Competitive Inhibition Assay Research Articles

Evaluation of Efficacy of Radioimmunotherapy with 90Y-Labeled Fully Human Anti-Transferrin Receptor Monoclonal Antibody in Pancreatic Cancer Mouse Models.

ObjectivePancreatic cancer is an aggressive tumor and the prognosis remains poor. Therefore, development of more effective therapy is needed. We previously reported that 89Zr-labeled TSP-A01, an antibody against transferrin receptor (TfR), is highly accumulated in a pancreatic cancer xenograft, but not in major normal organs. In the present study, we evaluated the efficacy of radioimmunotherapy (RIT) with 90Y-TSP-A01 in pancreatic cancer mouse models.MethodsTfR expression in pancreatic cancer cell lines (AsPC-1, BxPC-3, MIAPaCa-2) was evaluated by immunofluorescence staining. 111In-labeled anti-TfR antibodies (TSP-A01, TSP-A02) were evaluated in vitro by cell binding assay with the three cell lines and by competitive inhibition assay with MIAPaCa-2. In vivo biodistribution was evaluated in mice bearing BxPC-3 and MIAPaCa-2 xenografts. Tumor volumes of BxPC-3 and MIAPaCa-2 were sequentially measured after 90Y-TSP-A01 injection and histological analysis of tumors was conducted.ResultsMIAPaCa-2 cells showed the highest TfR expression, followed by AsPC-1 and BxPC-3 cells. 111In-TSP-A01 and 111In-TSP-A02 bound specifically to the three cell lines according to TfR expression. The dissociation constants for TSP-A01, DOTA-TSP-A01, TSP-A02, and DOTA-TSP-A02 were 0.22, 0.28, 0.17, and 0.22 nM, respectively. 111In-TSP-A01 was highly accumulated in tumors, especially in MIAPaCa-2, but this was not true of 111In-TSP-A02. The absorbed dose for 90Y-TSP-A01 was estimated to be 8.3 Gy/MBq to BxPC-3 and 12.4 Gy/MBq to MIAPaCa-2. MIAPaCa-2 tumors treated with 3.7 MBq of 90Y-TSP-A01 had almost completely disappeared around 3 weeks after injection and regrowth was not observed. Growth of BxPC-3 tumors was inhibited by 3.7 MBq of 90Y-TSP-A01, but the tumor size was not reduced.Conclusion 90Y-TSP-A01 treatment achieved an almost complete response in MIAPaCa-2 tumors, whereas it merely inhibited the growth of BxPC-3 tumors. 90Y-TSP-A01 is a promising RIT agent for pancreatic cancer, although further investigation is necessary to improve the efficacy for the radioresistant types like BxPC-3.

Evaluation of Tc-99m Labeled Dimeric GX1 Peptides for Imaging of Colorectal Cancer Vasculature.

This study aimed to evaluate the potential of PEGylated dimeric GX1 peptide as a radiotracer for imaging of colorectal cancer vasculature in a LoVo tumor xenografted mouse model. The [(99m)Tc]PEG-(GX1)2 peptide was synthesized and identified. Confocal immunofluorescence analysis, receptor binding assay, and competitive inhibition assay were performed to evaluate the binding specificity and the receptor binding affinity of PEG-(GX1)2 to Co-human umbilical vein endothelial cells (HUVECs). Single photon emission computed tomography imaging and biodistribution were performed to evaluate the targeting ability of PEG-(GX1)2 to colorectal cancer. The studies in vitro suggested that PEG-(GX1)2 co-localized with Factor VIII in the perinuclear cytoplasm of Co-HUVECs and bound specifically to Co-HUVECs with a high affinity. The studies in vivo demonstrated that the targeting efficacy of PEG-(GX1)2 was superior to GX1. PEGylation improved the affinity and the targeting ability of the GX1 peptide. PEG-(GX1)2 is a more promising probe for imaging of colorectal vasculature than GX1.

Development and characterization of neutralizing monoclonal antibodies against canine distemper virus hemagglutinin protein.

Canine distemper virus (CDV) causes a serious multisystemic disease in dogs and other carnivora. Hemagglutinin (H) protein-specific antibodies are mainly responsible for protective immunity against CDV infection. In the present study, six neutralizing MAbs to the H protein of CDV were newly obtained and characterized by immunizing BALB/c mice with a recent Chinese field isolate. Competitive binding inhibition assay revealed that they recognized four distinct antigenic regions of the H protein. Immunofluorescence assay and western blotting showed that all MAbs recognize the conformational rather than the linear epitopes of the H protein. Furthermore, in immunofluorescence and virus neutralization assays, two of the MAbs were found to react only with the recent Chinese field isolate and not with older CDV strains, including vaccine strain Onderstepoort, indicating there are neutralization-related antigenic variations between the recent Chinese field isolate and the older CDV strains examined in this study. The newly established MAbs are useful for differentiating the expanding CDV strains and could be used in immunotherapy and immunodiagnosis against infection with CDV.

Detection of clenbuterol hydrochloride residuals in pork liver using a customized surface plasmon resonance bioanalyzer.

A surface plasmon resonance (SPR) immunoassay with an immobilization of self-assembled molecular identification membrane for the detection of residual Clenbuterol Hydrochloride (CLB) in pork liver was systematically investigated and experimentally validated for its high performance. SPR immunoassay with a regular competitive inhibition assay cannot be directly verified to detect CLB residuals. In this study, the binding of Au film with mercaptopropionic acid was investigated using the known form of the strong S-Au covalent bonds formed by the chemical radical of the mercaptopropionic acid and the Au film. After that, the immunoglobulin IgG of swine (SwIgG-CLB) was bonded with the mercaptopropionic acid by covalent -CO-NH- amide bonding. The modified comprehensive analysis of how the membrane structure works was introduced together with the customized SPR bioanalyzer. In order to evaluate the performance of this biomembrane structure, the concentrations of CLB-contained solutions of 0 ng•mL-1, 10 ng•mL-1, 20 ng•mL-1, 33.3 ng•mL-1, and 40 ng•mL-1 were prepared by adding CLB reagents into the solutions of CLB antibody (Clenbuterol Hydrochloride Antibody, CLB-Ab), successively and then the response unit (RU) was measured individually. Using the data collected from the linear CCD array, the fitting curve was established with the R-Square value of 0.9929. Correspondingly, the recovery rate ranged from 88.48% to 103.21% was experimented and the limit of detection of CLB in 1.26 ng•mL-1 was obtained efficiently. It was concluded that the detection method associated with biomembrane properties is expected to contribute much to the determination of residual CLB in pork liver quantitatively by using the customized SPR bioanalyzer.

Selection of peptide mimics of HIV-1 epitope recognized by neutralizing antibody VRC01.

The ability to induce anti-HIV-1 antibodies that can neutralize a broad spectrum of viral isolates from different subtypes seems to be a key requirement for development of an effective HIV-1 vaccine. The epitopes recognized by the most potent broadly neutralizing antibodies that have been characterized are largely discontinuous. Mimetics of such conformational epitopes could be potentially used as components of a synthetic immunogen that can elicit neutralizing antibodies. Here we used phage display technology to identify peptide motifs that mimic the epitope recognized by monoclonal antibody VRC01, which is able to neutralize up to 91% of circulating primary isolates. Three rounds of biopanning were performed against 2 different phage peptide libraries for this purpose. The binding specificity of selected phage clones to monoclonal antibody VRC01 was estimated using dot blot analysis. The putative peptide mimics exposed on the surface of selected phages were analyzed for conformational and linear homology to the surface of HIV-1 gp120 fragment using computational analysis. Corresponding peptides were synthesized and checked for their ability to interfere with neutralization activity of VRC01 in a competitive inhibition assay. One of the most common peptides selected from 12-mer phage library was found to partially mimic a CD4-binding loop fragment, whereas none of the circular C7C-mer peptides was able to mimic any HIV-1 domains. However, peptides identified from both the 12-mer and C7C-mer peptide libraries showed rescue of HIV-1 infectivity in the competitive inhibition assay. The identification of epitope mimics may lead to novel immunogens capable of inducing broadly reactive neutralizing antibodies.

Preclinical evaluation of ⁸⁹Zr-labeled human antitransferrin receptor monoclonal antibody as a PET probe using a pancreatic cancer mouse model.

Pancreatic cancer is aggressive and its prognosis remains poor; thus, effective therapy is urgently needed. Transferrin receptor (TfR) is highly expressed in pancreatic cancer and is considered to be a good candidate for molecular-targeted therapy. We radiolabeled and evaluated fully human anti-TfR monoclonal antibodies as a new PET probe for evaluating the biodistribution of the anti-TfR antibody in pancreatic cancer. TfR expression was evaluated in four human pancreatic cancer (MIAPaCa-2, PANC-1, BxPC-3, and AsPC-1) and murine A4 cell lines. The binding of 125I-labeled anti-TfR antibodies (TSP-A01, TSP-A02, TSP-A03, and TSP-A04) to MIAPaCa-2 cells was compared. 125I-labeled, 67Ga-labeled, and 89Zr-labeled TSP-A01 were evaluated by cell binding, competitive inhibition, and internalization assays. Biodistribution studies of 125I-labeled and 89Zr-labeled TSP-A01 were conducted in mice bearing MIAPaCa-2 and A4 tumors. PET imaging with [89Zr]TSP-A01 was carried out. MIAPaCa-2 cells showed the highest TfR expression in vitro and in vivo, whereas A4 cells showed no expression. Of the four antibodies, [125I]TSP-A01 showed the highest binding to MIAPaCa-2 cells, but not to A4 cells. The dissociation constant of TSP-A01 was 0.29 nmol/l. Uptake of radiolabeled TSP-A01, especially [89Zr]TSP-A01, was significantly higher in MIAPaCa-2 tumors than in A4 tumors. PET with [89Zr]TSP-A01 clearly visualized MIAPaCa-2 xenografts but not A4 xenografts. [89Zr]TSP-A01 is a promising PET probe for evaluating the accumulation of anti-TfR antibody in pancreatic cancer and has the potential to facilitate the selection of appropriate patients who would benefit from anti-TfR antibody therapy.

Identification of a conserved B-cell epitope on duck hepatitis A type 1 virus VP1 protein.

BackgroundThe VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized.Methods and ResultsTo characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1–positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope.Conclusions and SignificanceWe identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.

Substrate specificity of aquatic extracellular peptidases assessed by competitive inhibition assays using synthetic substrates

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 75:271-281 (2015) - DOI: https://doi.org/10.3354/ame01755 Substrate specificity of aquatic extracellular peptidases assessed by competitive inhibition assays using synthetic substrates Andrew D. Steen1,*, Jasmine P. Vazin2, Shane M. Hagen3, Katherine H. Mulligan2,4, Steven W. Wilhelm2 1Department of Earth and Planetary Sciences, University of Tennessee, 306 EPS Building, Knoxville, TN 37996, USA 2Department of Microbiology, University of Tennessee, M409 Walters Life Sciences, Knoxville, TN 37996, USA 3Department of Civil and Environmental Engineering, University of Tennessee, 325 John D. Tickle Building, Knoxville, TN 37996, USA 4Department of Biology, University of North Carolina at Chapel Hill, Coker Hall, CB #3280, Chapel Hill, NC 27599, USA *Corresponding author: asteen1@utk.edu ABSTRACT: The identities and biochemical properties of extracellular enzymes present in natural environments are poorly constrained. We used a series of competitive inhibition experiments with samples from a freshwater environment (the Tennessee River at Knoxville, TN, USA) and a marine environment (Bogue Sound, NC, USA) to characterize the range of substrate specificities of naturally occurring enzymes that hydrolyze L-leucine 7-amido-4-methylcoumarin (Leu-AMC), L‑proline-AMC (Pro-AMC), and L-arginine-AMC (Arg-AMC)—putative substrates for leucyl-aminopeptidase, prolyl-aminopeptidase, and arginyl-aminopeptidase, respectively. Extracellular peptidases which hydrolyzed Arg-AMC and Leu-AMC demonstrated affinity for up to 8 other amino acids, whereas those hydrolyzing Pro-AMC in the Tennessee River, and Arg-AMC at Bogue Sound, were more specific to proline and arginine, respectively. Patterns of substrate affinity showed that Leu-AMC (at both sampling sites) and Arg-AMC (at Bogue Sound) were primarily hydrolyzed by enzymes other than leucyl-aminopeptidase and arginyl-aminopeptidase, respectively. The set of naturally occurring peptidases in both environments showed greater affinity towards a subset of amino acids. These amino acids were on average larger, yielded more free energy from oxidation to CO2, and tended to be depleted in aged organic matter. These relationships indicate that pathways of amino acid diagenesis are at least partially controlled by the substrate specificities of the peptidases involved in protein degradation. KEY WORDS: Extracellular enzymes · Peptidases · Protein degradation · Heterotrophy · Amino acids Full text in pdf format PreviousCite this article as: Steen AD, Vazin JP, Hagen SM, Mulligan KH, Wilhelm SW (2015) Substrate specificity of aquatic extracellular peptidases assessed by competitive inhibition assays using synthetic substrates. Aquat Microb Ecol 75:271-281. https://doi.org/10.3354/ame01755 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 75, No. 3. Online publication date: July 20, 2015 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2015 Inter-Research.

Surface Epitope Coverage Affects Binding Characteristics of Bisphenol‐A Functionalized Nanoparticles in a Competitive Inhibition Assay

The biomolecule interface is a key element in immunosensor fabrication, which can greatly influence the sensor performance. This paper explores the effects of surface epitope coverage of small molecule functionalized nanoparticle on the apparent affinity (avidity) of antibody in a competitive inhibition assay using bisphenol‐A (BPA) as a model target. An unconventional two‐antibody competitive inhibition ELISA (ci‐ELISA) using thiolated BPA modified gold nanoparticles (cysBPAv‐AuNP) as a competing reagent was devised for this study. It was shown that the antibody complexation with cysBPAv‐AuNPs required a minimum number of surface epitopes on the nanoparticle to form a sufficiently strong interaction and reliable detection. The binding of cysBPAv‐AuNP to anti‐BPA antibodies, for limited antibody binding sites, was enhanced by a greater number of epitope‐modified nanoparticles (cysBPAv‐AuNP) as well as with higher epitope coverage. Increasing the molar concentration of epitope present in an assay enhanced the binding between anti‐BPA antibodies and cysBPAv‐AuNP. This implies that, to increase the limit of detection of a competitive inhibition assay, a reduced molar concentration of epitope should be applied. This could be achieved by either lowering the epitope coverage on each cysBPAv‐AuNP or the assay molar concentration of cysBPAv‐AuNP or both of these factors.

Substrate specificity of aquatic extracellular peptidases assessed by competitive inhibition assays using synthetic substrates

Substrate specificity of aquatic extracellular peptidases assessed by competitive inhibition assays using synthetic substrates

Plasmonic sensors for the competitive detection of testosterone.

The ability to detect small molecules in a rapid and sensitive manner is of great importance in the field of clinical chemistry, and the advancement of novel biosensors is key to realising point-of-care analysis for essential targets. Testosterone is an example of such a small molecule, the detection of which is important in both clinical analysis, and in the sporting industry to prevent doping. As such, a portable, rapid and sensitive test for testosterone would be of great use across a variety of analytical fields. Here we report on a novel method of testosterone analysis, based on a competitive inhibition assay utilising functionalized gold nanoparticles. Two sensing platforms are directly compared for the detection of testosterone based on both classical SPR and LSPR. We provide an in-depth discussion on the optimum surface chemistries needed to create a stable detection conjugate before successfully detecting testosterone using our newly developed portable 4-channel SPR instrument. We provide the first detailed study into the comparison of SPR and LSPR for the analysis of a small molecule, and provide a simple and effective method of testosterone detection that could potentially be extended to a variety of different analytes.

Inhibitory effect of Lactobacilli (Isolated from Tarkhineh Dough) on E.coli and Lis.monocytogenes colonization

Lactobacilli are the most widely used probiotics that can inhibit the attachment of pathogenic bacteria through colonization and adhesion to epithelial cells or producing inhibitory compounds. In this study, we aimed to evaluate adhesion ability of Lactobacillus isolated from Tarkhineh Dough to Caco-2 cells in presence or absence of food-born pathogenic bacteria like E.coli and Lis.monocytogenes. In addition, we studied inhibitory effects of these isolates on above pathogenic bacteria. 16 strains of Lactobacillus isolates were grown under anaerobic conditions at 37oC for 24h. The Caco-2 cells were used in assay of Lactobacillus isolates adhesion. The inhibitory effects of these strains against E.coli and Lis. monocytogenes, were evaluated by diffusion method through measuring the ability of Caco-2 cells for adhesion or production of inhibitory compounds. TD16 showed the strongest attachment among the other isolates with more than 2×104 CFU/Well. In a competitive inhibition assay, TD3, TD4, TD12, and TD16 strains indicated more than 50% inhibitory effect on adhesion ability of E.coli and Lis. monocytogenes to Caco-2 cells. TD7 and TD14 revealed similar effects on attachment of Lis. monocytogenes. Moreover, the antagonist effects of Lactobacillus isolates which analysed through well diffusion method showed that TD5 had the strongest inhibitory effect on Lis. monocytogenes while TD3 and TD6 had the highest inhibitory effect on E. coli. Production of antimicrobial compounds like bacteriocin may be related to the inhibitory effects of Lactobacillus isolates against pathogenic bacteria, so application of probiotic strains such as Lactobacilli can be one of the therapeutic approaches in bacterial infections.

Proton-associated sucrose transport of mammalian solute carrier family 45: an analysis in Saccharomyces cerevisiae.

The members of the solute carrier 45 (SLC45) family have been implicated in the regulation of glucose homoeostasis in the brain (SLC45A1), with skin and hair pigmentation (SLC45A2), and with prostate cancer and myelination (SLC45A3). However, apart from SLC45A1, a proton-associated glucose transporter, the function of these proteins is still largely unknown, although sequence similarities to plant sucrose transporters mark them as a putative sucrose transporter family. Heterologous expression of the three members SLC45A2, SLC45A3 and SLC45A4 in Saccharomyces cerevisiae confirmed that they are indeed sucrose transporters. [(14)C]Sucrose-uptake measurements revealed intermediate transport affinities with Km values of approximately 5 mM. Transport activities were best under slightly acidic conditions and were inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone, demonstrating an H(+)-coupled transport mechanism. Na(+), on the other hand, had no effect on sucrose transport. Competitive inhibition assays indicated a possible transport also of glucose and fructose. Real-time PCR of mouse tissues confirmed mRNA expression of SLC45A2 in eyes and skin and of SLC45A3 primarily in the prostate, but also in other tissues, whereas SLC45A4 showed a predominantly ubiquitous expression. Altogether the results provide new insights into the physiological significance of SLC45 family members and challenge existing concepts of mammalian sugar transport, as they (i) transport a disaccharide, and (ii) perform secondary active transport in a proton-dependent manner.

P134 Sensitisation To Cross-reactive Carbohydrate Determinants In Britain's Bakers: The Implications For Health Surveillance

Introduction and objectives The diagnosis of baker’s asthma as part of health surveillance schemes in some UK supermarkets relies on determining sensitisation to wheat flour and/or alpha amylase. Recently, data have emerged suggesting that serum IgE analysis in bakers may be complicated by the presence of clinically irrelevant specific IgE to cross-reactive carbohydrate determinants (CCDs), which are complex-type Asn (N)-linked glycan structures commonly formed in plants. Potentially this might lead to false positive flour specific IgE assays, which would have an impact on bakers undergoing surveillance. The aim of this study was to identify the prevalence of CCD sensitisation in UK bakers and investigate the impact of CCD specific IgE within a health surveillance setting. Methods Serum samples from UK bakers attending our occupational asthma clinic (n = 209) were analysed for specific IgE to CCD (MUXF 3 ) using ImmunoCAP assay (Phadia). Any positive samples were further tested for specific IgE to grass pollen, and competitive inhibition assays were used to determine cross-reactivity between CCD, flour and grass pollen. Results Sensitisation rates to CCD in our population of UK bakers were low (7%) despite high sensitisation rates to grass pollen (48%) and flour (60%). Sensitisation to CCD was more prevalent in those sensitised to either flour or grass than in those not sensitised to flour (11.5% vs 0%, p Conclusions Our study demonstrated that a minority of bakers were sensitised to CCD and, interestingly, this was associated with being co-sensitised with both flour and grass. It is unlikely that CCDs have major implications for the health surveillance for UK bakeries. In the minority of bakers with CCD specific IgE, there was some suggestion that CCDs may play a role in the cross-reactivity between flour and grass pollen, although in others it was less likely. Within the clinical setting, it may be prudent to measure CCD specific IgE in bakers who are co-sensitised to both flour and grass pollen.

HIV-1 group O integrase displays lower susceptibility to raltegravir and has a different mutational pathway for resistance than HIV-1 group M.

IntroductionHIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase gene, e.g. positions L74I, S153A, G163Q and T206S. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on susceptibility to integrase inhibitors and emergence of resistance associated mutations.Viruses and MethodsWe cloned and purified integrase proteins from each of HIV-1 Group O clades A (HIV-O/A) and B (HIV-O/B), a HIV-O divergent strain (HIV-O/Div), and HIV-1 group M (subtype B, HIV-M/B) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of varying concentrations of several INSTIs. The inhibition constant (Ki) and IC50 were calculated and compared for HIV-M and HIV-O integrases. Selections for resistance-related mutations were performed using cord blood mononuclear cells and increasing concentration of INSTIs.ResultsHIV-O integrase and viruses were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. During selection, we observed different pathways of resistance depending on the drug and clade. Mutations selected in HIV-O can be classified as follows: (1) mutations described for HIV-M such as T97A, Q148R, V151A/I (RAL), T66I, E92Q, E157Q (EVG) and M50I, R263K (DTG) and (2) signature mutations for HIV-O (i.e. not described in HIV-M) F121C (HIV-O/B for RAL), V75I (HIV-O/A for RAL) and S153V (HIV-O/A for DTG). Only the HIV-O/Div selected the Q148R mutation for RAL and R263K+M50I for DTG, as previously described for HIV-M. None of the HIV-O viruses selected either N155H or Y143C. The selection of the specific S153V mutation could be explained at the nucleotide level: HIV-O at this position contains an alanine and substitution of alanine to valine (153AGGC→153VGTC) is easier than substitution of alanine to tyrosine (153AGGC→153YTAC), with only a transversion needed instead of one transition plus one transversion.ConclusionsThis is the first report of susceptibility and resistance in vitro to INSTIs for HIV-O. Our study confirmed the impact of HIV-O polymorphism, on susceptibility to INSTIs and the emergence of resistance mutations.

Targeted gene delivery via N-acetylglucosamine receptor mediated endocytosis.

Receptor-mediated endocytosis is a promising approach of gene delivery into the target cells via receptor-ligand interaction. Vimentins at the cell surface are recently known to bind N-acetylglucosamine (GlcNAc) residue, therefore, the cell surfaces of vimentin-expressing cells could be targeted by using the GlcNAc residue as a specific ligand for receptor-mediated gene delivery. Here, we have developed polymeric gene delivery vectors, based on poly(ethylene oxide)(PEO) and poly(aspartamide), namely poly[(aspartamide)(diethylenetriamine)]-b-[PEO-(GlcNAc)] (PADPG) and poly[(aspartamide)(diethylenetriamine)]-b-[PEO] (PADP) to elucidate the efficiency of GlcNAc ligand for gene delivery through receptor mediated endocytosis. To determine the efficiency of these polymeric vectors for specific gene delivery, the DNA condensation ability of PADPG and PADP and the subsequent formation of polymeric nanoparticles were confirmed by gel retardation assay and transmission electron microscopy respectively. Both PADPG and PADP had lower cytotoxicity than polyethylenimine 25 K (PEI 25 K). However, their transfection efficiency was comparatively lower than PEI 25 K due to hydrophilic property of PEO in the vectors. To observe the stability of polymeric nanoparticles, the transfection of PADPG and PADP was carried out in the presence of serum. Favorably, the interfering effect of serum on the transfection efficiency of PADPG and PADP was also very low. Finally, when the cell specificity of these polymeric vectors was investigated, PADPG had high gene transfection in vimentin-expressing cells than vimentin-deficiency cells. The high transfection efficiency of PADPG was attributed to the GlcNAc in the polymeric vector which interact specifically with vimentin in the cells for the receptor-mediated endocytosis. The competitive inhibition assay further proved the receptor-mediated endocytosis of PADPG. Thus, this study demonstrates that conjugation of GlcNAc is an effective and rational way to prepare a suitable vector for targeted gene delivery to vimentin-expressing cells.

HIV-1 group O integrase displays lower enzymatic efficiency and higher susceptibility to raltegravir than HIV-1 group M subtype B integrase.

HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase coding region. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on enzyme function and susceptibility to integrase inhibitors. Accordingly, we cloned and purified integrase proteins from each of HIV-1 group O clades A and B, an HIV-O divergent strain, and HIV-1 group M (HIV-M, subtype B), used as a reference. To assess enzymatic function of HIV-O integrase, we carried out strand transfer and 3' processing assays with various concentrations of substrate (DNA target and long terminal repeats [LTR], respectively) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of various concentrations of several INSTIs. The inhibition constant (Ki) and 50% effective concentration (EC50) values were calculated for HIV-O integrases and HIV-O viruses, respectively, and compared with those of HIV-M. The results showed that HIV-O integrase displayed lower activity in strand transfer assays than did HIV-M enzyme, whereas 3' processing activities were similar to those of HIV-M. HIV-O integrases were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. Molecular modeling suggests that two key polymorphic residues that are close to the integrase catalytic site, 74I and 153A, may play a role in these differences.

Synthesis of N-glycan units for assessment of substrate structural requirements of N-acetylglucosaminyltransferase III

N-Acetylglucosaminyltransferase (GnT) III is a glycosyltransferase which produces bisected N-glycans by transferring GlcNAc to the 4-position of core mannose. Bisected N-glycans are involved in physiological and pathological processes through the functional regulation of their carrier proteins. An understanding of the biological functions of bisected glycans will be greatly accelerated by use of specific inhibitors of GnT-III. Thus far, however, such inhibitors have not been developed and even the substrate-binding mode of GnT-III is not fully understood. To gain insight into structural features required of the substrate, we systematically synthesized four N-glycan units, the branching parts of the bisected and non-bisected N-glycans. The series of syntheses were achieved from a common core trimannose, giving bisected tetra- and hexasaccharides as well as non-bisected tri- and pentasaccharides. A competitive GnT-III inhibition assay using the synthetic substrates revealed a vital role for the Manβ(1–4)GlcNAc moiety. In keeping with previous reports, GlcNAc at the α1,3-branch is also involved in the interaction. The structural requirements of GnT-III elucidated in this study will provide a basis for rational inhibitor design.

Surface plasmon resonance technique for directly probing the interaction of DNA and graphene oxide and ultra-sensitive biosensing

The binding of DNA with graphene oxide (GO) is important for applications in disease diagnosis, genetic screening, and drug discovery. The standard assay methods are mainly limited to indirect observation via fluorescence labeling. Here we report the use of surface plasmon resonance for direct sensing of DNA/GO binding. We show that this can be used for ultra-sensitive detection of single-stranded DNA (ssDNA). Furthermore, the results provide a more direct probe of DNA/GO binding abilities and confirm that hydrogen bonding plays a key role in the interaction between GO and ssDNA. This enables to a novel biosensor for highly sensitive and selective detection of ssDNA based on indirect competitive inhibition assay (ICIA). We report development of such a sensor with a linear dynamic range of 10−14–10−6M, a detection limit of 10fM and a high level of stability during repeated regeneration.

One-step synthesis of linear and cyclic RGD conjugated gold nanoparticles for tumour targeting and imaging

We developed an easy one-step method for the synthesis of linear and cyclic arginine-glycine-aspartate (RGD) conjugated gold nanoparticles (GNPs). Compared with the previously reported multistep process, our method is simple, time-efficient, and cost-effective. Interestingly, the RGD peptides can function as reducing, protecting and tumour targeting agents and the prepared GNPs possess good stability and good biocompatibility. The uptake of RGD–GNPs by human tumour cells was assessed by dark-field light scattering imaging and transmission electron microscopy (TEM). Both types of RGD–GNPs can specifically target the melanoma A375 cells better than the breast cancer MCF-7 cells, which express the integrin αvβ3 at low level. We also compared the cellular uptake ability between the linear and cyclic RGD conjugated GNPs quantitatively. In order to validate the cellular uptake mechanism, the competitive inhibition assay was performed. Various important results indicated that the activity of the RGD peptides to specifically target tumour cells was not influenced noticeably after conjugation with GNPs.