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1.
Mufhandu HT Gray ES Madiga MC Tumba N Alexandre KB Khoza T Wibmer CK Moore PL Morris L Khati M 《Journal of virology》2012,86(9):4989-4999
Entry of human immunodeficiency virus type 1 (HIV-1) into cells is mediated by the virion surface envelope (Env) glycoproteins, making it a desirable target for antiretroviral entry inhibitors. We previously isolated a family of gp120 binding RNA aptamers and showed that they neutralized the infectivity of HIV-1. In this study, we assessed the activity of a shortened synthetic derivative of the B40 aptamer, called UCLA1, against a large panel of HIV-1 subtype C viruses. UCLA1 tightly bound to a consensus HIV-1 subtype C gp120 and neutralized isolates of the same subtype with 50% inhibitory concentrations (IC(50)s) in the nanomolar range. The aptamer had little toxicity in tests with cell lines and primary cells. Furthermore, it exhibited high therapeutic indices, suggesting that it may be effective at very low doses. Mapping of UCLA1 binding sites on gp120 revealed eight amino acid residues that modulated neutralization resistance. This included residues within the coreceptor binding site, at the base of the V3 loop, and in the bridging sheet within the conserved V1/V2 stem-loop of gp120. The aptamer was also shown to have synergistic effects with T20, a gp41 fusion inhibitor, and IgG1b12 (b12), an anti-CD4 binding site monoclonal antibody. These results suggest that UCLA1 may be suitable for development as a potent HIV-1 entry inhibitor. 相似文献
2.
Nqobile A. C. Ngubane Lionel Gresh Thomas R. Ioerger James C. Sacchettini Yanjia J. Zhang Eric J. Rubin Alexander Pym Makobetsa Khati 《PloS one》2013,8(11)
Bacterial cell wall components have been previously used as infection biomarkers detectable by antibodies. However, it is possible that the surface of the Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), also possesses molecules which might be non-antigenic. This makes the probing of biomarkers on the surface of M. tb cell wall difficult using antibodies. Here we demonstrate the use of phage display technology to identify peptides that bind to mycobacteria. We identified these clones using both random clone picking and high throughput sequencing. We demonstrate that random clone picking does not necessarily identify highly enriched clones. We further showed that the clone displaying the CPLHARLPC peptide which was identified by Illumina sequencing as the most enriched, binds better to mycobacteria than three clones selected by random picking. Using surface plasmon resonance, we showed that chemically synthesised CPLHARLPC peptide binds to a 15 KDa peptide from M.tb H37Rv whole cell lysates. These observations demonstrate that phage display technology combined with high-throughput sequencing is a powerful tool to identify peptides that can be used for investigating potential non-antigenic biomarkers for TB and other bacterial infections. 相似文献
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Nichole Cerutti Barry V. Mendelow Grant B. Napier Maria A. Papathanasopoulos Mark Killick Makobetsa Khati Wendy Stevens Alexio Capovilla 《The Journal of biological chemistry》2010,285(33):25743-25752
HIV-1 enters cells via interaction between the trimeric envelope (Env) glycoprotein gp120/gp41 and the host cell surface receptor molecule CD4. The requirement of CD4 for viral entry has rationalized the development of recombinant CD4-based proteins as competitive viral attachment inhibitors and immunotherapeutic agents. In this study, we describe a novel recombinant CD4 protein designed to bind gp120 through a targeted disulfide-exchange mechanism. According to structural models of the gp120-CD4 receptor complex, substitution of Ser60 on the CD4 domain 1 α-helix with Cys positions a thiol in proximity of the gp120 V1/V2 loop disulfide (Cys126–Cys196), satisfying the stereochemical and geometric conditions for redox exchange between CD4 Cys60 and gp120 Cys126, and the consequent formation of an interchain disulfide bond. In this study, we provide experimental evidence for this effect by describing the expression, purification, refolding, receptor binding and antiviral activity analysis of a recombinant two-domain CD4 variant containing the S60C mutation (2dCD4-S60C). We show that 2dCD4-S60C binds HIV-1 gp120 with a significantly higher affinity than wild-type protein under conditions that facilitate disulfide exchange and that this translates into a corresponding increase in the efficacy of CD4-mediated viral entry inhibition. We propose that targeted redox exchange between conserved gp120 disulfides and nucleophilic moieties positioned strategically on CD4 (or CD4-like scaffolds) conceptualizes a new strategy in the development of high affinity HIV-1 Env ligands, with important implications for therapy and vaccine development. More generally, this chalcogen substitution approach provides a general means of stabilizing receptor-ligand complexes where the structural and biophysical conditions for disulfide exchange are satisfied. 相似文献
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Neutralization of infectivity of diverse R5 clinical isolates of human immunodeficiency virus type 1 by gp120-binding 2'F-RNA aptamers 总被引:2,自引:0,他引:2
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Khati M Schüman M Ibrahim J Sattentau Q Gordon S James W 《Journal of virology》2003,77(23):12692-12698
Human immunodeficiency virus type 1 (HIV-1) has evolved a number of strategies to resist current antiretroviral drugs and the selection pressures of humoral and cellular adaptive immunity. For example, R5 strains, which use the CCR5 coreceptor for entry and are the dominant viral phenotype for HIV-1 transmission and AIDS pathogenesis, are relatively resistant to neutralization by antibodies, as are other clinical isolates. In order to overcome these adaptations, we raised nucleic acid aptamers to the SU glycoprotein (gp120) of the R5 strain, HIV-1(Ba-L). These not only bound gp120 with high affinity but also neutralized HIV-1 infectivity in human peripheral blood mononuclear cells (PBMCs) by more than 1,000-fold. Furthermore, these aptamers were able to neutralize the infectivity of R5 clinical isolates of HIV-1 derived from group M (subtypes A, C, D, E, and F) and group O. One aptamer defined a site on gp120 that overlaps partially with the conserved, chemokine receptor-binding, CD4-induced epitope recognized by monoclonal antibody 17b. In contrast to the antibody, the site is accessible to aptamer in the absence of CD4 binding. Neutralizing aptamers such as this could be exploited to provide leads in developing alternative, efficacious anti-HIV-1 drugs and lead to a deeper understanding of the molecular interactions between the virus and its host cell. 相似文献
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Pankaj Bhatt Saurabh Gangola Parul Chaudhary Priyanka Khati Govind Kumar Anita Sharma 《Bioremediation Journal》2019,23(1):42-52
During bacterial growth an increased mRNA level is usually linked with higher rate of metabolism related to biodegradation of an unusual compound. In this study, quantitative gene expression data derived from mRNA level reveal the presence of pesticide degrading genes in 6 bacterial isolates showing biodegradation of cypermethrin (SG2, SG4), sulfosulfuron (SA2, Sulfo3), and fipronil (FA3, FA4). A correlation existed between the level of esterase coding mRNA and mineralization of cypermethrin in SG2 and SG4. Similarly the level of EST coding mRNA increased with biodegradation of fipronil and sulfosulfuron in FA3, FA4, SA2, and Sulfo3. Expression of est gene was observed in all the bacterial strains, but their level of expression was different. Bacterial strains SG4 and Sulfo3 showed higher level of est gene expression as compared to SG2, FA3, FA4, and SA2 and was in the range of approximately 30- to 60-fold, respectively, in comparison to control. Expression of genes for aldehyde dehydrogenase was observed in SG4 and Sulfo3. We report co expression of aldh (1000?bp) and est (~550?bp) genes at the same time of pesticide induction/biodegradation. 相似文献
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对紫玉盘属8种,杯冠木属3种和Ellipeia属1种进行了叶的比较研究,以调查不同种之间解剖学的不同点,这对种的鉴别和了解它们的分类学意义可能是有用的。有意义的解剖学特征是:末端石细胞,表皮细胞的晶族,毛状体,在中脉和叶柄的薄壁组织内的短石细胞以及中脉和叶柄横切面的形状。有某些特征仅出现在某些种中,这对种的鉴别是有用的。结果亦显示这三个属的联系十分紧密。 相似文献
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Lia S. Rotherham Charlotte Maserumule Keertan Dheda Jacques Theron Makobetsa Khati 《PloS one》2012,7(10)
Background
Despite the enormous global burden of tuberculosis (TB), conventional approaches to diagnosis continue to rely on tests that have major drawbacks. The improvement of TB diagnostics relies, not only on good biomarkers, but also upon accurate detection methodologies. The 10-kDa culture filtrate protein (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) are potent T-cell antigens that are recognised by over 70% of TB patients. Aptamers, a novel sensitive and specific class of detection molecules, has hitherto, not been raised to these relatively TB-specific antigens.Methods
DNA aptamers that bind to the CFP-10.ESAT-6 heterodimer were isolated. To assess their affinity and specificity to the heterodimer, aptamers were screened using an enzyme-linked oligonucleotide assay (ELONA). One suitable aptamer was evaluated by ELONA using sputum samples obtained from 20 TB patients and 48 control patients (those with latent TB infection, symptomatic non TB patients, and healthy laboratory volunteers). Culture positivity for Mycobacterium tuberculosis (Mtb) served as the reference standard. Accuracy and cut-points were evaluated using ROC curve analysis.Results
Twenty-four out of the 66 aptamers that were isolated bound significantly (p<0.05) to the CFP-10.ESAT-6 heterodimer and six were further evaluated. Their dissociation constant (KD) values were in the nanomolar range. One aptamer, designated CSIR 2.11, was evaluated using sputum samples. CSIR 2.11 had sensitivity and specificity of 100% and 68.75% using Youden’s index and 35% and 95%, respectively, using a rule-in cut-point.Conclusion
This preliminary proof-of-concept study suggests that a diagnosis of active TB using anti-CFP-10.ESAT-6 aptamers applied to human sputum samples is feasible. 相似文献10.
An aptamer that neutralizes R5 strains of human immunodeficiency virus type 1 blocks gp120-CCR5 interaction 总被引:3,自引:0,他引:3
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We recently described the isolation and structural characterization of 2'-fluoropyrimidine-substituted RNA aptamers that bind to gp120 of R5 strains of human immunodeficiency virus type 1 and thereby potently neutralize the infectivity of phylogenetically diverse R5 strains. Here we investigate the physical basis of their antiviral action. We show that both N-linked oligosaccharides and the variable loops V1/V2 and V3 are not required for binding of one aptamer, B40, to gp120. Using surface plasmon resonance binding analyses, we show that the aptamer binds to the CCR5-binding site on gp120 in a relatively CD4-independent manner, providing a mechanistic explanation for its neutralizing potency. 相似文献