Knowledge and awareness of human papillomavirus infection and human papillomavirus vaccine among Kazakhstani women attending gynecological clinics.

Cervical cancer remains one of the top causes of cancer-related morbidity and mortality all over the world. Currently, however, there are no published studies to assess the knowledge of HPV and cervical cancer in Kazakhstan. This study aimed to assess the awareness of HPV, the knowledge of HPV as a cause of cervical cancer, and the awareness of HPV vaccination among Kazakhstani women visiting gynecological clinics across the country. In addition, the study aimed to identify the factors associated with the awareness of HPV and the HPV vaccine and knowledge of HPV as a major cause of cervical cancer. This was a cross-sectional survey-based study with 2,272 women aged between 18-70 years attending gynecological clinics, who were administered paper-based questionnaires. Data analysis included descriptive statistics consisting of mean values, standard deviations, and frequencies, where applicable. Differences in categorical variables between groups were analyzed using the Chi-square test with a significance value of <0.005. Crude odds ratio (OR) and adjusted odds ratio (AOR) with 95% corresponding confidence intervals were calculated in regression analysis using univariate and multivariable logistic regression models. The mean age of participants was 36.33±10.09 years. More than half (53%) of the participants had been screened for cervical cancer. Among those who were aware of HPV, 46% knew that HPV causes cervical cancer and 52% were aware of the HPV vaccine. The key factors related to outcome variables were age, ethnicity, education, family, number of deliveries, and menarche. From a subgroup analysis, results from the HPV test and Pap smear test were factors related to dependent variables such as awareness of HPV and awareness of HPV vaccination.

The Distribution and Prevalence of High-Risk HPV Genotypes Other than HPV-16 and HPV-18 among Women Attending Gynecologists' Offices in Kazakhstan.

Cervical cancer represents a great burden to public health of women. This study aimed to obtain a nationwide genotyping survey and analysis of high risk-HPV including those that are caused by HPV types other than HPV-16 and HPV-18, among women in Kazakhstan. This study was conducted based on the collection of survey and cervical swabs of 1645 women across the country. The samples were genotyped for high-risk HPV types based on real-time PCR methods. Collected data was analyzed with the focus on high-risk HPV types other than HPV-16 and -18. Infection was present in 22% of women who participated in the study. The most prevalent types were HPV-31 among single infections and HPV-68 among multiple infections. Conclusively, despite the lack of attention high-risk HPV types beyond HPV-16 and -18 get in attempts of cervical cancer prevention in Kazakhstan, their prevalence is high and plays a large role in cervical cancer epidemiological situation.

Genetic factors associated with obesity risks in a Kazakhstani population.

OBJECTIVES: There is limited published literature on the genetic risks of chronic inflammatory related disease (eg, obesity and cardiovascular disease) among the Central Asia population. The aim is to determine potential genetic loci as risk factors for obesity for the Kazakhstani population. SETTING: Kazakhstan. PARTICIPANTS: One hundred and sixty-three Kazakhstani nationals (ethnic groups: both Russians and Kazakhs) were recruited for the cross-sectional study. Linear regression models, adjusted for confounding factors, were used to examine the genetic associations of single nucleotide polymorphisms (SNPs) in 19 genetic loci with obesity (73 obese/overweight individuals and 90 controls). RESULTS: Overall, logistic regression analyses revealed genotypes C/T in CRP (rs1205), A/C in AGTR1 (rs5186), A/G in CBS (rs234706), G/G in FUT2 (rs602662), A/G in PAI-1 (rs1799889), G/T (rs1801131) and A/G (rs1801133) in MTHFR genes significantly decrease risk of overweight/obesity. After stratification for ethnicity, rs234706 was significantly associated with overweight/obesity in both Russians and Kazakhs, while rs1800871 was significant in Kazakhs only. CONCLUSIONS: This study revealed that variations in SNPs known to be associated with cardiovascular health can also contribute to the risks of developing obesity in the population of Kazakhstan.

Prevalence of high-risk human papillomavirus infection among Kazakhstani women attending gynecological outpatient clinics.

OBJECTIVES: To conduct a nationwide high-risk human papillomavirus (HR-HPV) infection genotyping analysis of women attending gynecological clinics and identify factors associated with HR-HPV infection. METHODS: A cross-sectional survey-based study with 759 participants. Demographics, lifestyle, and medical history data were collected by questionnaire completed by gynecologists during patients' visits. Cervical swabs were used for HPV genotyping using AmpliSens kit. Data analysis included descriptive statistics consisting of mean values, standard deviations, and frequencies, where applicable. Ordinal logistic regression was performed to identify factors associated with HPV infection status. RESULTS: The mean age of participants was 36.51 ± 10.09 years. The majority of participants were aged 26-35 years. Less than half of the women (39%) were HPV positive; 26% had single HR-HPV, and 13% had multiple HR-HPV infection. The most prevalent HR-HPV genotypes were HPV-16 (54%), HPV-51 (7%), HPV-68 (7%), and HPV-18 (6%). Ordinal logistic regression demonstrated that older age, not being single, and having a history of sexually transmitted infections, decrease the odds of HPV infection. CONCLUSION: This study identified high prevalence of HR-HPV among Kazakhstani women. Our results showed that adding HPV testing to compulsory cervical cancer screening in Kazakhstan could improve the screening program and decrease cervical cancer rates.

Factors associated with cervical cancer screening behaviour of women attending gynaecological clinics in Kazakhstan: A cross-sectional study.

OBJECTIVE: Although cervical cancer could be prevented through medical screening, it remains one of the top causes of cancer-related morbidity and mortality all over the world. A number of factors may contribute to cervical cancer screening behaviour of women. The aim of this study was to investigate factors related to cervical cancer screening behaviour of women in Kazakhstan. METHODS: This was a cross-sectional survey-based study with a total of 1189 participants. Women attending gynaecological clinics aged between 18 and 70 years were administered paper-based questionnaires about their awareness of cervical cancer, the associated risk factors, and cervical cancer screening. Student t test or Wilcoxon rank-sum test and chi-square test or Fisher's exact test, where appropriate, were used to determine associations with categorical independent variables. RESULTS: The mean age of participants was 36.5 ± 10.1 years. Less than half (45.7%) of the participants had been screened for cervical cancer. The key factors related to the cervical cancer screening behaviour of women in this study included age, having a larger number of children, regular menstrual function, awareness of Pap smear test, and free screening programme for cervical cancer, and the causal association of human papillomavirus with cervical cancer. CONCLUSION: This study revealed several significant factors predicting screening behaviour in Kazakhstani women. To improve the rate of screening, there is a need to increase public knowledge and awareness of cervical cancer and opportunities for the free screening programme in the female population of Kazakhstan.

Association of rs12722 COL5A1 with pulmonary tuberculosis: a preliminary case-control study in a Kazakhstani population.

Lung cavitation is the classic hallmark of TB, which facilitates the disease development and transmission. It involves the degradation of lung parenchyma which is mainly made up of collagen fibers by metalloproteinases (MMPs) produced by activated monocyte-derived cells, neutrophils and stromal cells. The following population-based preliminary case-control study of adults with TB (50) and controls (112) without TB was used to investigate possible association between rs1800012 in COL1A1, rs12722 in COL5A1 genes and pulmonary TB in Kazakhstan. We examined 162 samples (50 cases and 112 controls) to study the associations between TB disease status and demographic variables along with single nucleotide polymorphisms related to COLA1 and COL5A1. The unadjusted χ2 and multivariable logistic regression was performed to find out relationships between SNP and other predictors. Preliminary findings suggest that there is a statistically significant association of age (AOR = 0.97, 95% CI:0.94-0.99, p value = 0.049), social status (AOR = 2.41, 95% CI:1.16-5.02, p value = 0.018), HIV status (AOR = 7.12, 95% CI:1.90-26.7, p value = 0.004) and heterozygous rs12722 SNP (AOR = 2.47, 95% CI:1.17-5.19, p value = 0.018) polymorphism of COL5A1 gene with TB susceptibility. The association of collagen genes with TB pathogenesis indicates that anti TB programs can include development of new drug regimens that include MMP inhibitors which has been found to be helpful in collagen remodeling and repair. Therapeutic targeting of MMPs will prevent extracellular matrix and collagen degradation and granuloma maturation.

Cervical cancer screening and prevention in Kazakhstan and Central Asia.

OBJECTIVE: To examine the state of cervical screening and prevention in Central Asian states, specifically Kazakhstan. RESULTS: In the five Central Asian countries that were formerly part of the Soviet Union (Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan, and Uzbekistan), cervical cancer incidence and mortality rates are far higher than those in most Western and high-income nations, and are increasing. Cervical cancer screening is available in all five countries, but is mainly opportunistic. Only Kazakhstan has a structured cytological screening program, from which screening coverage analysis is possible. CONCLUSION: Despite significant decreases in cervical cancer incidence and mortality in developed countries, the problem is still of great concern in these Central Asian countries and is attributed to poorly organized screening and the absence of vaccination programs.

The Efficacy of Therapeutic DNA Vaccines Expressing the Human Papillomavirus E6 and E7 Oncoproteins for Treatment of Cervical Cancer: Systematic Review.

Cervical cancer is recognized as a serious public health problem since it remains one of the most common cancers with a high mortality rate among women despite existing preventative, screening, and treatment approaches. Since Human Papillomavirus (HPV) was recognized as the causative agent, the preventative HPV vaccines have made great progress over the last few years. However, people already infected with the virus require an effective treatment that would ensure long-term survival and a cure. Currently, clinical trials investigating HPV therapeutic vaccines show a promising vaccine-induced T-cell mediated immune response, resulting in cervical lesion regression and viral eradication. Among existing vaccine types (live vector, protein-based, nucleic acid-based, etc.), deoxyribonucleic acid (DNA) therapeutic vaccines are the focus of the study, since they are safe, cost-efficient, thermostable, easily produced in high purity and distributed. The aim of this study is to assess and compare existing DNA therapeutic vaccines in phase I and II trials, expressing HPV E6 and E7 oncoproteins for the prospective treatment of cervical cancer based on clinical efficacy, immunogenicity, viral clearance, and side effects. Five different DNA therapeutic vaccines (GX-188E, VGX-3100, pNGVL4a-CRT/E7(detox), pNGVL4a-Sig/E7(detox)/HSP70, MEDI0457) were well-tolerated and clinically effective. Clinical implementation of DNA therapeutic vaccines into treatment regimen as a sole approach or in combination with conservative treatment holds great potential for effective cancer treatment.

"Chasing hope": Parents' perspectives on complementary and alternative interventions for children with autism in Kazakhstan.

LAY ABSTRACT: The article reports the findings of a qualitative research study on how and why parents of autistic children in Kazakhstan utilize complementary and alternative medicine. We found that parents turn to complementary and alternative medicine because of the lack of professional care options available to them and in pursuit for hope and opportunities for their children with ASD.

Association of genetic variations in the vitamin D pathway with susceptibility to tuberculosis in Kazakhstan.

Tuberculosis (TB) poses an important health challenge and a significant economic burden for Kazakhstan and in Central Asia. Recent findings show a number of immunological related processes and host Mycobacterium tuberculosis defense are impacted by a variety of genes of the human host including those that play a part in the vitamin D metabolism. We investigated the genetic variation of genes in the vitamin D metabolic pathway of a cohort 50 TB cases in Kazakhstan and compared them to 34 controls living in the same household with someone infected with TB. We specifically analyzed 11 SNPs belonging to the following genes: DHCR7, CYP2R1, GC-1, CYP24A1, CYP27A1, CYP27B1, VDR and TNFα. These genes play a number of different roles including synthesis, activation, delivery and binding of the activated vitamin D. Our preliminary results indicate significant association of VDR (vitamin D receptor) SNPs (rs1544410, BsmI, with OR = 0.425, CI 0.221-0.816, p = 0.009 and rs731236, TaqI with OR = 0.443, CI 0.228-0.859, p = 0.015) and CYP24A1 (rs6013897 with OR = 0.436, CI 0.191-0.996, p = 0.045) with TB. Interaction of genetic variation of VDR and CYP24A1 may impact susceptibility to TB. The findings provided initial clues to understand individual genetic differences in relation to susceptibility and protection to TB.

Human Papillomavirus Infection and Cervical Cancer: Epidemiology, Screening, and Vaccination-Review of Current Perspectives.

Viral infections contribute as a cause of 15-20% of all human cancers. Infection by oncogenic viruses can promote different stages of carcinogenesis. Among many types of HPV, around 15 are linked to cancer. In spite of effective screening methods, cervical cancer continues to be a major public health problem. There are wide differences in cervical cancer incidence and mortality by geographic region. In addition, the age-specific HPV prevalence varies widely across different populations and showed two peaks of HPV positivity in younger and older women. There have been many studies worldwide on the epidemiology of HPV infection and oncogenic properties due to different HPV genotypes. However, there are still many countries where the population-based prevalence has not yet been identified. Moreover, cervical cancer screening strategies are different between countries. Organized cervical screening programs are potentially more effective than opportunistic screening programs. Nevertheless, screening programs have consistently been associated with a reduction in cervical cancer incidence and mortality. Developed countries have achieved such reduced incidence and mortality from cervical cancer over the past 40 years. This is largely due to the implementation of organized cytological screening and vaccination programs. HPV vaccines are very effective at preventing infection and diseases related to the vaccine-specific genotypes in women with no evidence of past or current HPV infection. In spite of the successful implementation of the HPV vaccination program in many countries all over the world, problems related to HPV prevention and treatment of the related diseases will continue to persist in developing and underdeveloped countries.

First year medical students' learning style preferences and their correlation with performance in different subjects within the medical course.

BACKGROUND: Students commencing their medical training arrive with different educational backgrounds and a diverse range of learning experiences. Consequently, students would have developed preferred approaches to acquiring and processing information or learning style preferences. Understanding first-year students' learning style preferences is important to success in learning. However, little is understood about how learning styles impact learning and performance across different subjects within the medical curriculum. Greater understanding of the relationship between students' learning style preferences and academic performance in specific medical subjects would be valuable. METHODS: This cross-sectional study examined the learning style preferences of first-year medical students and how they differ across gender. This research also analyzed the effect of learning styles on academic performance across different subjects within a medical education program in a Central Asian university. A total of 52 students (57.7% females) from two batches of first-year medical school completed the Index of Learning Styles Questionnaire, which measures four dimensions of learning styles: sensing-intuitive; visual-verbal; active-reflective; sequential-global. RESULTS: First-year medical students reported preferences for visual (80.8%) and sequential (60.5%) learning styles, suggesting that these students preferred to learn through demonstrations and diagrams and in a linear and sequential way. Our results indicate that male medical students have higher preference for visual learning style over verbal, while females seemed to have a higher preference for sequential learning style over global. Significant associations were found between sensing-intuitive learning styles and performance in Genetics [ß = -0.46, B = -0.44, p < 0.01] and Anatomy [ß = -0.41, B = -0.61, p < 0.05] and between sequential-global styles and performance in Genetics [ß = 0.36, B = 0.43, p < 0.05]. More specifically, sensing learners were more likely to perform better than intuitive learners in the two subjects and global learners were more likely to perform better than sequential learners in Genetics. CONCLUSION: This knowledge will be helpful to individual students to improve their performance in these subjects by adopting new sensing learning techniques. Instructors can also benefit by modifying and adapting more appropriate teaching approaches in these subjects. Future studies to validate this observation will be valuable.

Evidence of evolutionary constraints that influences the sequence composition and diversity of mitochondrial matrix targeting signals.

Mitochondrial targeting signals (MTSs) are responsible for trafficking nuclear encoded proteins to their final destination within mitochondria. These sequences are diverse, sharing little amino acid homology and vary significantly in length, and although the formation of a positively-charged amphiphilic alpha helix within the MTS is considered to be necessary and sufficient to mediate import, such a feature does not explain their diversity, nor how such diversity influences target sequence function, nor how such dissimilar signals interact with a single, evolutionarily conserved import mechanism. An in silico analysis of 296 N-terminal, matrix destined MTSs from Homo sapiens, Mus musculus, Saccharomyces cerevisiae, Arabidopsis thaliana, and Oryza sativa was undertaken to investigate relationships between MTSs, and/or, relationships between an individual targeting signal sequence and the protein that it imports. We present evidence that suggests MTS diversity is influenced in part by physiochemical and N-terminal characteristics of their mature sequences, and that some of these correlated characteristics are evolutionarily maintained across a number of taxa. Importantly, some of these associations begin to explain the variation in MTS length and composition.

CSF-1 receptor-dependent colon development, homeostasis and inflammatory stress response.

The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) directly regulates the development of Paneth cells (PC) and influences proliferation and cell fate in the small intestine (SI). In the present study, we have examined the role of CSF-1 and the CSF-1R in the large intestine, which lacks PC, in the steady state and in response to acute inflammation induced by dextran sulfate sodium (DSS). As previously shown in mouse, immunohistochemical (IHC) analysis of CSF-1R expression showed that the receptor is baso-laterally expressed on epithelial cells of human colonic crypts, indicating that this expression pattern is shared between species. Colons from Csf1r null and Csf1(op/op) mice were isolated and sectioned for IHC identification of enterocytes, enteroendocrine cells, goblet cells and proliferating cells. Both Csf1r(-/-) and Csf1(op/op) mice were found to have colon defects in enterocytes and enteroendocrine cell fate, with excessive goblet cell staining and reduced cell proliferation. In addition, the gene expression profiles of the cell cycle genes, cyclinD1, c-myc, c-fos, and c-myb were suppressed in Csf1r(-/-) colonic crypt, compared with those of WT mice and the expression of the stem cell marker gene Lgr5 was markedly reduced. However, analysis of the proliferative responses of immortalized mouse colon epithelial cells (lines; Immorto-5 and YAMC) indicated that CSF-1R is not a major regulator of colonocyte proliferation and that its effects on proliferation are indirect. In an examination of the acute inflammatory response, Csf1r(+/-) male mice were protected from the adverse affects of DSS-induced colitis compared with WT mice, while Csf1r(+/-) female mice were significantly less protected. These data indicate that CSF-1R signaling plays an important role in colon homeostasis and stem cell gene expression but that the receptor exacerbates the response to inflammatory challenge in male mice.

Enhanced annealing of mismatched oligonucleotides using a novel melting curve assay allows efficient in vitro discrimination and restriction of a single nucleotide polymorphism.

BACKGROUND: Many SNP discrimination strategies employ natural restriction endonucleases to discriminate between allelic states. However, SNPs are often not associated with a restriction site and therefore, a number of attempts have been made to generate sequence-adaptable restriction endonucleases. In this study, a simple, sequence-adaptable SNP discrimination mechanism between a 'wild-type' and 'mutant' template is demonstrated. This model differs from other artificial restriction endonuclease models as cis- rather than trans-orientated regions of single stranded DNA were generated and cleaved, and therefore, overcomes potential issues of either inefficient or non-specific binding when only a single variant is targeted. RESULTS: A series of mismatch 'bubbles' that spanned 0-5-bp surrounding a point mutation was generated and analysed for sensitivity to S1 nuclease. In this model, generation of oligonucleotide-mediated ssDNA mismatch 'bubbles' in the presence of S1 nuclease resulted in the selective degradation of the mutant template while maintaining wild-type template integrity. Increasing the size of the mismatch increased the rate of mutant sequence degradation, until a threshold above which discrimination was lost and the wild-type sequence was degraded. This level of fine discrimination was possible due to the development of a novel high-resolution melting curve assay to empirically determine changes in Tm (~5.0°C per base-pair mismatch) and to optimise annealing conditions (~18.38°C below Tm) of the mismatched oligonucleotide sets. CONCLUSIONS: The in vitro 'cleavage bubble' model presented is sequence-adaptable as determined by the binding oligonucleotide, and hence, has the potential to be tailored to discriminate between any two or more SNPs. Furthermore, the demonstrated fluorometric assay has broad application potential, offering a rapid, sensitive and high-throughput means to determine Tm and annealing rates as an alternative to conventional hybridisation detection strategies.

Development of pluronic micelle-encapsulated doxorubicin and formaldehyde-releasing prodrugs for localized anticancer chemotherapy.

The chemotherapeutic agent doxorubicin forms drug-DNA adducts that are enhanced by formaldehyde-releasing prodrugs such as AN-9. One of the major limitations of doxorubicin is dose-limiting cardiotoxicity; therefore, the use of a targeting strategy that enables drug delivery and release at tumor sites is of great interest. The major aim of this study was to use the Pluronic-ultrasound delivery system to encapsulate doxorubicin and formaldehyde-releasing prodrugs within Pluronic micelles, and then use ultrasound to trigger controlled drug release from micelles. Pluronic micelles themselves were not stable upon dilution and required the use of a stabilizing agent DSPE-PEG2000 to form stable "mixed micelles." Following the separation of free doxorubicin, approximately 60% of doxorubicin remained encapsulated within mixed micelles with a retention half-life of approximately 12 h. The formaldehyde-releasing prodrugs, however, were not retained within mixed micelles, but could potentially be administered separately to doxorubicin-loaded micelles to achieve tumor-localized formation of doxorubicin-DNA adducts. The use of low-frequency, high-power ultrasound (20 kHz, 100 W/cm2) released 7-10% of doxorubicin from mixed micelles. Collectively, these results indicate that the Pluronic-ultrasound system could be used to deliver and release doxorubicin with the potential of forming cytotoxic DNA adducts at tumor sites with coadministrated formaldehyde-releasing prodrugs.

Colony stimulating factor-1 dependence of paneth cell development in the mouse small intestine.

BACKGROUND & AIMS: Paneth cells (PCs) secrete defensins and antimicrobial enzymes that contribute to innate immunity against pathogen infections within the mucosa of the small intestine. We examined the role of colony stimulating factor-1 (CSF-1) in PC development. METHODS: CSF-1-deficient and CSF-1 receptor (CSF-1R)-deficient mice and administration of neutralizing anti-CSF-1R antibody were used to study the requirement of CSF-1 for the development of epithelial cells of the small intestine. CSF-1 transgenic reporter mice and mice that express only the membrane-spanning, cell-surface CSF-1 isoform were used to investigate regulation by systemic versus local CSF-1. RESULTS: Mice deficient in CSF-1 or CSF-1R had greatly reduced numbers of mature PCs. PCs express the CSF-1R, and administration of anti-CSF-1R antibody to neonatal mice significantly reduced the number of PCs. Analysis of transgenic CSF-1 reporter mice showed that CSF-1-expressing cells are in close proximity to PCs. CSF-1/CSF-1R-deficient mice also had reduced numbers of the proliferating epithelial cell progenitors and lamina propria macrophages. Expression of the membrane-spanning, cell-surface CSF-1 isoform in CSF-1-deficient mice completely rescued the deficiencies of PCs, proliferating progenitors, and lamina propria macrophages. CONCLUSIONS: These results indicate local regulation by CSF-1 of PC development, either directly, in a juxtacrine/paracrine manner, or indirectly, by lamina propria macrophages. Therefore, CSF-1R hyperstimulation could be involved in hyperproliferative disorders of the small intestine, such as Crohn's disease and ulcerative colitis.

Mitochondrial gene therapy: an evaluation of strategies for the treatment of mitochondrial DNA disorders.

Mitochondrial DNA (mtDNA) disorders include a vast range of pathological conditions, despite each sharing a mutual inability to produce ATP efficiently as a result of defective oxidative phosphorylation. There is no clear consensus regarding an effective therapeutic approach, and consequently the current treatment strategies are largely supportive rather than curative. This is almost certainly the result of there being virtually no defined genotype-phenotype relationships among the mtDNA disorders; hence an identical mutation may be responsible for multiple phenotypes, or the same phenotype may be produced by different mutations. In light of this, the development of gene therapy to treat mtDNA disorders offers a promising approach, as it potentially circumvents the complication of the aforementioned genotype-phenotype inconsistency and ultimately the current inability to treat individual disorders with sufficient efficacy. Such an approach will ultimately require the combination of efficient mitochondrial targeting, and an effective therapeutic molecule. Although promising proof-of-principle developments in this field have been demonstrated, the realization of a successful therapeutic mitochondrial gene therapy strategy has not come to fruition. This review critiques the key approaches under development by discussing the theory underlying each strategy, and detailing the current progress made. We also emphasize the potential hurdles that must be acknowledged and overcome if the potential of a therapeutic gene therapy to treat mitochondrial DNA disorders is to be realized.

Differential intracellular distribution of DNA complexed with polyethylenimine (PEI) and PEI-polyarginine PTD influences exogenous gene expression within live COS-7 cells.

BACKGROUND: Polyethylenimine (PEI) is one of the most efficient and versatile non-viral vectors available for gene delivery. Despite many advantages over viral vectors, PEI is still limited by lower transfection efficiency compared to its viral counterparts. Considerable investigation is devoted to the modification of PEI to incorporate virus-like properties to improve its efficacy, including the incorporation of the protein transduction domain (PTD) polyarginine (Arg); itself demonstrated to facilitate membrane translocation of molecular cargo. There is, however, limited understanding of the underlying mechanisms of gene delivery facilitated by both PEI and PEI-bioconjugates such as PEI-polyarginine (PEI-Arg) within live cells, which once elucidated will provide valuable insights into the development of more efficient non-viral gene delivery vectors. METHODS: PEI and PEI-Arg were investigated for their ability to facilitate DNA internalization and gene expression within live COS-7 cells, in terms of the percentage of cells transfected and the relative amount of gene expression per cell. Intracellular trafficking of vectors was investigated using fluorescent microscopy during the first 5 h post transfection. Finally, nocodazole and aphidicolin were used to investigate the role of microtubules and mitosis, respectively, and their impact on PEI and PEI-Arg mediated gene delivery and expression. RESULTS: PEI-Arg maintained a high cellular DNA uptake efficiency, and facilitated as much as 2-fold more DNA internalization compared to PEI alone. PEI, but not PEI-Arg, displayed microtubule-facilitated trafficking, and was found to accumulate within close proximity to the nucleus. Only PEI facilitated significant gene expression, whereas PEI-Arg conferred negligible expression. Finally, while not exclusively dependent, microtubule trafficking and, to a greater extent, mitotic events significantly contributed to PEI facilitated gene expression. CONCLUSION: PEI polyplexes are trafficked by an indirect association with microtubules, following endosomal entrapment. PEI facilitated expression is significantly influenced by a mitotic event, which is increased by microtubule organization center (MTOC)-associated localization of PEI polyplexes. PEI-Arg, although enhancing DNA internalization per cell, did not improve gene expression, highlighting the importance of microtubule trafficking for PEI vectors and the impact of the Arg peptide to intracellular trafficking. This study emphasizes the importance of a holistic approach to investigate the mechanisms of novel gene delivery vectors.

Oncogenic properties of HIV-Tat in colorectal cancer cells.

With the advent of Highly-Active-Anti-Retroviral-Therapy (HAART), HIV patients can expect to live beyond 10-15 years following diagnosis. An unexpected result of increased survival is the emergence of opportunistic, oncogenic virus-associated cancers such as Burkitt's lymphoma (Epstein-Barr Virus), cervical cancer (Human Papilloma Virus) and Kaposi's sarcoma (Kaposi's sarcoma-associated herpesvirus) in this immuno-compromised population. Furthermore, there are reports of colorectal cancers (CRC) in long-term HIV-AIDS survivors. Compared to the general, non-immuno-compromised population, long-term AIDS patients have 4 and 3.3-fold increased risk of developing colorectal and anorectal cancer respectively. Unlike oncogenic virus-associated cancers, CRC is not known to have a viral etiology. Our study aimed to investigate one aspect of HIV infection and colorectal carcinogenesis. We proposed that the HIV transactivator protein Tat; a protein with known oncogenic properties that is secreted and can re-enter non-infected cells may have a role in CRC. Using two CRC cell lines, LIM1215 and LIM2537 we found that Tat inhibits epithelial cyto-differentiation, blocks apoptosis in vitro and accelerates tumour formation in vivo. In addition, Tat significantly increases in vitro migration in the absence of foetal calf serum. These properties underpin CRC, and as HIV infection is initiated in the gut lymphoid system, these data provide a basis for the increased incidence of CRC in long term AIDS patients.