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Plasmodium resistance to Artemisinin Combination-based Therapies (ACT) in Southeast Asia is a major public health concern that is sporadically appearing in Africa. Senegal has shifted from malaria control to elimination plans. Given notable progresses obtained through robust strategic plans, it is still crucial to assess genetic variability of the Plasmodium falciparum artemisinin resistance gene marker Kelch13 (PfKelch13) in circulating field isolates. We here report an analysis of PfKelch13-propeller polymorphism in clinical isolates collected nine years after ACT introduction in five Senegalese regions with different malaria transmission settings. Sequencing of PfKelch13-propeller domain from 280 clinical isolates reveals that 16% (45/280) of the parasite population harbored variants. Dynamics of PfKelch13 variants reveals emerging, persistent but also disappearing mutations over time. In addition to the malaria epidemiology, our survey also shows the dynamics of PfKelch13 variants in different malaria transmission settings in Senegal. Despite the absence of PfKelch13 associated artemisinin resistance mutations, a shift from 86% to 68% of PfKelch13WT was observed when comparing parasites collected prior vs. post ACT intensive usage in Dakar a low malaria transmission area. All together, our data confirms the need to closely monitor PfKelch13 polymorphism to anticipate and or prevent emergence of P. falciparum resistance in Senegal.

Background Several genetic polymorphisms were reported to be prevalent among populations living in tropical endemic regions and induce protection against malaria. In this study, we investigated the prevalence of key malaria-protective polymorphisms in G6PD and HBB genes in a Senegalese population. Methods We performed a retrospective study in 323 samples from patients recruited in hospitals located in three different endemic areas where malaria episodes were confirmed. Patients enrolled were classified in two groups: severe (153 patients) and uncomplicated malaria (170 patients). PCR and DNA sequencing assessed host genetic polymorphisms in HBB and G6PD. Using a multivariate regression and additive model, estimates the impact of human HBB and G6PD polymorphisms on malaria incidence were performed. Results We identified 12 SNPs in HBB gene. Among them, 6 SNPs (rs7946748, rs7480526, rs10768683, rs35209591, HbS (rs334) and rs713040) were detected with high frequencies in global population. The MAF of the sickle cell HbS polymorphism was estimated to 0.026, 0.069 and 0.035; and HbC polymorphism was estimated to be 0, 0.009, 0.029, in SM, UM and CTR group respectively. The MAF of G6PD deficiency polymorphisms such as G6PD-202 G>A were 0.022, 0.032 and 0.018 in SM, UM and CTR, respectively. Analysis of selected HbS polymorphism showed significant association with protection against severe malaria with a significant p-value = 0.033 (OR 0.38, 95%CI: 0.16–0.91). Surprisingly, HbC polymorphism is not a protective variant in our population. Finally, we found that the selected SNPs were associated to biological parameters such as PNE, PNB and lymphocytes. Conclusion Our data report at the first time the prevalence of HBB and G6PD mutations in senegalese population. These deficiencies are very common in West Africa endemic regions such as Gambia, Mali and Burkina Faso. Our findings show the important role of genetic factors in malaria outcome and these genetic markers could be good tools for malaria diagnosis and prognosis. Keywords : severe malaria, HBB, G6PD, Polymorphisms, Senegal, severe malaria.

The availability of next-generation sequencing techniques is about to strongly modify the strategies of control of the viral safety of biologicals products. It is now possible to use the tools of metagenomics, which is the study of the microbial genetic sequences recovered directly from a given sample. In this setting, the sequence of all the nucleic acids species of the sample are determined and compared with those in databases. We describe the pipeline we are currently using and show comparison of its analytical sensitivity with that of polymerase chain reaction together with results of time course experiments in infected cells. We propose to test cell supernatants for viral genomes associated to capsids and testing cells for viral RNA transcripts as the hallmark for virus replication, and we suggest rules of interpretation of the results. We also discuss the applicability of next-generation sequencing for the control of raw materials.

Background Although mobilization of hematopoietic stem cells and hematopoietic progenitor cells can be achieved with a combination of granulocyte colony-stimulating factor and plerixafor (AMD3100), improving approaches for hematopoietic progenitor cell mobilization is clinically important.Design and Methods Heparan sulfate proteoglycans are ubiquitous macromolecules associated with the extracellular matrix that regulates biology of hematopoietic stem cells. We studied the effects of a new family of synthetic oligosaccharides mimicking heparan sulfate on hematopoietic stem cell mobilization. These oligosaccharides were administered intravenously alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 in mice. Mobilized hematopoietic cells were counted and phenotyped at different times and the ability of mobilized hematopoietic stem cells to reconstitute long-term hematopoiesis was determined by competitive transplantation into syngenic lethally irradiated mice followed by secondary transplantation.Results Mimetics of heparan sulfate induced rapid mobilization of B-lymphocytes, T-lymphocytes, hematopoietic stem cells and hematopoietic progenitor cells. They increased the mobilization of hematopoietic stem cells and hematopoietic progenitor cells more than 3-fold when added to the granulocyte colony-stimulating factor/AMD3100 association. Hematopoietic stem cells mobilized by mimetics of heparan sulfate or by the granulocyte colony-stimulating factor/AMD3100/mimetics association were as effective as hematopoietic stem cells mobilized by the granulocyte colony-stimulating factor/AMD3100 association for primary and secondary hematopoietic reconstitution of lethally irradiated mice.Conclusions This new family of mobilizing agents could alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 mobilize a high number of hematopoietic stem cells that were able to maintain long-term hematopoiesis. These results strengthen the role of heparan sulfates in the retention of hematopoietic stem cells in bone marrow and support the use of small glyco-drugs based on heparan sulfate in combination with granulocyte colony-stimulating factor and AMD3100 to improve high stem cell mobilization, particularly in a prospect of use in human therapeutics.

Heparan sulfate is a polysaccharide belonging to the glycaminoglycan family. It interacts with numerous proteins of the extracellular matrix, in particular cellular growth factors. The number of experimental protein– heparin sulfate complexes obtained by crystallography or nuclear magnetic resonance is limited. Alternatively, computational approaches can be employed. Generally, they restrain the conformation of the glycosidic rings and linkages in order to reduce the complexity of the problem. Modeling the interaction between protein and heparan sulfate is indeed challenging because of the large size of the fragment needed for a strong binding, the flexibility brought by the glycosidic rings and linkages and the high density of negative charges. We propose a two-step method based on molecular docking and molecular dynamics simulation. Molecular docking allows exploring the positioning of a rigid heparin sulfate fragment on the protein surface. Molecular dynamics refine selected docking models by explicitly representing solvent molecules and not restraining the polysaccharide backbone. The interaction of a hexamer of heparin sulfate was studied in interaction with fibroblast growth factor 2 and stromal cell-derived factor 1α. This approach shed light on the plasticity of the growth factors interacting with heparan sulfate. This approach can be extended to the study of other protein/glycosaminoglycan complexes.

Heparan sulfate is abundantly present in the extracellular matrix. As other glycosaminoglycans, it is synthesized in the Golgi apparatus and then exposed on the cell surface. The glucuronidase activity of human heparanase plays a major role in the structural remodeling of the extracellular matrix, which underlies cell migration, hence tumor invasion. Heparanase is therefore a major target for anti-cancer treatment. Several inhibitors of its enzymatic activity have been synthesized. However, their design is limited by the absence of experimental structure of the protein. Homology modeling is proposed based on the structure of the endoxylanase from Penicillium simplicissimum co-crystallized with a series of xylan oligosaccharide. The new heparanase model is consistent with the few experimental data suited for the validation of such work. Furthermore, the presence of natural substrates in the template structure allowed us to propose a binding model for a hydrolyzed heparin sulfate pentasaccharide. Several lysine residues have been identified to play a key role in binding to the anionic polysaccharide substrate. In addition, two phenylalanine residues are also potentially important for the interaction with the substrate. The enzymatic mechanism investigated in the light of this new model allows for the proposal of several amino acids that can influence the protonation state of the nucleophile and the proton donor. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 21–34, 2012.

Chicken embryo fibroblasts (CEF) express several growth arrest-specific (GAS) gene products in G0. In contact-inhibited cells, the expression of the most abundant of these proteins, the p20K lipocalin, is activated at the transcriptional level by C/EBPbeta. In this report, we describe the role of C/EBPbeta in CEF proliferation. We show that the expression of a dominant negative mutant of C/EBPbeta (designated Delta184-C/EBPbeta) completely inhibited p20K expression at confluence and stimulated the proliferation of CEF without inducing transformation. Mouse embryo fibroblasts nullizygous for C/EBPbeta had a proliferative advantage over cells with one or two functional copies of this gene. C/EBP inhibition enhanced the expression of the three major components of AP-1 in cycling CEF, namely c-Jun, JunD, and Fra-2, and stimulated AP-1 activity. In contrast, the over-expression of C/EBPbeta caused a dramatic reduction in the levels of AP-1 proteins. Therefore, C/EBPbeta is a negative regulator of AP-1 expression and activity in CEF. The expression of cyclin D1 and cell proliferation were stimulated by the dominant negative mutant of C/EBPbeta but not in the presence of TAM67, a dominant negative mutant of c-Jun and AP-1. CEF over-expressing c-Jun, and to a lesser extent JunD and Fra-2, did not growth arrest at high cell density and did not express p20K. Therefore, AP-1 interfered with the action of C/EBPbeta at high cell density, indicating that these factors play opposing roles in the control of GAS gene expression and CEF proliferation.

The NF-kappaB/Rel family of transcription factors regulates a wide variety of genes whose products play a fundamental role in inflammatory and immune responses. The implication of NF-kappaB/Rel proteins and their IkappaB regulatory subunits in the control of cellular growth and oncogenesis, was suggested by the induction of fatal lymphomas in birds by the v-rel oncoprotein, and the rearrangement and amplification of several genes encoding the NF-kappaB/Rel/IkappaB signal transduction factors in human malignancies, primarily of lymphoid origin. Hodgkin's disease (HD) is a lymphoma characterized by a low frequency of malignant Hodgkin and Reed-Sternberg (H/RS) cells in a reactive background of non-neoplastic cells. The peculiar activated phenotype of Hodgkin and Reed-Sternberg cells and their pattern of cytokine secretion are believed to be a consequence of constitutive activation of the NF-kappaB transcription factor. Here, we report the detection of mutations of the IkBa gene, in two HD-derived cell lines and in two out of eight biopsy samples from patients with relapsed Hodgkin's disease. The presence of defective IkappaBalpha is thus likely to explain the constitutive activation of NF-kappaB in these cells and suggests that IkappaBalpha is a tumour suppressor controlling the oncogenic activation of NF-kappaB in Hodgkin and Reed-Sternberg cells.

The CEF-4/9E3 gene is expressed aberrantly in chicken embryo fibroblasts transformed by the Rous sarcoma virus. This aberrant expression is dependent on transcriptional activation and on the stabilization of the CEF-4 mRNA. The characterization of the CEF-4 promoter indicated that three distinct regulatory elements corresponding to an AP-1 binding site, a PRDII/ kappaB domain and a CAAT box are involved in the activation by pp60v-src. Several v-src responsive genes are controlled by AP-1 and members of the Ets family but few appear to be dependent on NF-kappaB. In this study we characterize the expression of genes regulated by NF-kappaB in normal and RSV-transformed CEF. Run-on transcription analysis indicated that pp60v-src induces the transcription of several genes controlled by NF-kappaB but at different levels. While the transcription of CEF-4 was strongly stimulated, that of NF-kappaB1, c-rel, p53 or IkappaB-alpha was activated more modestly by pp60v-src. In addition the CEF-4 mRNA was the only mRNA species to accumulate significantly in transformed CEF. The ectopic expression of RelA or Rel resulted in the stimulation of the transcription of several known targets of NF-kappaB. However, the mRNA for IkappaB-alpha was the only mRNA species to accumulate considerably in the RelA- or Rel-expressing cells. Hence for most kappaB-controlled genes, transcriptional activation was not sufficient to obtain a significant increase in mRNA expression. Likewise, RelA or Rel enhanced the transcription of the CEF-4 gene without a significant accumulation of the CEF-4 mRNA. However, transformation by v-src caused a massive accumulation of the CEF-4 mRNA but not of other mRNA species in the RelA- and Rel-expressing cells. Transient expression assays, run-on transcription and Northern blotting analyses indicated that the effect of pp60v-src on CEF-4 expression was mediated predominantly at the post-transcriptional level in these cells. Therefore transcriptional and post-transcriptional mechanisms determine the restricted pattern of activation of kappaB-controlled genes in RSV-transformed CEF.

Protein H16, which we have identified previously in mammalian cell lines, binds in vitro to two single stranded DNA sites on the late strand of the early promoter of SV40. It has no other single strand binding site in the SV40 genome and does not bind to double stranded DNA. In vitro, H16 can be shown to stimulate strongly the activity of purified RNA polymerase II. Here we have purified this 70 kDa protein from cultured monkey cells and have sequenced three of its tryptic peptides. The analysis indicates that H16 is the simian homolog of human protein K, a nuclear RNA-binding protein found in heterogeneous nuclear ribonucleoprotein (hnRNP) particles, which contains a KH domain present in several proteins including the fragile X mental retardation gene product (FMR1). The binding affinities of protein K/H16 for RNA and DNA were subsequently compared in detail. They showed that under conditions where K/H16 binds strongly to its single stranded DNA site, it binds very weakly to the corresponding RNA sequence. This result suggests a possible shuttling of the protein from RNA to DNA during processes which involve opening of the DNA double helix.

Background: Heparan sulphate (HS) containing proteoglycans regulate the activity of many proteins (growth factors, cytokines, adhesion molecules…) involved in pathologies like cancer, inflammation, cardiovascular, metabolic and neurodegenerative diseases, as well as in viral infection. HS mimicking oligosaccharides can interfere with protein/HS interactions and thus modulate the resulting biological effects. The optimization of fully synthetic HS mimetics exhibiting anti-tumour and anti-metastatic properties is currently in progress. Methods: Compound selection was performed using classical in vitro assays (binding and proliferation) and a cellular angiogenesis assay (Angiokit®). In vivo, Swiss nude mice subcutaneously (SC) xenografted with A-673 human rhabdomyosarcoma tumor cells were used to compare compounds showing significant anti-tumour activity. Compounds were intraperitonealy (IP) injected on a daily basis. The most potent compound was then evaluated on syngeneic mouse models: C57 BL/6 mice intravenously (IV) injected with B16-F10 malignant melanoma cells and Balb/c mice bearing orthotopic (OT) Renca kidney carcinoma. Results: Binding assays to FGF-2, PDGF-BB, VEGF-B and SDF-1α in Biacore experiments and growth factor-dependent proliferation tests led to the identification of several inhibitors. Optimized compounds exhibiting anti-angiogenic activities were selected in Angiokit® assays. EP80061 was selected for further studies on the basis of significant activity in this assay. In A-673 SC mouse model, EP80061 was also the most powerful compound. When IP injected at 30 mg/kg/inj. for 28 consecutive days, it induced a marked tumour growth delay. At D22, the optimal T/C value (46%) was reached with a significantly lower tumour volume (p=0.0006, 1672 ± 760 and 765 ± 402 mm3 for vehicle and EP80061 treated mice, respectively). EP80061 induced also a very significant decrease in lung metastasis development in B16-F10 IV injected mice treated at 30 mg/kg/inj. (p Conclusion: Current preclinical results support EP80061 as a lead candidate for further developments of small-glyco drugs as anticancer compounds. 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 5459.