User fees and access to ARV treatment for persons living with HIV/AIDS: implementation and challenges in Burkina Faso, a limited-resource country.

Access to antiretroviral (ARV) treatment remains a crucial problem for patients living with HIV/AIDS (PLWHA) in limited-resources countries. Some African countries have adopted the principle of providing ARV free of charge, but Burkina Faso opted for a direct out-of-pocket payment at the point of care delivery, with subsidized payments and mechanisms for the poorest populations to receive these services free of charge. Our objectives were to determine the proportion of PLWHA who pay for ARV and to identify the factors associated with ARV access in Burkina Faso. A cross-sectional study was performed in 13 public health facilities, 10 Nongovernmental Organizations and association health facilities, and three faith-based health facilities. In each facility, 20 outpatients receiving ARV were interviewed during a routine clinic visit. A multivariate analysis by logistic regression was performed. Among the expected 520 patients receiving ARV, 499 (96.0%) were surveyed. The majority of patients (79%) did not pay for their ARV treatment, thereby limiting cost recovery from patient payments. In a multivariate analysis, level of education and income were associated with free access to ARV. Patients with no education more frequently received free ARV than those who had received some level of education (OR 2.7, 95% CI [1.3-5.6]). Patients without any income or with less than US$10 per month were more likely to receive free ARV (OR 2.6 [95% CI 1.3-5.2]) than those who earned more than US$10 per month. However, 16% of patients without any income and 21% of those without employment paid for ARV, and the costs of drugs for opportunistic infections, food, and transport remained a burden for 85%, 91%, and 74%, respectively, of those who did not pay for ARV. Free access to a minimum care package for every PLWHA would enhance access to ARV.

Physical analysis of transcription preinitiation complex assembly on a class II gene promoter.

Transcription of protein-encoding genes by human RNA polymerase II requires multiple ancillary proteins (transcription factors). Interactions between these proteins and the promoter DNA of a viral class II gene (the major late transcription unit of adenovirus) were investigated by enzymatic and chemical footprinting. The experiments indicated that the assembly of functionally active RNA polymerase II-containing transcription preinitiation complexes requires a complete set of transcription factors, and that both specific protein-DNA and protein-protein interactions are involved. This allows individual steps along the transcription reaction pathway to be tested directly, thus providing a basis for understanding basic transcription initiation mechanisms as well as the regulatory processes that act on them.

Satisfaction with health services for STIs, HIV, AIDS among a high-risk population in West Africa.

The goal of this study was to develop an instrument and assess the degree of satisfaction regarding HIV/STIs services for women working in, associated with, or living in or nearby, prostitution environments. This study took place in seven West-African countries (Benin, Burkina Faso, Ghana, Mali, Niger, Senegal, Togo) participating in the West Africa Aids Program (AIDS3). A validated six-dimension questionnaire was used to interview 698 women. The main inclusion criterion was having had recourse to adapted services offered through the AIDS3 program in the last six months. Results showed that women surveyed are satisfied overall. Two dimensions scored low: 'Technical skills perceived' and 'Accessibility'. Regression analyses showed that those most satisfied were women who had used the adapted services many times and women connected with community groups. Although these results are consistent with results published previously in other contexts, they now allow the AIDS3 program to consider the voices of women rarely listened to: West-African women living and working in prostitution environments.

Tumor-suppression function of transcription factor USF2 in prostate carcinogenesis.

Although the transcription factor USF2 has been implicated in the regulation of cellular growth and proliferation, it is unknown whether alterations in USF2 contribute to tumorigenesis and tumor development. We examined the role of USF2 in prostate tumorigenesis. Western blot analysis revealed markedly decreased USF2 levels in three androgen-independent prostate cancer cell lines, PC-3, DU145, and M12, as compared to nontumorigenic prostate epithelial cells or the androgen-dependent cell line, LNCaP. Ectopic expression of USF2 in PC-3 cells did not affect the cell proliferation rate of PC-3 cells on plastic surfaces. However, it dramatically decreased anchorage-independent growth of PC-3 cells in soft agar (90-98% inhibition) and the invasion capability (80% inhibition) of PC-3 cells in matrix gel assay. Importantly, expression of USF2 in PC-3 cells inhibited the tumorigenicity of PC-3 cells in an in vivo nude mice xenograft model (80-90% inhibition). These results suggest that USF2 has tumor-suppression function. Consistent with its function in tumor suppression, we found that the USF2 protein is present in normal prostate epithelial cells but absent in 18 of 42 (43%) human prostate cancer tissues (P = 0.015). To further examine the functional role of USF2 in vivo, we generated mice with genetic deletion of USF2 gene. We found that USF2-null mice displayed marked prostate hyperplasia at a young age, suggesting that USF2 is involved in the normal growth and differentiation of prostate. Together, these studies demonstrate that USF2 has tumor-suppressor function and plays a role in prostate carcinogenesis.

Functional domains of the transcription factor USF2: atypical nuclear localization signals and context-dependent transcriptional activation domains.

USF is a family of basic helix-loop transcriptional factors that recognizes DNA-binding sites similar to those of the Myc oncoproteins. Here, various functional domains in the mouse USF2 protein were identified and characterized. Indirect immunofluorescence studies with transiently transfected cells revealed that both the basic region and the highly conserved USF-specific region (USR) are involved in the nuclear localization of USF2. Cotransfection assays with deletion mutants containing the DNA-binding domain of either USF2 or GAL4 identified two distinct transcriptional activation domains in USF2, the USR and the exon 5-encoded region. Activity of the exon 5 activation domain was detectable in both assay systems. Within USF2, however, its potency varied with the conformation induced by the surrounding regions, especially that encoded by alternatively spliced exon 4. In contrast, the USR activated transcription only in its natural context upstream of the USF2 basic region and only with reporter constructs containing the adenovirus major late minimal promoter but not the E1b minimal promoter. However, insertion of an initiator element downstream of the TATA box rescued the activity of the USR on the E1b-driven reporters. The USR therefore represents a new type of activation domain whose function depends very strongly on the core promoter context.

DNA-binding and transcriptional properties of human transcription factor TFIID after mild proteolysis.

The existence of separable functions within the human class II general transcription factor TFIID was probed for differential sensitivity to mild proteolytic treatment. Independent of whether TFIID was bound to DNA or free in solution, partial digestion with either one of a variety of nonspecific endoproteases generated a protease-resistant protein product that retained specific DNA recognition, as revealed by DNase I footprinting. However, in contrast to native TFIID, which interacts with the adenovirus major late (ML) promoter over a very broad DNA region, partially proteolyzed TFIID interacted with only a small region of the ML promoter immediately surrounding the TATA sequence. This novel footprint was very similar to that observed with the TATA factor purified from yeast cells. Partially proteolyzed human TFIID could form stable complexes that were resistant to challenge by exogenous templates. It could also nucleate the assembly of transcription complexes on the ML promoter with an efficiency comparable to that of native TFIID, yielding similar levels of transcription initiation. These results suggest a model in which the human TFIID protein is composed of at least two different regions or polypeptides: a protease-resistant "core," which by itself is sufficient for promoter recognition and basal transcriptional levels, and a protease-sensitive "tail," which interacts with downstream promoter regions and may be involved in regulatory processes.

Stability of transcription complexes on class II genes.

Commitment of a TATA box-driven class II gene to transcription requires binding of only one transcription factor, TFIID. Additional factors (TFIIB, TFIIE, and RNA polymerase II) do not remain associated with the TFIID-promoter complex during the course of transcription. This indicates that there are two intermediates along the transcription reaction pathway which may be potential targets for the regulation of gene expression.

The cellular transcription factor USF cooperates with varicella-zoster virus immediate-early protein 62 to symmetrically activate a bidirectional viral promoter.

The mechanisms governing the function of cellular USF and herpesvirus immediate-early transcription factors are subjects of considerable interest. In this regard, we identified a novel form of coordinate gene regulation involving a cooperative interplay between cellular USF and the varicella-zoster virus immediate-early protein 62 (IE 62). A single USF-binding site defines the potential level of IE 62-dependent activation of a bidirectional viral early promoter of the DNA polymerase and major DNA-binding protein genes. We also report a dominant negative USF-2 mutant lacking the DNA-binding domain that permits the delineation of the biological role of both USF-1 and USF-2 in this activation process. The symmetrical stimulation of the bidirectional viral promoter by IE 62 is achieved at concentrations of USF-1 (43 kDa) or USF-2 (44 kDa) already existing in cells. Our observations support the notion that cellular USF can intervene in and possibly target promoters for activation by a herpesvirus immediate-early protein.

The basic helix-loop-helix protein upstream stimulating factor regulates the cardiac ventricular myosin light-chain 2 gene via independent cis regulatory elements.

Previous studies have documented that 250 bp of the rat cardiac ventricular myosin light-chain 2 (MLC-2v) promoter is sufficient to confer cardiac muscle-specific expression on a luciferase reporter gene in both transgenic mice and primary cultured neonatal rat myocardial cells. Utilizing ligation-mediated PCR to perform in vivo dimethyl sulfate footprinting, the present study has identified protein-DNA interaction within the position from -176 to -165. This region, identified as MLE1, contains a core sequence, CACGTG, which conforms to the consensus E-box site and is identical to the upstream stimulating factor (USF)-binding site of the adenovirus major late promoter. Transient assays of luciferase reporter genes containing point mutations of the site demonstrate the importance of this cis regulatory element in the transcriptional activation of this cardiac muscle gene in ventricular muscle cells. The protein complex that occupies this site is capable of binding to HF-1a and PRE B sites which are known to be required for cardiac muscle-specific expression of rat MLC-2v and alpha-myosin heavy-chain genes, respectively. This study provides direct evidence that USF, a member of the basic helix-loop-helix leucine zipper family, binds to MLE1, HF-1a, and PRE B sites and suggests that it is a component of protein complexes that may coordinately control the expression of MLC-2v and alpha-myosin heavy-chain genes. The current study also provides evidence that USF can positively and negatively regulate the MLC-2v gene via independent cis regulatory elements.

Autoregulation of the human C/EBP alpha gene by stimulation of upstream stimulatory factor binding.

The human C/EBP alpha gene promoter shares significant sequence homology with that of the mouse but has a different mechanism of autoregulation. Activation of the murine promoter by direct binding of C/EBP alpha to a site within 200 bp of the transcriptional start was shown to elevate activity by approximately threefold (R. J. Christy, K. H. Kaestner, D. E. Geiman, and M. D. Lane, Proc. Natl. Acad. Sci. USA 88:2593-2597, 1991; K. Legraverend, P. Antonson, P. Flodby, and K. G. Xanthapoulos, Nucleic Acids Res. 21:1735-1742, 1993). Unlike its murine counterpart, the human C/EBP alpha gene promoter does not contain a cis element that binds the C/EBP alpha protein. Neither C/EBP alpha nor C/EBP beta (NF-Il-6) binds the human C/EBP alpha promoter within 437 bp. However, cotransfection studies show that C/EBP alpha stimulates transcription of a reporter gene driven by 437 bp of the C/EBP alpha promoter. Our studies show that the human C/EBP alpha protein stimulates USF to bind to a USF consensus element within C/EBP alpha promoter and activates it by two- to threefold. We propose that the human gene employs the ubiquitously expressed DNA-binding protein factor USF to carry out autoregulation. Autoregulation of the human C/EBP alpha promoter was abolished by deletion of the USF binding site, CACGTG. Expression of human C/EBP beta following transfection did not stimulate USF binding. These studies suggest a mechanism whereby tissue-specific autoregulation can be achieved via a trans-acting factor that is expressed in all cell types. Thus, direct binding of the C/EBP alpha protein to the promoter of the C/EBP alpha gene is not required for autoregulation.

Upstream stimulatory factor regulates major histocompatibility complex class I gene expression: the U2DeltaE4 splice variant abrogates E-box activity.

The tissue-specific expression of major histocompatibility complex class I genes is determined by a series of upstream regulatory elements, many of which remain ill defined. We now report that a distal E-box element, located between bp -309 and -314 upstream of transcription initiation, acts as a cell type-specific enhancer of class I promoter activity. The class I E box is very active in a neuroblastoma cell line, CHP-126, but is relatively inactive in the HeLa epithelial cell line. The basic helix-loop-helix leucine zipper proteins upstream stimulatory factor 1 (USF1) and USF2 were shown to specifically recognize the class I E box, resulting in the activation of the downstream promoter. Fine mapping of USF1 and USF2 amino-terminal functional domains revealed differences in their abilities to activate the class I E box. Whereas USF1 contained only an extended activation domain, USF2 contained both an activation domain and a negative regulatory region. Surprisingly, the naturally occurring splice variant of USF2 lacking the exon 4 domain, U2DeltaE4, acted as a dominant-negative regulator of USF-mediated activation of the class I promoter. This latter activity is in sharp contrast to the known ability of U2DeltaE4 to activate the adenovirus major late promoter. Class I E-box function is correlated with the relative amount of U2DeltaE4 in a cell, leading to the proposal that U2DeltaE4 modulates class I E-box activity and may represent one mechanism to fine-tune class I expression in various tissues.

Cell-type-dependent activity of the ubiquitous transcription factor USF in cellular proliferation and transcriptional activation.

USF1 and USF2 are basic helix-loop-helix transcription factors implicated in the control of cellular proliferation. In HeLa cells, the USF proteins are transcriptionally active and their overexpression causes marked growth inhibition. In contrast, USF overexpression had essentially no effect on the proliferation of the Saos-2 osteosarcoma cell line. USF1 and USF2 also lacked transcriptional activity in Saos-2 cells when assayed by transient cotransfection with USF-dependent reporter genes. Yet, there was no difference in the expression, subcellular localization, or DNA-binding activity of the USF proteins in HeLa and Saos-2 cells. Furthermore, Gal4-USF1 and Gal4-USF2 fusion proteins activated transcription similarly in both cell lines. Mutational analysis and domain swapping experiments revealed that the small, highly conserved USF-specific region (USR) was responsible for the inactivity of USF in Saos-2 cells. In HeLa, the USR serves a dual function. It acts as an autonomous transcriptional activation domain at promoters containing an initiator element and also induces a conformational change that is required for USF activity at promoters lacking an initiator. Taken together, these results suggest a model in which the transcriptional activity of the USF proteins, and consequently their antiproliferative activity, is tightly controlled by interaction with a specialized coactivator that recognizes the conserved USR domain and, in contrast to USF, is not ubiquitous. The activity of USF is therefore context dependent, and evidence for USF DNA-binding activity in particular cells is insufficient to indicate USF function in transcriptional activation and growth control.

In vitro susceptibility of Candida albicans clinical isolates to eight antifungal agents in Ouagadougou (Burkina Faso).

INTRODUCTION: In recent years, the infection Candida albicans infection worldwide has risen, and the incidence of resistance to traditional antifungal therapies is also increasing. The aim of this study was to evaluate in vitro susceptibility of C. albicans clinical isolates to eight antifungal agents in Ouagadougou. MATERIALS AND METHODS: A cross-sectional study was conducted from January 2013 to December 2015 at Yalgado Ouédraogo University Teaching Hospital. Two hundred seven strains have been isolated from 347 symptomatic patients received in different clinical services. Samples were cultured on Sabouraud Dextrose Agar supplemented with Cloramphenicol. Isolates were diagnosed as C. albicans using germ tube test, chlamydospore formation on Corn Meal Agar, and Api-Candida test (Biomérieux). Antifungal susceptibility testing was performed by disk diffusion method and isolates classified as susceptible, susceptible dose-dependent and resistant. RESULTS: Three hundred forty-seven (347) patients are included in this study. Two hundred and six (206) out of 347 collected samples (59.36%) were found positive for C. albicans. The strains were mostly isolated from vulvovaginal (49%) and oral infections (40.3%). The highest resistance rates of azoles were obtained with fluconazole (66.5%), itraconazole (52.3%) and ketoconazole (22.9%) when all clinical isolates were included. The resistance rates of fluconazole, itraconazole and ketoconazole remain highest for vulvovaginal and oral isolates. The rate of resistance to the polyene amphotericin B was 32.0% for all clinical isolates and was 56.4% for vulvovaginal strains. Resistance rate to nystatin was 6.3% for all clinical isolates. Cross-resistance analysis with data of all clinical strains revealed that the incidence of resistance to ketoconazole and itraconazole in fluconazole-resistant isolates was significantly higher than recorded for fluconazole-susceptible isolates. CONCLUSION: In vitro C. albicans antifungal susceptibility test in this study showed relatively high resistance to commonly and widely used azoles (fluconazole, ketoconazole). Most C. albicans clinical isolates were susceptible to nystatin.

Spatial organization of RNA polymerase II transcription in the nucleus.

In eukaryotic cells, mRNA synthesis is carried out by large, multifunctional complexes that are also involved in coordinating transcription with other nuclear processes. This survey focuses on the distribution and structural arrangement of these complexes within the nucleus, in relationship with the discrete positioning of particular chromosomal loci. To better understand the link between the spatial organization of the nucleus and the regulation of gene expression, it is necessary to combine information from biochemical studies with results from microscopic observations of preserved nuclear structures. Recent experimental approaches have made this possible. The subnuclear locations of specific chromosome loci, RNA transcripts, RNA polymerases, and transcription and pre-mRNA-processing factors can now be observed with computer-assisted microscopy and specific molecular probes. The results indicate that RNA polymerase II (RNAPII) transcription takes place at discrete sites scattered throughout the nucleoplasm, and that these sites are also the locations of pre-mRNA processing. Transcribing polymerases appear to be grouped into clusters at each transcription site. Cell cycle-dependent zones of transcription and processing factors have been identified, and certain subnuclear domains appear specialized for expression or silencing of particular genes. The arrangement of transcription in the nucleus is dynamic and depends on its transcriptional activity, with the RNAPII itself playing a central role in marshalling the large complexes involved in gene expression.

[Genetic diversity of P. falciparum and pathogenesis of the severe malarial anaemia in children under 5 years old in the province of Boulgou, Burkina Faso]. / Diversité génétique de Plasmodium falciparum et pathogénie de l'anémie palustre sévere chez des enfants de moins de 5 ans de la province du Boulgou, Burkina Faso.

The clinical presentation of malaria mainly the severe form may be related to Plasmodium falciparum msp-2 (merozoite surface protein 2) specific family To verify this hypothesis, during the high malaria transmission season in 2001; we analyzed the allelic polymorphism of the msp-2 gene of P. falciparum in children under 5 years old with different presentation of malaria in the regional Hospital and at community level in the Boulgou Province (Burkina Faso). A total of 405 children (107 severe malarial anaemia cases, 102 severe malaria cases without severe anaemia and 196 non severe malaria cases) were enrolled in the study. The frequencies of the FC27 were 89.2% in severe malarial anaemia children group, then 89.7% and 86.9% respectively in severe malaria non anaemic children cases and non severe malaria cases (P = 0.4). The frequencies of the 3D7 were 72.5%; 84.1% and 77% respectively severe malaria non anaemic children, severe malarial anaemia cases and non severe malaria cases (P = 0.7). The complexity of the FC27 genotypes was significantly higher in children with severe malaria (with and without severe anaemia) compared to the non severe malarial children (P << 0.001). No significant difference was pointed up in the complexity of the 3D7 genotypes.

Loss of USF transcriptional activity in breast cancer cell lines.

USF is a family of transcription factors that are structurally related to the Myc oncoproteins and also share with Myc a common DNA-binding specificity. USF overexpression can prevent c-Myc-dependent cellular transformation and also inhibit the proliferation of certain transformed cells. These antiproliferative activities suggest that USF inactivation could be implicated in carcinogenesis. To explore this possibility, we compared the activities of the ubiquitous USF1 and USF2 proteins in several cell lines derived from either normal breast epithelium or breast tumors. The DNA-binding activities of USF1 and USF2 were present at similar levels in all cell lines. In the non-tumorigenic MCF-10A cells, USF in general, and USF2 in particular, exhibited strong transcriptional activities. In contrast, USF1 and USF2 were completely inactive in three out of six transformed breast cell lines investigated, while the other three transformed cell lines exhibited loss of USF2 activity. Analyses in cells cultured from healthy tissue confirmed the transcriptional activity of USF in normal human mammary epithelial cells. These results demonstrate that a partial or complete loss of USF function is a common event in breast cancer cell lines, perhaps because, like Myc overexpression, it favors rapid proliferation.

Plasmids for the in vitro analysis of RNA polymerase II-dependent transcription based on a G-free template.

Described are a series of plasmids containing combinations of adenovirus-2 major late promoter elements, including consensus TATA box and initiator, upstream of G-free transcription cassettes of various lengths. These provide an assortment of tools for investigating both basal and regulated transcription mechanisms by in vitro transcription methods.

The USF proteins regulate transcription of the follicle-stimulating hormone receptor but are insufficient for cell-specific expression.

Expression of the FSH receptor (FSHR) is limited to granulosa cells of the ovary and Sertoli cells of the testis. Previous studies showed that an E box in the proximal promoter of the FSHR gene is required for transcription and that the predominant E box binding proteins are the ubiquitous transcription factors, upstream stimulatory factor 1 (USF1) and USF2. Through cotransfection analysis, we have shown that both wild-type and dominant negative forms of the USF proteins regulate the rat FSHR promoter and that transcriptional activation of FSHR required several domains within the amino-terminal portion of the USF proteins. Analysis of the FSHR promoter region using in vivo genomic footprinting indicated that the E box is occupied by proteins in Sertoli cells but not in cells that fail to express the receptor, despite the presence of the USF proteins. To help delineate the regions of the rat FSHR gene required for correct spatial and temporal expression, transgenic mice harboring two constructs containing variable amounts of 5'-flanking sequence (5,000 bp and 100 bp) were generated. Examination of 16 different transgenic lines revealed varied transgene expression profiles with multiple lines having different amounts of ectopic expression and two lines failing to express the transgene. In addition, little or no expression was observed in Sertoli cells. These studies indicate that additional regulatory sequences outside the region from -5,000 to +123 bp are needed for proper expression in Sertoli cells.

[Plasma lipids profile of non treated HIV infected adults in Ouagadougou (Burkina Faso)]. / Profil lipidique au cours de l'infection par le VIH à Ouagadougou - Burkina Faso: intérêt des marqueurs lipidiques dans le suivi de l'évolution de l'infection à VIH.

Many authors reported metabolic perturbations in connection with HIV infection. The aim of this studies was to determinate plasma lipids profile in non treated HIV infected adults in Ouagadougou (Burkina Faso). The results obtain with 187 HIV infected patients showed a significative (p < 10(-6)) high level of LDL, triglycerid, atherogenic indice; HDL was decreased. The atherogenic risk is increased with lymphocytes CD4 depletion. Plasma lipids levels must be consider in the choice of antiretroviral treatment.