Amanda Skepu

RBBP6 Interacts with Multifunctional Protein YB-1 through Its RING Finger Domain, Leading to Ubiquitination and Proteosomal Degradation of YB-1

RBBP6 (retinoblastoma binding protein 6) is a 250-kDa multifunctional protein that interacts with both p53 and pRb and has been implicated in mRNA processing. It has also been identified as a putative E3 ubiquitin ligase due to the presence of a RING finger domain, although no substrate has been identified up to now. Using the RING finger domain as bait in a yeast two-hybrid screen, we identified YB-1 (Y-box binding protein 1) as a binding partner of RBBP6, localising the interaction to the last 62 residues of YB-1. We showed, furthermore, that both full-length RBBP6 and the isolated RING finger domain were able to ubiquitinate YB-1, resulting in its degradation in the proteosome. As a result, RBBP6 was able to suppress the levels of YB-1 in vivo and to reduce its transactivational ability. In the light of the important role that YB-1 appears to play in tumourigenesis, our results suggest that RBBP6 may be a relevant target for therapeutic drugs aimed at modifying the activity of YB-1.

Label-free in vitro toxicity and uptake assessment of citrate stabilised gold nanoparticles in three cell lines.

BackgroundReliable in vitro toxicity testing is needed prior to the commencement of in vivo testing necessary for hazard identification and risk assessment of nanoparticles. In this study, the cytotoxicity and uptake of 14 nm and 20 nm citrate stabilised gold nanoparticles (AuNPs) in the bronchial epithelial cell line BEAS-2B, the Chinese hamster ovary cell line CHO, and the human embryonic kidney cell line HEK 293 were investigated.MethodsCytotoxicity of the AuNPs was assessed via traditional XTT-, LDH-, and ATP-based assays, followed by cell impedance studies. Dark-field imaging and hyperspectral imaging were used to confirm the uptake of AuNPs into the cells.ResultsInterference of the AuNPs with the XTT- and ATP-based assays was overcome through the use of cell impedance technology. AuNPs were shown to be relatively non-toxic using this methodology; nevertheless CHO cells were the most sensitive cell type with 20 nm AuNPs having the highest toxicity. Uptake of both 14 nm and 20 nm AuNPs was observed in all cell lines in a time- and cell type-dependent manner.ConclusionsUsing the cell impedance and dark-field hyperspectral imaging technologies, it was possible to study the toxicity of AuNPs in different cell lines and show that these cells could internalize AuNPs with their subsequent intracellular aggregation. It was also possible to show that this toxicity would not correlate with the level of uptake but it would correlate with cell-type and the size of the AuNPs. Therefore, these two label-free methodologies used in this study are suitable for in vitro studies on the effects of AuNPs, and could present themselves as appropriate and valuable methodologies for future nanoparticle toxicity and uptake studies.

De-regulation of the RBBP6 isoform 3/DWNN in human cancers

Retinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. The human RBBP6 gene codes for three protein isoforms and isoform 3 consists of the domain with no name domain only whilst the other two isoforms, 1 and 2 comprise of additional zinc, RING, retinoblastoma and p53 binding domains. In this study, the localization of RBBP6 using RBBP6 variant 3 mRNA-specific probe was performed to investigate the expression levels of the gene in different tumours and find a link between RBBP6 and human carcinogenesis. Using FISH, real-time PCR and Western blotting analysis our results show that RBBP6 isoform 3 is down-regulated in human cancers. RBBP6 isoform 3 knock-down resulted in reduced G2/M cell cycle arrest whilst its over-expression resulted in increased G2/M cell cycle arrest using propidium iodide DNA staining. The results further demonstrate that the RBBP6 isoform 3 may be the cell cycle regulator and involved in mitotic apoptosis not the isoform 1 as previously reported for mice. In conclusion, these findings suggest that RBBP6 isoform 3 is a cell cycle regulator and may be de-regulated in carcinogenesis.

Gold nanoparticle based Tuberculosis immunochromatographic assay: The quantitative ESE Quanti analysis of the intensity of test and control lines

A rapid dual channel lateral flow assay for the detection of Mycobacterium Tuberculosis antibodies (MTB 38 kDa monoclonal antibody) in human blood was developed. The MTB 6-14-38 kDa fusion antigen and anti-Protein A were used as the capture proteins for test and control lines respectively. Protein A labeled 40 nm gold nanoparticles were used as the detection conjugate. Whole blood and serum were spiked with MTB 38 kDa monoclonal antibody to make a positive sample model. The developed lateral flow was used to test MTB 38 kDa monoclonal antibody, and a detection limit of 5 ng/ml was used as a cut-off concentration of the analytes. The effect of the analyte concentration on the MTB lateral flow assay was studied using the variation of the intensity obtained from a ESE Quanti reader. There was a direct correlation between the analyte (MTB 38 kDa monoclonal antibody) concentration and the intensity of the test line. The intensity increased with an increase in the concentration of MTB 38 kDa monoclonal antibody, while in contrast, an increase in analyte concentration decreased the intensity of the control line.

Facile Attachment of TAT Peptide on Gold Monolayer Protected Clusters: Synthesis and Characterization

High affinity thiolate-based polymeric capping ligands are known to impart stability onto nanosized gold nanoparticles. Due to the stable gold-sulfur bond, the ligand forms a protective layer around the gold core and subsequently controls the physicochemical properties of the resultant nanogold mononuclear protected clusters (AuMPCs). The choice of ligands to use as surfactants for AuMPCs largely depends on the desired degree of hydrophilicity and biocompatibility of the MPCs, normally dictated by the intended application. Subsequent surface modification of AuMPCs allows further conjugation of additional biomolecules yielding bilayer or multilayered clusters suitable for bioanalytical applications ranging from targeted drug delivery to diagnostics. In this study, we discuss our recent laboratory findings on a simple route for the introduction of Trans-Activator of Transcription (TAT) peptide onto the surface of biotin-derivatised gold MPCs via the biotin-strepavidin interaction. By changing the surface loading of biotin, controlled amounts of TAT could be attached. This bioconjugate system is very attractive as a carrier in intercellular delivery of various delivery cargoes such as antibodies, proteins and oligonucleotides.

Peptide-functionalized nanoparticles for the selective induction of apoptosis in target cells

AIM The study developed a prohibitin (PHB) targeted nanotherapy for selective induction of apoptosis in target cells. METHODS Gold nanoparticles (AuNPs) were bifunctionalized with adipose homing and proapoptotic peptides. The efficacy and mode of cell death induced by the AuNPs were investigated in vitro on three cancer cell lines. RESULTS The antiproliferative activity of PHB-targeted bifunctionalized AuNPs was more pronounced on cells that express the PHB receptor, and demonstrated receptor-mediated targeting and selectivity. The bifunctionalized AuNPs induced cell death by apoptosis. CONCLUSION The PHB-targeted nanotherapy under study could potentially be used for treatment of diseases that are characterized by overexpression of PHB. As such, further investigations will be conducted in vivo.

Targeted destruction of HIV positive cells

T protein modeling and virtual drug screening were performed to identify new inhibitors of Herpes virus DNA polymerase, a key enzyme in the viral replication cycle. Twelve potential inhibitors were identified, purchased and evaluated by plaque assays. Two compounds (Nos 2 and 9) were particularly active against HSV-1, HSV-2 and varicella-zoster virus (VZV) and one compound (No 3) inhibited more specifically human Cytomegalovirus (HCMV). These compounds exhibited activity against wild-type viruses and strains resistant to current antiviral agents, i.e. nucleoside and pyrophosphate analogues, with IC50 values between 3 and 10 μM. Furthermore, compounds 2 and 3 had good cellular permeability and metabolic stability as determined by parallel artificial membrane permeability assay (PAMPA) and microsomal stability assay, respectively. Derivatives of these compounds were also synthesized to evaluate their activity against representative strains of HSV-1, HSV-2, VZV and HCMV as well as their toxicity on different cell lines. One fluoro derivative of compound 2 (No 20) retained excellent activity against HSV-1, HSV-2 and VZV with a therapeutic index near 10 in Vero cells. In conclusion, we discovered a new class of non-nucleosidic Herpes virus inhibitors with in vitro activity against drug-resistant clinical isolates that warrant further pre-clinical studies.Human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) are global health problems. Since AIDS is not only a vital medical problem, but also a socioeconomic complication, increasing peoples knowledge and replacing their fatalistic belief by a non-fatalistic belief is important to decrease prevalence of the problems. The objective of the study is to examine beliefs about HIV/AIDS of pregnant women and to study the relationship between their belief and HIV/AIDS prevention behaviors. A cross-sectional structured and semi-structured interview with mixed method approach based survey was used. Convenience sampling technique was used to select study participants. Data was analyzed using SPSS V.15. Our result demonstrated that, out of 422 respondents 336 (79.6%) and 86 (20.4%) had a non-fatalistic and a fatalistic belief, respectively. Majority of the respondents 407 (96.4%) believe that HIV can be transmitted from mother to child and small proportion 15 (3.6%) don’t believe the transmission of HIV from mother to child. The findings showed that 20% of the women were fatalistic and that their beliefs about AIDS may affect prevention behaviors. Fatalistic believe is one of the factors influencing HIV/AIDS prevention behaviors, but it is a vital factor for health professionals to consider when developing future HIV/AIDS prevention strategies among fatalistic people.Method: SPARSHA NEPAL a community-led NGO of people living with HIV in Nepal initiated a “Community-based ART” from February 2005 with just 12 patients. In the beginning SPARSHA only had 1 Health Assistant and a weekly visit by Doctor along with basic medication service. With an overall management and implementation by the community of PLHIV, SPARSHA has managed to increase the number of patients on ART along with developing and empowering itself on crucial factors such as medical and technical know-how with the support from various Governmental and non-governmental partners and funding agencies.Methodology: Survey data of indicators of the four Information-Motivation-Behavioral skills (IMB) model’s latent constructs prevention information or knowledge, prevention motivation and prevention behavioral skills, as well as past exposure to violent living conditions (PEVLC)prevention was collected from students attending an HBCU. Exploratory principal component factor analysis and Cronbach’s alpha test were performed to identify the factorial structure of the PEVLC questionnaire and reliability of the violent exposure subscales, respectively. Confirmatory factor analysis was performed to estimate the overall model fit indices and the magnitude of effects of prevention motivation, PEVLC prevention and prevention information or knowledge on the prevention behavior of the students.I early stages of HIV infection, even if the infection is controlled and asymptomatic, several macrophages are loosened from granuloma, carry the mycobacteria within them, and help disseminate the infection through haematogenous route. By this time mycobacteremia may or may not cause clinical symptoms. In the current communication, we report findings of a study carried out on a cohort of 60 HIV-1 infected patients attending antiretroviral clinic at All India Institute of Medical Sciences, New Delhi, India. After taking informed consent from the caes blood and sputum samples were collected and processed, inoculated for culture examination under aseptic conditions, incubated at 37°C. Mycobacteremia was detectable in 40.7% of subjects whereas sputum cultures were positive in 48%. The rate of mycobacteremia was slightly higher in asymptomatic patients (44.4%, 8 of 18) than symptomatic patients (39%, 16 of 41). However, sputum positivity was higher in symptomatic cases (50%, 16 of 32) as compared to asymptomatic (44.4%, 8 of 18). Six (33.3%) asymptomatic cases had mycobacterial growth in both blood and sputum samples. Our study shows that HIV-1 positive cases, (symptomatic/asymptomatic), must be investigated for tuberculosis and blood culture has equal TB detection rate, if not higher, than sputum culture. Blood culture examination is desirable, specially in those patients who are asymptomatic and can not expectorate.I Russia 780,000 HIV-infected people registered and 130,000 people died. In St. Petersburg, recorded 50,000 cases from 1987 to 2011 and 8,500 people died. The main causes of death are generalized tuberculosis, rarely other opportunistic diseases and chronic viral hepatitis (CVH) cirrhosis. Every year in Russia revealed 60-65,000 “new cases” of HIV-infection and in St. Petersburg 2500 3000 per year. In St. Petersburg based on cumulative data from 2006, CVH with cirrhosis diagnosed in 200,000 patients, including patients with HIV infection. CVH patients with HIV infection have a rapidly progressive course disease development with a greater risk of liver failure, cirrhosis and hepatocellular carcinoma (HCC).Results: Overall, 26.8% (95% Confidence Interval [CI]: 23.4-30.2) of pregnant women did not know their HIV status. 9.2% of these women had high risk behaviors, 15% no health coverage, 24.2% were more than 35 years old, 64.4% were non-Hispanic White and 37.8% were college graduates. After controlling for cofounders, pregnant women with more than 35 years old (Odd Ratio [OR] =2.5, 95%CI: 1.4-4.4), self-identify as non-Hispanic White (OR=2.1, 95%CI: 1.2-3.8), no health coverage (OR=2.6, 95%CI: 1.4-4.8), and college degree (OR=1.6, 95%CI: 1.1-2.6) were significantly more likely to be unaware of their HIV status.H stigma persists to be a major public health challenge in Ethiopia. This study examines knowledge about HIV/ AIDS and factors explaining stigmatizing attitude towards people living with the virus based on demographic and health survey data collected from 15,786 women in 2011. The result shows that considerable percentage of rural women had inadequate knowledge about HIV/AIDS. The likelihood of having adequate knowledge about HIV/AIDS was significantly higher for relatively better educated women and lower in Afar, Somali, and Gambella regions and Dire Dawa City. Women with higher level of education and better access to media had lower likelihood of stigmatizing people living with the virus. Besides, respondents with adequate knowledge about HIV/AIDS had lower likelihood of stigmatization. The results entail that HIV/AIDS stigma in Ethiopia is partly explained by the level of knowledge of the people about HIV/AIDS and socio-cultural factors that shape their perception of the epidemic and infected persons. Hence, awareness raising campaign that considers the socio-cultural context in which stigma occurs is required to eliminate stigmatization.

Targeted destruction of HIV-positive cells

HIV/AIDS is now a global epidemic that has become the leading infectious killer of adults worldwide. Although antiretroviral (ARV) therapy has dramatically improved the quality of life and increased the life expectancy of those infected with HIV but frequency of dosing and drug toxicity as well as the development of viral resistance pose additional limitations. The rapidly expanding field of nanotechnology has vast potential to radically advance the treatment and prevention of HIV/AIDS. Nanoparticles can provide improved drug delivery, by virtue of their small size, robustness, safety, multimodality or multifunctionality.

Abstract 1265: RbBP6 Isoform 3 (DWNN) is a p53 stabilizer in arsenic trioxide-induced apoptosis in human cancer cell lines

Domain With No Name (DWNN) has a ubiquitin-like fold and shares 22% similarity with ubiquitin. This suggests its significant importance in cell homeostasis and protein degradation in a human body. The presence of a ubiquitin-like fold suggests a role similar to ubiquitin and RbBP6 may be involved in ubiquitin ligase-like activities. Furthermore RbBP6 interacts with both p53 and pRb which are key components of cell cycle regulation. These two tumour suppressors are key to the understanding the function of the RbBP6. It has been found that upon arsenic trioxide-induced treatment RbBP6 isoform 3 (DWNN) is up-regulated. In this study the role of the DWNN on p53-dependent apoptosis was investigated. Over-expressions of RbBP6 isoforms was achieved by transfection of recombinant isoforms using Metafectene Easy. Apoptosis was induced using arsenic trioxide and staurosporine. Apoptosis was then measured using APOPercentage and flow cytometry. The expression of RbBP6 gene products and p53 was determined using both real-time PCR and western blotting. Up-regulation of RbBP6 was observed in As2O3-induced apoptosis in cancer cells. Over-expression of the RbBP6 isoform 1 intensified As2O3-induced apoptosis. MTT assay showed an IC50 of 40µM for As2O3. When this concentration was used to induce apoptosis in human cancer cells and this resulted in induced expression of RbBP6 isoform 1 not the isoform 3. Over-expression of RbBP6 isoform 3 though resulted in stabilization of p53 protein. The apoptosis that was observed was also accompanied by the expression of p53. This study suggests that the DWNN may not be directly involved in the prosecution of apoptosis but may be involved through p53 stabilization. This study suggests that the RbBP6 may be a p53 stabilizer in contrast to mdm2, which is a negative regulator of p53. It will be very important to also determine the relationship between RbBP6 and pRb in both cell cycle and apoptosis Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1265.