenitor cells (EPC) is essential not only in hematology and stem cell research but also virology, in light of the strict erythrotropism of the clinically important human parvovirus B19.

Methodology/Principal Findings

We cultured EPC directly from ordinary blood samples, without ex vivo stem cell mobilization or CD34+ cell in vitro preselection. Profound increase in the absolute cell number and clustering activity were observed during culture. The cells obtained expressed the EPC marker combination CD36, CD71 and glycophorin, but none of the lymphocyte, monocyte or NK markers. The functionality of the generated EPC was examined by an in vitro infection assay with human parvovirus B19, tropic for BFU-E and CFU-E cells. Following infection (i) viral DNA replication and mRNA production were confirmed by quantitative PCR, and (ii) structural and nonstructural proteins were expressed in >50% of the cells. As the overall cell number increased 100–200 fold, and the proportion of competent EPC (CD34+ to CD36+) rose from <0.5% to >50%, the in vitro culture procedure generated the EPC at an efficiency of >10 000-fold. Comparative culturing of unselected PBMC and ex vivo-preselected CD34+ cells produced qualitatively and quantitatively similar yields of EPC.

Conclusions/Significance

This approach yielding EPC directly from unmanipulated peripheral blood is gratifyingly robust and will facilitate the study of myeloid infectious agents such as the B19 virus, as well as the examination of erythropoiesis and its cellular and molecular mechanisms.     

Interactions between Cells and Collagen V Molecules or Single Chains Involve Distinct Mechanisms

Acid-soluble and pepsin-treated collagen V were prepared from fetal human bones or human placenta, respectively, to be tested for potential cell adhesion promoting activity. Out of 14 different collagen I-adhering cell lines, 10 showed distinct adhesion to collagen V. In all cases adhesion was followed by spreading. The activities of intact and pepsin-solubilized collagen V were similar, suggesting that the cell binding sites are restricted to the triple-helical domain of the molecules. Cell adhesion was also induced by the unfolded form of collagen V and after separation of the α chains by heparin affinity chromatography. Isolated α2(V) chains, rich in RGD sequences, were more efficient than isolated α1(V) chains. However, cell adhesion to native or denatured collagen V did not proceed by the same molecular mechanisms as shown by cell adhesion inhibition experiments. Cell adhesion to native collagen V was insensitive to the presence of RGD-containing synthetic peptides while adhesion to denatured collagen V was inhibited by the peptides. Furthermore, the results strongly suggested a major role for α1α1 and α2β1 integrins in the RGD-independent cell adhesion to native collagen V. These data indicate that collagen V is a specific adhesive substrate for different cell types. It also suggests that distinct sets of RGD-dependent and RGD-independent receptors mediate cell attachment to unfolded and native collagen V, respectively. This mechanism is shared by at least the interstitial collagens I and VI, which supports the hypothesis that when included in the triple-helical conformation of collagens, RGD sequences are either not accessible to cells or exhibit specific conformations recognized by different integrins.     

Analysis of the α4β1 Integrin–Osteopontin Interaction

The integrin α4β1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via α4β1 using GST fusion proteins. We show that α4β1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the α4β1 binding region in OPN lies between amino acid residues 125 and 168 (aa125–168). This region contains the central RGD motif of OPN, which also interacts with integrins αvβ3, αvβ5, αvβ1, α8β1, and α5β1. Mutating the RGD motif to RAD had no effect on the interaction with α4β1. To define the binding site the region incorporating aa125–168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via α4β1, aa132–146, and aa153–168; of these only a synthetic peptide, SVVYGLR (aa162–168), derived from aa153–168 was able to inhibit α4β1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of α4β1, and the primary α4β1 binding site within OPN.     

Zoledronate Attenuates Angiogenic Effects of Angiotensin II-Stimulated Endothelial Progenitor Cells via RhoA and MAPK Signaling

Background

New vessel formation plays a pivotal role in the pathogenesis of neovascular-related diseases. Endothelial progenitor cells (EPCs) were found to contribute to neovascular-related diseases and interference with EPC neovascularization may be a novel target for these diseases. Zoledronate (Zol) was reported to exhibit anti-angiogenic effect. Basing on these evidences, we proposed that Zol may affect EPC function to exert novel anti-angiogenic effect. In this study, we therefore investigated the effects of Zol on multiple aspects of EPC function and explored the underlying mechanisms involved.

Methodology/Principal Findings

EPCs were cultured from bone marrow derived mononuclear cells. The potential effects of Zol on Angiotensin II (Ang II)-stimulated EPC proliferation, migration, adhesion, in vitro tube formation were investigated. The results showed that Ang II (1 µM) enhanced EPC migration, adhesion, in vitro tube formation but had no effect on cell proliferation. Zol (75 and 100 µM) inhibited proliferation of EPCs and 50 µM geranylgeranyol (GGOH) could reverse the decrease of EPC proliferation. We found for the first time that Zol (50–100 µM) dose dependently attenuated migration, adhesion, and in vitro tube formation of EPCs stimulated by Ang II. GGOH could reverse the attenuation of EPC function induced by Zol. However, Zol did not induce EPC apoptosis. In addition, the underlying mechanisms were determined. The results revealed that Zol markedly down-regulated active RhoA stimulated by Ang II and inhibited the phosphorylation of Erk1/2 and JNK. Moreover, RhoA silencing resulted in a notable inhibition of EPC in vitro tube formation, suggesting that RhoA suppression played a pivotal role in Zol antiangiogenic effect.

Conclusions/Significance

These findings suggested that Zol attenuated the promotion of EPC function stimulated by Ang II and exhibited novel antiangiogenic effect via RhoA and MAPK signaling. Thus, Zol may be served as a novel therapeutic agent for neovascular-related diseases treatment.     

Direct Detection of α-Synuclein Dimerization Dynamics: Single-Molecule Fluorescence Analysis

The aggregation of α-synuclein (α-Syn) is linked to Parkinson’s disease. The mechanism of early aggregation steps and the effect of pathogenic single-point mutations remain elusive. We report here a single-molecule fluorescence study of α-Syn dimerization and the effect of mutations. Specific interactions between tethered fluorophore-free α-Syn monomers on a substrate and fluorophore-labeled monomers diffusing freely in solution were observed using total internal reflection fluorescence microscopy. The results showed that wild-type (WT) α-Syn dimers adopt two types of dimers. The lifetimes of type 1 and type 2 dimers were determined to be 197 ± 3 ms and 3334 ± 145 ms, respectively. All three of the mutations used, A30P, E46K, and A53T, increased the lifetime of type 1 dimer and enhanced the relative population of type 2 dimer, with type 1 dimer constituting the major fraction. The kinetic stability of type 1 dimers (expressed in terms of lifetime) followed the order A30P (693 ± 14 ms) > E46K (292 ± 5 ms) > A53T (226 ± 6 ms) > WT (197 ± 3 ms). Type 2 dimers, which are more stable, had lifetimes in the range of several seconds. The strongest effect, observed for the A30P mutant, resulted in a lifetime 3.5 times higher than observed for the WT type 1 dimer. This mutation also doubled the relative fraction of type 2 dimer. These data show that single-point mutations promote dimerization, and they suggest that the structural heterogeneity of α-Syn dimers could lead to different aggregation pathways.     

RGD-Targeted Ultrasound Contrast Agent for Longitudinal Assessment of Hep-2 Tumor Angiogenesis In Vivo

Objective

To prepare arginine-glycine-aspartate (RGD)-targeted ultrasound contrast microbubbles (MBs) and explore the feasibility of their use in assessing dynamic changes in αvβ3 integrin expression in a murine model of tumor angiogenesis.

Methods

RGD peptides were conjugated to the surfaces of microbubbles via biotin-avidin linkage. Microbubbles bearing RADfK peptides were prepared as controls. The RGD-MBs were characterized using an Accusizer 780 and optical microscopy. The binding specificity of the RGD-MBs for ανβ3-expressing endothelial cells (bEnd.3) was demonstrated in vitro by a competitive inhibition experiment. In an in vivo study, mice bearing tumors of three different stages were intravenously injected with RGD-MBs and subjected to targeted, contrast-enhanced, high-frequency ultrasound. Subsequently, tumors were harvested and sectioned for immunofluorescence analysis of ανβ3 expression.

Results

The mean size of the RGD-MBs was 2.36 ± 1.7 μm. The RGD-MBs showed significantly higher adhesion levels to bEnd.3 cells compared to control MBs (P < 0.01). There was rarely binding of RGD-MBs to αvβ3-negative MCF-7 cells. Adhesion of the RGD-MBs to the bEnd.3 cells was significantly inhibited following treatment with anti-alpha(v) antibodies. The quantitative acoustic video intensity for high-frequency, contrast-enhanced ultrasound imaging of subcutaneous human laryngeal carcinoma (Hep-2) tumor xenografts was significantly higher in small tumors (19.89 ± 2.49) than in medium tumors (11.25 ± 2.23) and large tumors (3.38 ± 0.67) (P < 0.01).

Conclusions

RGD-MBs enable noninvasive in vivo visualization of changes in tumor angiogenesis during tumor growth in subcutaneous cancer xenografts.     

Receptor Binding Sites and Antigenic Epitopes on the Fiber Knob of Human Adenovirus Serotype 3

The adenovirus fiber knob causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s), the interaction of labeled cell membrane proteins to synthetic peptides covering the adenovirus type 3 (Ad3) fiber knob was studied. Peptide P6 (amino acids [aa] 187 to 200), to a lesser extent P14 (aa 281 to 294), and probably P11 (aa 244 to 256) interacted specifically with cell membrane proteins, indicating that these peptides present cell receptor binding sites. Peptides P6, P11, and P14 span the D, G, and I β-strands of the R-sheet, respectively. The other reactive peptides, P2 (aa 142 to 156), P3 (aa 153 to 167), and P16 (aa 300 to 319), probably do not present real receptor binding sites. The binding to these six peptides was inhibited by Ad3 virion and was independent of divalent cations. We have also screened the antigenic epitopes on the knob with recombinant Ad3 fiber, recombinant Ad3 fiber knob, and Ad3 virion-specific antisera by enzyme-linked immunosorbent assay. The main antigenic epitopes were presented by P3, P6, P12 (aa 254 to 269), P14, and especially the C-terminal P16. Peptides P14 and P16 of the Ad3 fiber knob were able to inhibit Ad3 infection of cells.     

Mechanism of Membrane Permeation Induced by Synthetic β-Hairpin Peptides

We have investigated the membrane destabilizing properties of synthetic amphiphilic cationic peptides, MAX1 and MAX35, which have the propensity to form β-hairpin structures under certain conditions, and a control non-β-hairpin-forming peptide MAX8V16E. All three peptides bind to liposomes containing a mixture of zwitterionic POPC and negatively charged POPS lipids as determined by Zeta potential measurements. Circular dichroism measurements indicated folding of MAX1 and MAX35 in the presence of the POPC/POPS liposomes, whereas no such folding was observed with MAX8V16E. There was no binding or folding of these peptides to liposomes containing only POPC. MAX1 and MAX35 induced release of contents from negatively charged liposomes, whereas MAX8V16E failed to promote solute release under identical conditions. Thus, MAX1 and MAX35 bind to, and fold at the surface of negatively charged liposomes adopting a lytic conformation. We ruled out leaky fusion as a mechanism of release by including 2 mol % PEG-PE in the liposomes, which inhibits aggregation/fusion but not folding of MAX or MAX-induced leakage. Using a concentration-dependent quenching probe (calcein), we determined that MAX-induced leakage of liposome contents was an all-or-none process. At MAX1 concentrations, which cause release of ∼50% of the liposomes that contain small (R_h <1.5 nm) markers, only ∼15% of those liposomes release a fluorescent dextran of 40 kDa. A multimeric model of the pore is presented based on these results. Atomistic molecular dynamics simulations show that barrels consisting of 10 β-hairpin MAX1 and MAX35 peptides are relatively more stable than MAX8V16E barrels in the bilayer, suggesting that barrels of this size are responsible for the peptides lytic action.     

VGF Peptide Profiles in Type 2 Diabetic Patients’ Plasma and in Obese Mice

To address the possible involvement of VGF peptides in obesity and diabetes, we studied type 2 diabetes (T2D) and obese patients, and high-fat diet induced obese mice. Two VGF peptides (NAPP-19 and QQET-30) were identified in human plasma by HPLC-ESI-MS. The VGF C-terminus, the above two cleaved peptides, and the TLQP-21 related peptide/s were studied using ELISA and immunohistochemistry. In euglycemic patients, plasma NAPPE and TLQP like peptides were significantly reduced with obesity (74±10 vs. 167±28, and 92±10 vs. 191±19 pmol/ml, mean+SEM, n = 10 and 6, obese vs. normal BMI, respectively, p<0.03). Upon a standard glucose load, a distinct response was shown for VGF C-terminus, TLQP and QQET-like (ERVW immunoreactive) peptides in euglycemic normal BMI patients, but was virtually abolished in euglycemic obese, and in T2D patients independently of BMI. High-fat diet induced obese mice showed reduced plasma VGF C-terminus, NAPPE and QQET-like (ERVW) peptide/s (3±0.2 vs. 4.6±0.3, 22±3.5 vs. 34±1.3, and 48±7 vs. 100±7 pmol/ml, mean+SEM, n = 8/group, obese vs. slim, respectively, p<0.03), with a loss of the response to glucose for all VGF peptides studied. In immunohistochemistry, TLQP and/or VGF C-terminus antibodies labelled VGF containing perikarya in mouse celiac ganglia, pancreatic islet cells and thin beaded nerve fibres in brown adipose tissues, with fewer in white adipose tissue. Upon the glucose load, tyrosine hydroxylase and VGF C-terminus immunoreactive axons became apparent in pancreatic islets of slim animals, but not in obese animals. Alltogether, a significant loss of VGF peptide immunoreactivity and/or their response to glucose was demonstrated in obese patients, with or without T2D, in parallel with a similar loss in high-fat diet induced obese mice. An involvement of VGF in metabolic regulations, including those of brown and/or white adipose tissues is underlined, and may point out specific VGF peptides as potential targets for diagnosis and/or treatment.     

Increased Circulating Endothelial Apoptotic Microparticle to Endothelial Progenitor Cell Ratio Is Associated with Subsequent Decline in Glomerular Filtration Rate in Hypertensive Patients

Background

Recent research indicates hypertensive patients with microalbuminuria have decreased endothelial progenitor cells (EPCs) and increased levels of endothelial apoptotic microparticles (EMP). However, whether these changes are related to a subsequent decline in glomerular filtration rate (GFR) remains unclear.

Methods and Results

We enrolled totally 100 hypertensive out-patients with eGFR ≥30 mL/min/1.73 m². The mean annual rate of GFR decline (△GFR/y) was −1.49±3.26 mL/min/1.73 m² per year during the follow-up period (34±6 months). Flow cytometry was used to assess circulating EPC (CD34⁺/KDR⁺) and EMP levels (CD31⁺/annexin V⁺) in peripheral blood. The △GFR/y was correlated with the EMP to EPC ratio (r = −0.465, p<0.001), microalbuminuria (r = −0.329, p = 0.001), and the Framingham risk score (r = −0.245, p = 0.013). When we divided the patients into 4 groups according to the EMP to EPC ratio, there was an association between the EMP to EPC ratio and the ΔGFR/y (mean ΔGFR/y: 0.08±3.04 vs. −0.50±2.84 vs. −1.25±2.49 vs. −4.42±2.82, p<0.001). Multivariate analysis indicated that increased EMP to EPC ratio is an independent predictor of ΔeGFR/y.

Conclusions

An increased circulating EMP to EPC ratio is associated with subsequent decline in GFR in hypertensive patients, which suggests endothelial damage with reduced vascular repair capacity may contribute to further deterioration of renal function in patients with hypertension.     

Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin

The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs) are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE) and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin α_Vβ₃ blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. α_Vβ₃ blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving α_Vβ₃. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants.     

Effects of Aging on Genioglossus Motor Units in Humans

The genioglossus is a major upper airway dilator muscle thought to be important in obstructive sleep apnea pathogenesis. Aging is a risk factor for obstructive sleep apnea although the mechanisms are unclear and the effects of aging on motor unit remodeled in the genioglossus remains unknown. To assess possible changes associated with aging we compared quantitative parameters related to motor unit potential morphology derived from EMG signals in a sample of older (n = 11; >55 years) versus younger (n = 29; <55 years) adults. All data were recorded during quiet breathing with the subjects awake. Diagnostic sleep studies (Apnea Hypopnea Index) confirmed the presence or absence of obstructive sleep apnea. Genioglossus EMG signals were analyzed offline by automated software (DQEMG), which estimated a MUP template from each extracted motor unit potential train (MUPT) for both the selective concentric needle and concentric needle macro (CNMACRO) recorded EMG signals. 2074 MUPTs from 40 subjects (mean±95% CI; older AHI 19.6±9.9 events/hr versus younger AHI 30.1±6.1 events/hr) were extracted. MUPs detected in older adults were 32% longer in duration (14.7±0.5 ms versus 11.1±0.2 ms; P  =  0.05), with similar amplitudes (395.2±25.1 µV versus 394.6±13.7 µV). Amplitudes of CNMACRO MUPs detected in older adults were larger by 22% (62.7±6.5 µV versus 51.3±3.0 µV; P<0.05), with areas 24% larger (160.6±18.6 µV.ms versus 130.0±7.4 µV.ms; P<0.05) than those detected in younger adults. These results confirm that remodeled motor units are present in the genioglossus muscle of individuals above 55 years, which may have implications for OSA pathogenesis and aging related upper airway collapsibility.     

Discovery of Immunodominant B Cell Epitopes within Surface Pneumococcal Virulence Proteins in Pediatric Patients with Invasive Pneumococcal Disease

The identification of immunodominant B cell epitopes within surface pneumococcal virulence proteins in pediatric patients with invasive pneumococcal disease (IPD) is a valuable approach to define novel vaccine candidates. To this aim, we evaluated sera from children with IPD and age-matched controls against 141 20-mer synthetic peptides covering the entire sequence of major antigenic fragments within pneumococcal virulence proteins; namely, choline-binding protein D (CbpD), pneumococcal histidine triad proteins (PhtD and PhtE), pneumococcal surface protein A (PspA), plasminogen and fibronectin binding protein B (PfbB), and zinc metalloproteinase B (ZmpB). Ten immunodominant B cell epitopes were identified: CbpD-pep4 (amino acids (aa) 291–310), PhtD-pep11 (aa 88–107), PhtD-pep17 (aa 172–191), PhtD-pep19 (aa 200–219), PhtE-pep32 (aa 300–319), PhtE-pep40 (aa 79–98), PfbB-pep76 (aa 180–199), PfbB-pep79 (aa 222–241), PfbB-pep90 (aa 484–503), and ZmpB-pep125 (aa 431–450). All epitopes were highly conserved among different pneumococcal serotypes, and four of them were located within the functional zinc-binding domain of the histidine triad proteins PhtD and PhtE. Peptides CbpD-pep4, PhtD-pep19, and PhtE-pep40 were broadly recognized by IPD patient sera with prevalences of 96.4%, 92.9%, and 71.4%, respectively, whereas control sera exhibited only minor reactivities (<10.7%). Their specificities for IPD were 93.3%, 95%, and 96.7%; their sensitivities were 96.4%, 92.9%, and 71.4% and their positivity likelihood ratios for IPD were 14.5, 18.6, and 21.4, respectively. Furthermore, purified antibodies against CbpD-pep4, PhtD-pep19, and PhtE-pep40 readily bound on the surfaces of different pneumococcal serotypes, as assessed by FACS and immunofluorescence analysis. The identified immunodominant B cell epitopes provide a better understanding of immune response in IPD and are worth evaluation in additional studies as potential vaccine candidates.     

Electric Field-Induced Changes in Lipids Investigated by Modulated Excitation FTIR Spectroscopy

The effect of electric fields on dry oriented multibilayers of dimyristoylphosphatidylcholine (DMPC) was investigated by transmission Fourier transform infrared electric field modulated excitation (E-ME) spectroscopy. A periodic rectangular electric potential (0–150 V, 1.25 Hz, 28.4°C ± 0.2°C) was applied across the sample. To discriminate electric field-induced effects from possible temperature-induced effects resulting from a current flow (<1 pA) across the sample, corresponding temperature-modulated excitation (T-ME) measurements within the temperature uncertainty limits of ±0.2°C at 28.4°C were performed. T-ME induced reversible gauche defects in the hydrocarbon chains, whereas E-ME resulted in reversible compression of dry DMPC bilayers. Periodic variation of the tilt angle of the hydrocarbon chains is suggested. The degree of absorbance modulation in the CH-stretching region was found to be in the order of 1:700, corresponding to a variation of the bilayer thickness of Δz = 0.0054 nm. Using a series connection of capacitors as equivalent circuit of the cell resulted in E = (1.2 ± 0.7) × 10⁷ V/m for the electric field in DMPC. Young's elasticity modulus of DMPC could be calculated to be E = 2.2 × 10⁶ Pa ± 1.8 × 10⁶ Pa, which is in good agreement with published data obtained by electric field-dependent capacitance measurements.     

Diaryl azo derivatives as anti-diabetic and antimicrobial agents: synthesis,in vitro,kinetic and docking studies

In the present study, a series of azo derivatives (TR-1 to TR-9) have been synthesised via the diazo-coupling approach between substituted aromatic amines with phenol or naphthol derivatives. The compounds were evaluated for their therapeutic applications against alpha-glucosidase (anti-diabetic) and pathogenic bacterial strains E. coli (gram-negative), S. aureus (gram-positive), S. aureus (gram-positive) drug-resistant strain, P. aeruginosa (gram-negative), P. aeruginosa (gram-negative) drug-resistant strain and P. vulgaris (gram-negative). The IC₅₀ (µg/mL) of TR-1 was found to be most effective (15.70 ± 1.3 µg/mL) compared to the reference drug acarbose (21.59 ± 1.5 µg/mL), hence, it was further selected for the kinetic studies in order to illustrate the mechanism of inhibition. The enzyme inhibitory kinetics and mode of binding for the most active inhibitor (TR-1) was performed which showed that the compound is a non-competitive inhibitor and effectively inhibits the target enzyme by binding to its binuclear active site reversibly.