Annotating the human genome

2 Abstract2.1 En françaisLe séquençage du génome humain est un pré-requis fondamental à la compréhension de la biologie de l'être humain. Ce projet achevé, les scientifiques ont dû faire face à une tâche aussi importante, comprendre cette suite de 3 milliards de lettres qui compose notre génome. Le consortium ENCODE (ENCyclopedia Of Dna Elements) fût formé comme une suite logique au projet du génome humain. Son rôle est d'identifier tous les éléments fonctionnels de notre génome incluant les régions transcrites, les sites d'attachement des facteurs de transcription, les sites hypersensibles à la DNAse I ainsi que les marqueurs de modification des histones. Dans le cadre de ma thèse doctorale, j'ai participé à 2 sous-projets d'ENCODE. En premier lieu, j'ai eu la tâche de développer et d'optimiser une technique de validation expérimentale à haut rendement de modèles de gènes qui m'a permis d'estimer la qualité de la plus récente annotation manuelle. Ce nouveau processus de validation est bien plus efficace que la technique RNAseq qui est actuellement en train de devenir la norme. Cette technique basée sur la RT-PCR, m'a notamment permis de découvrir de nouveaux exons dans 10% des régions interrogées. En second lieu j'ai participé à une étude ayant pour but d'identifier les extrémités de tous les gènes des chromosomes humains 21 et 22. Cette étude à permis l'identification à large échelle de transcrits chimères comportant des séquences provenant de deux gènes distincts pouvant être à une grande distance l'un de autre.2.2 In EnglishThe completion of the human genome sequence js the prerequisite to fully understand the biology of human beings. This project achieved, scientists had to face another challenging task, understanding the meaning of the 3 billion letters composing this genome. As a logical continuation of the human genome project, the ENCODE (ENCyclopedia Of DNA Elements) consortium was formed with the aim of annotating all its functional elements. These elements include transcribed regions, transcription binding sites, DNAse I hypersensitive sites and histone modification marks. In the frame of my PhD thesis, I was involved in two sub-projects of ENCODE. Firstly I developed and optimized an high throughput method to validate gene models, which allowed me to assess the quality of the most recent manually-curated annotation. This novel experimental validation pipeline is extremely effective, far more so than transcriptome profiling through RNA sequencing, which is becoming the norm. This RT-PCR-seq targeted-approach is likewise particularly efficient in identifying novel exons, as we discovered about 10% of loci with unannotated exons. Secondly, I participated to a study aiming to identify the gene boundaries of all genes in the human chromosome 21 and 22. This study led to the identification of chimeric transcripts that are composed of sequences coming form two distinct genes that can be map far away from each other.

Clinical follow-up of women infected with human papillomavirus-16, either alone or with other human papillomavirus types: identification of different risk groups.

OBJECTIVES: Evaluation of the clinical impact of multiple infections of the cervix by human papillomavirus, including human papillomavirus-16, compared with single human papillomavirus-16 infection. STUDY DESIGN: One hundred sixty-nine women were classified in 3 categories depending on their human papillomavirus profile: human papillomavirus-16 only, human papillomavirus-16 and low-risk type(s), and human papillomavirus-16 and other high-risk type(s). Cervical brush samples were analyzed for human papillomavirus DNA by polymerase chain reaction and reverse line blot hybridization. All women were evaluated with colposcopy during 24 months or more. Management was according to the Bethesda recommendations. RESULTS: Women infected with human papillomavirus-16 and other high-risk human papillomavirus type(s) presented more progression or no change in the grade of dysplasia, compared with women of the other groups (relative risk [RR], 1.39; 95% confidence interval [CI], 1.07-1.82; P = .02 at 6 months; RR, 2.10; 95% CI, 1.46-3.02; P < .001 at 12 months; RR, 1.82; 95% CI, 1.21-2.72; P = .004 at 24 months). CONCLUSION: Coinfection of women with human papillomavirus-16 and other high-risk human papillomavirus type(s) increases the risk of unfavorable evolution.

Molecular characterization and phenotypic expression of mutations in genes for gonadotropins and their receptors in humans.

Gonadotropin hormones undergo important dynamic changes during life. Their rise during puberty stimulates gonadal steroid secretion, triggering the development of secondary sexual characteristics and the acquisition of fertility. The full spectrum of possible mutations and polymorphisms in the human gonadotropins and in their receptor genes has been described in recent years. Patients harboring these mutations display a very wide range of phenotypes affecting all aspects of the reproductive axis. An important insight provided by the careful study of these patients lies in the striking gender differences in the phenotypes associated with a given mutation. As a result, the careful study of these rare patients has allowed us to better define the respective roles of luteinizing hormone and follicle-stimulating hormone in normal human pubertal development and in the achievement of full fertility potential in either males or females. In this work, we describe briefly the known mutations in the genes for both gonadotropins and their receptors, and discuss their genotype/phenotype correlations in light of these important gender differences.

Myotonic dystrophy transgenic mice exhibit pathologic abnormalities in diaphragm neuromuscular junctions and phrenic nerves

Myotonic dystrophy Type 1 (DM-1) is caused by abnormal expansion of a (CTG) repeat located in the DM protein kinase gene. Respiratory problems have long been recognized to be a major feature of this disorder. Because respiratory failure can be associated with dysfunction of phrenic nerves and diaphragm muscle, we examined the diaphragm and respiratory neural network in transgenic mice carrying the human genomic DM-1 region with expanded repeats of more than 300 CTG, a valid model of the human disease. Morphologic and morphometric analyses revealed distal denervation of diaphragm neuromuscular junctions in DM-1 transgenic mice indicated by a decrease in the size and shape complexity of end-plates and a reduction in the concentration of acetyl choline receptors on the postsynaptic membrane. More importantly, there was a significant reduction in numbers of unmyelinated, but not of myelinated, fibers in DM-1 phrenic nerves; no morphologic alternations of the nerves or loss of neuronal cells were detected in medullary respiratory centers or cervical phrenic motor neurons. Because neuromuscular junctions are involved in action potential transmission and the afferent phrenic unmyelinated fibers control the inspiratory activity, our results suggest that the respiratory impairment associated with DM-1 may be partially due to pathologic alterations in neuromuscular junctions and phrenic nerves.

Activin enhances skin tumourigenesis and malignant progression by inducing a pro-tumourigenic immune cell response.

Activin is an important orchestrator of wound repair, but its potential role in skin carcinogenesis has not been addressed. Here we show using different types of genetically modified mice that enhanced levels of activin in the skin promote skin tumour formation and their malignant progression through induction of a pro-tumourigenic microenvironment. This includes accumulation of tumour-promoting Langerhans cells and regulatory T cells in the epidermis. Furthermore, activin inhibits proliferation of tumour-suppressive epidermal γδ T cells, resulting in their progressive loss during tumour promotion. An increase in activin expression was also found in human cutaneous basal and squamous cell carcinomas when compared with control tissue. These findings highlight the parallels between wound healing and cancer, and suggest inhibition of activin action as a promising strategy for the treatment of cancers overexpressing this factor.

Schistosomiasis vaccine development: progress and prospects

The undisputed, worldwide success of chemotherapy notwithstanding, schistosomiasis continues to defy control efforts in as much rapid reinfection demands repeated treatment, sometimes as often as once a year. There is thus a need for a complementary tool with effect for the longer term, notably a vaccine. International efforts in this direction have been ongoing for several decades but, until the recombinant DNA techniques were introduced, antigen production remained an unsurmountable bottleneck. Although animal experiments have been highly productive and are still much needed, they probably do not reflect the human situation adequately and real progress can not be expected until more is known about human immune responses to schistosome infection. It is well-known that irradiated cercariae consistently produce high levels of protection in experimental animals but, for various reasons, this proof of principle cannot be directly exploited. Research has instead been focussed on the identification and testing of specific schistosome antigens. This work has been quite successful and is already at the stage where clinical trials are called for. Preliminary results from coordinated in vitro laboratory and field epidemiological studies regarding the protective potential of several antigens support the initiation of such trials. A series of meetings, organized earlier this year in Cairo, Egypt, reviewed recent progress, selecteded suitable vaccine candidates and made firm recommendations for future action including pledging support for large-scale production according to good manufacturing practice (GMP) and Phase I trials. Scientists at the American Centers for Disease Control and Prevention (CDC) have drawn up a detailed research plan. The major financial support will come from USAID, Cairo, which has established a scientific advisory group of Egyptian scientists and representatives from current and previous international donors such as WHO, NIAID, the European Union and the Edna McConnell Clark Foundation.

The impact of agglomeration effects and accessibility on wages

This paper contributes to the empirical literature on the effects of agglomeration and road accessibility on productivity of firms by looking at the case of Spain. We approach productivity indirectly by using individual wages allocated at the NUTS III level. We use a repeated cross-section of individual micro-data for the years 1995, 2002 and 2006. The availability of interprovincial travel time data for each of the three years allows controlling for transport improvements over the period by using a market potential variable. Additionally, agglomeration is approached by employment density and we control for localization economies, human capital externalities and a large set of individual and workplace characteristics. Estimating by instrumental variables, our results show a positive and significant effect of market accessibility on wages and non linear effect for employment density.

Prevalence, species differentiation, haemolytic activity, and antibiotic susceptibility of aeromonads in untreated well water

The use of untreated water for drinking and other activities have been associated with intestinal and extraintestinal infections in humans due to Aeromonas species. In the present study aeromonads were isolated from 48.7% of 1,000 water samples obtained from wells and other miscellaneous sources. Aeromonas species were detected in 45% of samples tested in spring, 34.5% in summer, 48% in autumn and 60% of samples tested in winter. Speciation of 382 strains resulted in 225 (59%) being A. hydrophila, 103 (27%) A. caviae, 42 (11%) A. sobria and 11 (3%) atypical aeromonads. Of 171 Aeromonas strains tested for their haemolytic activity, 53%, 49%, 40% and 37% were positive in this assay using human, horse, sheep and camel erythrocytes respectively. The results obtained indicate that potentially enteropathogenic Aeromonas species are commonly present in untreated drinking water obtained from wells in Libya (this may also apply to other neighbouring countries) which may pose a health problem to users of such water supplies. In addition, ceftriaxone and ciprofloxacin are suitable drugs that can be used in the treatment of Aeromonas-associated infections, particularly in the immunocompromised, resulting from contact with untreated sources of water.

E2F1 mediates DNA damage and apoptosis through HCF-1 and the MLL family of histone methyltransferases.

E2F1 is a key positive regulator of human cell proliferation and its activity is altered in essentially all human cancers. Deregulation of E2F1 leads to oncogenic DNA damage and anti-oncogenic apoptosis. The molecular mechanisms by which E2F1 mediates these two processes are poorly understood but are important for understanding cancer progression. During the G1-to-S phase transition, E2F1 associates through a short DHQY sequence with the cell-cycle regulator HCF-1 together with the mixed-lineage leukaemia (MLL) family of histone H3 lysine 4 (H3K4) methyltransferases. We show here that the DHQY HCF-1-binding sequence permits E2F1 to stimulate both DNA damage and apoptosis, and that HCF-1 and the MLL family of H3K4 methyltransferases have important functions in these processes. Thus, HCF-1 has a broader role in E2F1 function than appreciated earlier. Indeed, sequence changes in the E2F1 HCF-1-binding site can modulate both up and down the ability of E2F1 to induce apoptosis indicating that HCF-1 association with E2F1 is a regulator of E2F1-induced apoptosis.

High occurrence of hepatitis E virus in samples from wastewater treatment plants in Switzerland and comparison with other enteric viruses.

Hepatitis E virus (HEV) is responsible for many enterically transmitted viral hepatitides around the world. It is currently one of the waterborne diseases of global concern. In industrialized countries, HEV appears to be more common than previously thought, even if it is rarely virulent. In Switzerland, seroprevalence studies revealed that HEV is endemic, but no information was available on its environmental spread. The aim of this study was to investigate -using qPCR- the occurrence and concentration of HEV and three other viruses (norovirus genogroup II, human adenovirus-40 and porcine adenovirus) in influents and effluents of 31 wastewater treatment plants (WWTPs) in Switzerland. Low concentrations of HEV were detected in 40 out of 124 WWTP influent samples, showing that HEV is commonly present in this region. The frequency of HEV occurrence was higher in summer than in winter. No HEV was detected in WWTP effluent samples, which indicates a low risk of environmental contamination. HEV occurrence and concentrations were lower than those of norovirus and adenovirus. The autochthonous HEV genotype 3 was found in all positive samples, but a strain of the non-endemic and highly pathogenic HEV genotype I was isolated in one sample, highlighting the possibility of environmental circulation of this genotype. A porcine fecal marker (porcine adenovirus) was not detected in HEV positive samples, indicating that swine are not the direct source of HEV present in wastewater. Further investigations will be necessary to determine the reservoirs and the routes of dissemination of HEV.

Experimental evidence and ecological perspectives for the adaptation of Schistosoma mansoni Sambon, 1907 (Digenea: Schistosomatidae) to a wild host, the water-rat, Nectomys squamipes Brants, 1827 (Rodentia: Sigmodontinae)

Due to the semi aquatic habits and the overlap of the geographical distribution of the water-rat, Nectomys spp., with schistosomiasis endemic areas, these wild rodents are very likely to acquire Schistosoma mansoni infection in their daily activities. The role of the water-rat in the S. mansoni cycle would be substantiated if one could prove that these rodents acquire the parasite during their own activity time, a completely independent time schedule of human activities. To pursue this goal, we performed two field experiments in the municipality of Sumidouro, State of Rio de Janeiro, Brazil, a schistosomiasis endemic area where N. squamipes is found naturally infected. One experiment was devised as a series of observations of activity time of the water-rat. The other experiment was a test of the occurrence of late transmission of S. mansoni to the water-rat. The daily activity pattern showed that the water-rat is active chiefly just after sunset. At both diurnal and late exposition essays the water-rat sentinels got infected by S. mansoni. These findings clarify ecological and behavioral components necessary to the adaptation of S. mansoni to the water-rat as a non human definitive host and the existence of a transmission cycle involving this animals as a reservoir.

Antileishmanial IgG and IgE antibodies recognize predominantly carbohydrate epitopes of glycosylated antigens in visceral leishmaniasis

The specificity of human antileishmanial IgG and IgE antibodies to glycosylated antigens of Leishmania chagasi was evaluated. An ELISA was performed with soluble leishmanial antigen (SLA) and a panel of 95 sera including samples from patients with subclinical infection (SC) and visceral leishmaniasis (VL), subjects cured of visceral leishmaniasis (CVL), and from healthy individuals from endemic areas (HIEA). Antileishmanial IgG were verified for 18 (40%) of 45 SC subjects (mean absorbance of 0.49 ± 0.17). All nine sera from VL patients had such antibody (0.99 ± 0.21), while 11 (65%) of 17 CVL individuals were seropositive (0.46 ± 0.05). Only three (12%) of 24 HIEA controls reacted in IgG-ELISA. Antileishmanial IgE was detected in 26 (58%) of 45 SC patients (0.35 ± 0.14), and in all VL patients (0.65 ± 0.29). These antibodies were also detected in 13(76%) of 17 CVL subjects (0.42 ± 0.14) while all HIEA controls were seronegative. There was no correlation between antileishmanial IgG and IgE antibody absorbances. Mild periodate oxidation at acid pH of SLA carbohydrates drastically diminished its antigenicity in both IgG and IgE-ELISA, affecting mainly the antigens of 125, 102, 94, and 63 kDa as demonstrated by western immunoblotting.

Improvement of the HIV/AIDS vaccine candidate NYVAC-C by deletion of the viral type I IFN inhibitor and/or acquisition of replication competence

Background: Recombinant viruses based on the attenuated vaccinia virus strain NYVAC are promising HIV vaccine candidates as phase I/II clinical trials have shown good safety and immunogenicity profiles. However, this NYVAC strain is non-replicating in most human cell lines and encodes viral inhibitors of the immune system. Methods: With the aim to increase the immune potency of the current NYVAC-C vector (expressing the codon optimized clade C HIV-1 genes encoding gp120 and Gag-Pol-Nef polyprotein), we have generated and characterized three NYVAC-C-based vectors by, 1) deletion of the viral type I IFN inhibitor gene (NYVAC-CdeltaB19R), 2) restoration of virus replication competence in human cells by re-inserting K1L and C7L host range genes (NYVAC-C-KC) and, 3) combination of both strategies (NYVACC- KC-deltaB19R). Results: Insertion of the KC fragment restored the replication competence of the viruses in human cells (HeLa cells and primary dermal fibroblasts and keratinocytes), increased the expression of HIV antigens by more than 3-fold compared to the non-replicating homologs, inhibited apoptosis induced by the parental NYVAC-C and retained attenuation in a newborn mouse model. In adult mice, replication-competent viruses showed a limited capacity to replicate in tissues surrounding the inoculation site (ovaries and lymph nodes). After infection of keratinocytes, PBMCs and dendritic cells these viruses induced differential modulation in specific host cell signal transduction pathways, triggering genes important in immune modulation. Conclusion: We have developed improved NYVAC-C-based vectors with enhanced HIV-1 antigen expression, with the ability to replicate in cultured human cells and partially in some tissues, with an induced expression of cellular genes relevant to immune system activation, and which trigger IFN-dependent and independent signalling pathways, while maintaining a safety phenotype. These new vectors are promising new HIV vaccine candidates. These studies were performed within the Poxvirus Tcell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

Trypanosoma rangeli sialidase: lack of activity in stocks from Panama and other regions

A total of 33 crude and cloned Trypanosoma rangeli stocks found as natural infections in human from Panama and other endemic areas of Central and South America were evaluated as producers of sialidase (SA) activity through the MU-NANA fluorescence test. Negative results were observed in 6 of the isolates: Panama (4), Honduras (1), and Brazil (1). In addition, an immunoblotting analysis confirm the presence of the SA antigen in these stocks without enzymatic activity. These findings must be considered in the interpretation of the biological significance of T. rangeli SA and in the proper characterization and identification of this parasite.