PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans.

Using genetically matched azole-susceptible (AS) and azole-resistant (AR) clinical isolates of Candida albicans, we recently demonstrated that CDR1 overexpression in AR isolates is due to its enhanced transcriptional activation and mRNA stability. This study examines the molecular mechanisms underlying enhanced CDR1 mRNA stability in AR isolates. Mapping of the 3' untranslated region (3' UTR) of CDR1 revealed that it was rich in adenylate/uridylate (AU) elements, possessed heterogeneous polyadenylation sites, and had putative consensus sequences for RNA-binding proteins. Swapping of heterologous and chimeric lacZ-CDR1 3' UTR transcriptional reporter fusion constructs did not alter the reporter activity in AS and AR isolates, indicating that cis-acting sequences within the CDR1 3' UTR itself are not sufficient to confer the observed differential mRNA decay. Interestingly, the poly(A) tail of the CDR1 mRNA of AR isolates was approximately 35-50 % hyperadenylated as compared with AS isolates. C. albicans poly(A) polymerase (PAP1), responsible for mRNA adenylation, resides on chromosome 5 in close proximity to the mating type-like (MTL) locus. Two different PAP1 alleles, PAP1-a/PAP1-alpha, were recovered from AS (MTL-a/MTL-alpha), while a single type of PAP1 allele (PAP1-alpha) was recovered from AR isolates (MTL-alpha/MTL-alpha). Among the heterozygous deletions of PAP1-a (Deltapap1-a/PAP1-alpha) and PAP1-alpha (PAP1-a/Deltapap1-alpha), only the former led to relatively enhanced drug resistance, to polyadenylation and to transcript stability of CDR1 in the AS isolate. This suggests a dominant negative role of PAP1-a in CDR1 transcript polyadenylation and stability. Taken together, our study provides the first evidence, to our knowledge, that loss of heterozygosity at the PAP1 locus is linked to hyperadenylation and subsequent increased stability of CDR1 transcripts, thus contributing to enhanced drug resistance.

A methodology to event reconstruction from trace images

The widespread use of digital imaging devices for surveillance (CCTV) and entertainment (e.g., mobile phones, compact cameras) has increased the number of images recorded and opportunities to consider the images as traces or documentation of criminal activity. The forensic science literature focuses almost exclusively on technical issues and evidence assessment [1]. Earlier steps in the investigation phase have been neglected and must be considered. This article is the first comprehensive description of a methodology to event reconstruction using images. This formal methodology was conceptualised from practical experiences and applied to different contexts and case studies to test and refine it. Based on this practical analysis, we propose a systematic approach that includes a preliminary analysis followed by four main steps. These steps form a sequence for which the results from each step rely on the previous step. However, the methodology is not linear, but it is a cyclic, iterative progression for obtaining knowledge about an event. The preliminary analysis is a pre-evaluation phase, wherein potential relevance of images is assessed. In the first step, images are detected and collected as pertinent trace material; the second step involves organising and assessing their quality and informative potential. The third step includes reconstruction using clues about space, time and actions. Finally, in the fourth step, the images are evaluated and selected as evidence. These steps are described and illustrated using practical examples. The paper outlines how images elicit information about persons, objects, space, time and actions throughout the investigation process to reconstruct an event step by step. We emphasise the hypothetico-deductive reasoning framework, which demonstrates the contribution of images to generating, refining or eliminating propositions or hypotheses. This methodology provides a sound basis for extending image use as evidence and, more generally, as clues in investigation and crime reconstruction processes.

Meta-analysis of genome-wide association data identifies a risk locus for major mood disorders on 3p21.1.

The major mood disorders, which include bipolar disorder and major depressive disorder (MDD), are considered heritable traits, although previous genetic association studies have had limited success in robustly identifying risk loci. We performed a meta-analysis of five case-control cohorts for major mood disorder, including over 13,600 individuals genotyped on high-density SNP arrays. We identified SNPs at 3p21.1 associated with major mood disorders (rs2251219, P = 3.63 x 10(-8); odds ratio = 0.87; 95% confidence interval, 0.83-0.92), with supportive evidence for association observed in two out of three independent replication cohorts. These results provide an example of a shared genetic susceptibility locus for bipolar disorder and MDD.

Different epitopes of the LACK protein are recognized by V beta 4 V alpha 8 CD4+ T cells in H-2b and H-2d mice susceptible to Leishmania major.

After inoculation of Leishmania major, a rapid production of IL-4 by LACK-specific CD4+ T cells has been shown to drive Th2 cell development in susceptible mice i.e. BALB/c and C57BL/6 mice rendered susceptible by neutralization of IFN-gamma at the onset of infection. Here, we showed that peptide AA 156-173 induced an early IL-4 mRNA expression not only in BALB/c mice but also in resistant B10.D2 mice when IFN-gamma is neutralized. Epitope mapping of LACK protein demonstrated that peptide containing AA 293-305 induced early IL-4 mRNA transcripts in susceptible H-2b mice i.e. BALB/b and resistant C57BL/6 mice when IFN-gamma is neutralized. Stringently, the early IL-4 response to the H-2d (AA 156-173) or the H-2b (AA 293-305) epitopes occurred in V beta 4 V alpha 8 CD4+ T cells from either H-2d or H-2b susceptible mice, respectively.

Neutrophil-derived CCL3 is essential for the rapid recruitment of dendritic cells to the site of Leishmania major inoculation in resistant mice.

Neutrophils are rapidly and massively recruited to sites of microbial infection, where they can influence the recruitment of dendritic cells. Here, we have analyzed the role of neutrophil released chemokines in the early recruitment of dendritic cells (DCs) in an experimental model of Leishmania major infection. We show in vitro, as well as during infection, that the parasite induced the expression of CCL3 selectively in neutrophils from L. major resistant mice. Neutrophil-secreted CCL3 was critical in chemotaxis of immature DCs, an effect lost upon CCL3 neutralisation. Depletion of neutrophils prior to infection, as well as pharmacological or genetic inhibition of CCL3, resulted in a significant decrease in DC recruitment at the site of parasite inoculation. Decreased DC recruitment in CCL3(-/-) mice was corrected by the transfer of wild type neutrophils at the time of infection. The early release of CCL3 by neutrophils was further shown to have a transient impact on the development of a protective immune response. Altogether, we identified a novel role for neutrophil-secreted CCL3 in the first wave of DC recruitment to the site of infection with L. major, suggesting that the selective release of neutrophil-secreted chemokines may regulate the development of immune response to pathogens.

New insights into the long-term evolutionary history of the avian MHC

SummaryGene duplication and neofunctidnalization are important processes in the evolution of phenotypic complexity. They account for important evolutionary novelties that confer ecological adaptation, such as the major histocompatibility complex (MHC), a multigene family with a central role in vertebrates' adaptive immune system. Multigene families, which evolved in large part through duplication, represent promising systems to study the still strongly depbated relative roles of neutral and adaptive processes in the evolution of phenotypic complexity. Detailed knowledge on ecological function and a well-characterized evolutionary history place the mammals' MHC amongst ideal study systems. However mammalian MHCs usually encompass several million base pairs and hold a large number of functional and non-functional duplicate genes, which makes their study complex. Avian MHCs on the other hand are usually way more compact, but the reconstruction of. their evolutionary history has proven notoriously difficult. However, no focused attempt has been undertaken so far to study the avian MHC evolutionary history in a broad phylogenetic context and using adequate gene regions.In the present PhD, we were able to make important contributions to the understanding of the long-term evolution of the avian MHC class II Β (MHCI1B). First, we isolated and characterized MHCIIB genes in barn owl (Tyto alba?, Strigiformes, Tytonidae), a species from an avian lineage in which MHC has not been studied so far. Our results revealed that with only two functional MHCIIB genes the MHC organization of barn owl may be similar to the 'minimal essential' MHC of chicken (Gallus gallus), indicating that simple MHC organization may be ancestral to birds. Taking advantage of the sequence information from barn owl, we studied the evolution of MHCIIB genes in 13 additional species of 'typical' owls (Strigiformes, Strigidae). Phylogenetic analyses revealed that according to their function, in owls the peptide-binding region (PBR) encoding exon 2 and the non-PBR encoding exon 3 evolve by different patterns. Exon 2 exhibited an evolutionary history of positive selection and recombination, while exon 3 traced duplication history and revealed two paralogs evolving divergently from each other in owls, and in a shorebird, the great snipe {Gallinago media). The results from exon 3 were the first ever from birds to demonstrate gene orthology in species that diverged tens of millions of years ago, and strongly questioned whether the taxa studied before provided an adequate picture of avian MHC evolution. In a follow-up study, we aimed at explaining a striking pattern revealed by phylogenetic trees analyzing the owl sequences along with MHCIIB sequences from other birds: One owl paralog (termed DAB1) grouped with sequences of passerines and falcons, while the other (DAB2) grouped with wildfowl, penguins and birds of prey. This could be explained by either a duplication event preceding the evolution of these bird orders, or by convergent evolution of similar sequences in a number of orders. With extensive phylogenetic analyses we were able to show, that indeed a duplication event preceeded the major avian radiation -100 my ago, and that following this duplication, the paralogs evolved under positive selection. Furthermore, we showed that the divergently evolving amino acid residues in the MHCIIB-encoded β-chain potentially interact with the MHCI I α-chain, and that molecular coevolution of the interacting residues may have been involved in the divergent evolution of the MHCIIB paralogs.The findings of this PhD are of particular interest to the understanding of the evolutionary history of the avian MHC and, by providing essential information on long-term gene history in the avian MHC, open promising perspectives for advances in the understanding of the evolution of multigene families in general, and for avian MHC organization in particular. Amongst others I discuss the importance of including protein structure in the phylogenetic study of multigene families, and the roles of ecological versus molecular selection pressures. I conclude by providing a population genomic perspective on avian MHC, which may serve as a basis for future research to investigate the relative roles of neutral processes involving effective population size effects and of adaptation in the evolution of avian MHC diversity and organization.RésuméLa duplication de gènes et leur néo-fonctionnalisation sont des processus importants dans l'évolution de la complexité phénotypique. Ils sont impliqués dans l'apparition d'importantes nouveautés évolutives favorisant l'adaptation écologique, comme c'est le cas pour le complexe majeur d'histocompatibilité

Impact of a pain protocol including hypnosis in major burns.

BACKGROUND: Pain is a major issue after burns even when large doses of opioids are prescribed. The study focused on the impact of a pain protocol using hypnosis on pain intensity, anxiety, clinical course, and costs. METHODS: All patients admitted to the ICU, aged >18 years, with an ICU stay >24h, accepting to try hypnosis, and treated according to standardized pain protocol were included. Pain was scaled on the Visual Analog Scale (VAS) (mean of daily multiple recordings), and basal and procedural opioid doses were recorded. Clinical outcome and economical data were retrieved from hospital charts and information system, respectively. Treated patients were matched with controls for sex, age, and the burned surface area. FINDINGS: Forty patients were admitted from 2006 to 2007: 17 met exclusion criteria, leaving 23 patients, who were matched with 23 historical controls. Altogether patients were 36+/-14 years old and burned 27+/-15%BSA. The first hypnosis session was performed after a median of 9 days. The protocol resulted in the early delivery of higher opioid doses/24h (p<0.0001) followed by a later reduction with lower pain scores (p<0.0001), less procedural related anxiety, less procedures under anaesthesia, reduced total grafting requirements (p=0.014), and lower hospital costs per patient. CONCLUSION: A pain protocol including hypnosis reduced pain intensity, improved opioid efficiency, reduced anxiety, improved wound outcome while reducing costs. The protocol guided use of opioids improved patient care without side effects, while hypnosis had significant psychological benefits.

Second cranio-facial malignancies in hereditary retinoblastoma survivors previously treated with radiation therapy: clinic and radiologic characteristics and survival outcomes.

INTRODUCTION: Hereditary retinoblastoma survivors have an increased risk for cranio-facial second primary tumours (SPT), especially after treatment with external beam radiotherapy (EBRT). This multicentre study evaluates the clinical and imaging characteristics and outcomes of cranio-facial SPTs in irradiated retinoblastoma survivors. PATIENTS AND METHODS: Clinical and radiological data of 42 hereditary retinoblastoma patients with 44 second and third malignancies were reviewed. Radiological data included anatomic location and computed tomography (CT) and magnetic resonance (MR) characteristics. Cox regression and likelihood ratio chi-square test were used to evaluate differences in patients' survival rates. RESULTS: Cranio-facial SPTs were diagnosed at a median age of 13 years. Histological types included osteosarcomas (43%), rhabdomyosarcomas (20%) (57% embryonal, 43% alveolar) and a variety of other types of SPT (37%). Predilection sites were: temporal fossa (39%), ethmoid sinus (23%), orbit (18%), maxillary sinus (16%) and intracranial dura mater (4%). Most of the osteosarcomas (78%) and rhabdomyosarcomas (80%) occurred in patients treated with EBRT in the first year-of-life. Treatment of SPTs with a microscopically complete surgical resection led to a significantly better 5-year overall survival (OS) (P=0.017) and event-free survival (EFS) (P=0.012) compared to patients treated without surgery or incomplete resection (OS: 83% versus 52%; EFS: 80% versus 47%). CONCLUSIONS: Osteosarcomas and rhabdomyosarcomas are the most common cranio-facial SPTs in irradiated hereditary retinoblastoma survivors, which develop in specific locations and occur predominantly in patients irradiated in their first year-of-life. Microscopically complete surgical resection of SPTs is a major prognostic factor, suggesting the potential benefit of early detection by imaging.

Release of extracellular particles by recombinant vaccinia virus over-expressing the major envelope protein p37K.

During the replication cycle of vaccinia virus, four different forms of viral particles are produced. The two extracellular enveloped forms, cell-associated enveloped virus and extracellular enveloped virus, are responsible for cell-to-cell transmission and long-range spread of infection both in vivo and in vitro. Despite the biological importance of the enveloped forms, the mechanism of envelopment and the components involved in this process have been analysed only recently. Therefore the individual steps and the rate-limiting factors of the envelopment process are still unknown. The protein p37K, an unglycosylated but acylated envelope protein of molecular mass 37 kDa, has been shown to be essential for envelopment. However, this study shows that over-expression of p37K by vaccinia virus recombinants reduces rather than increases the yield of infectious enveloped virus which is mainly due to the enveloped virions exhibiting a strongly diminished specific infectivity.

Evolution of vitellogenin genes: comparative analysis of the nucleotide sequences downstream of the transcription initiation site of four Xenopus laevis and one chicken gene.

Electron microscopic analysis of heteroduplexes between the most distantly related Xenopus vitellogenin genes (A genes X B genes) has revealed the distribution of homologous regions that have been preferentially conserved after the duplication events that gave rise to the multigene family in Xenopus laevis. DNA sequence analysis was limited to the region downstream of the transcription initiation site of the Xenopus genes A1, B1 and B2 and a comparison with the Xenopus A2 and the major chicken vitellogenin gene is presented. Within the coding regions of the first three exons, nucleotide substitutions resulting in amino acid changes accumulate at a rate similar to that observed in globin genes. This suggests that the duplication event which led to the formation of the A and B ancestral genes in Xenopus laevis occurred about 150 million years ago. Homologous exons of the A1-A2 and B1-B2 gene pairs, which formed about 30 million years ago, show a quite similar sequence divergence. In contrast, A1-A2 homologous introns seem to have evolved much faster than their B1-B2 counterparts.