Application of Molecular Diagnostic Techniques for Viral Testing.

Nucleic acid amplification techniques are commonly used currently to diagnose viral diseases and manage patients with this kind of illnesses. These techniques have had a rapid but unconventional route of development during the last 30 years, with the discovery and introduction of several assays in clinical diagnosis. The increase in the number of commercially available methods has facilitated the use of this technology in the majority of laboratories worldwide. This technology has reduced the use of some other techniques such as viral culture based methods and serological assays in the clinical virology laboratory. Moreover, nucleic acid amplification techniques are now the methods of reference and also the most useful assays for the diagnosis in several diseases. The introduction of these techniques and their automation provides new opportunities for the clinical laboratory to affect patient care. The main objectives in performing nucleic acid tests in this field are to provide timely results useful for high-quality patient care at a reasonable cost, because rapid results are associated with improvements in patients care. The use of amplification techniques such as polymerase chain reaction, real-time polymerase chain reaction or nucleic acid sequence-based amplification for virus detection, genotyping and quantification have some advantages like high sensitivity and reproducibility, as well as a broad dynamic range. This review is an up-to-date of the main nucleic acid techniques and their clinical applications, and special challenges and opportunities that these techniques currently provide for the clinical virology laboratory.

Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species

In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

New approaches in antimalarial drug discovery and development: a review

Malaria remains a major world health problem following the emergence and spread of Plasmodium falciparum that is resistant to the majority of antimalarial drugs. This problem has since been aggravated by a decreased sensitivity of Plasmodium vivax to chloroquine. This review discusses strategies for evaluating the antimalarial activity of new compounds in vitro and in animal models ranging from conventional tests to the latest high-throughput screening technologies. Antimalarial discovery approaches include the following: the discovery of antimalarials from natural sources, chemical modifications of existing antimalarials, the development of hybrid compounds, testing of commercially available drugs that have been approved for human use for other diseases and molecular modelling using virtual screening technology and docking. Using these approaches, thousands of new drugs with known molecular specificity and active against P. falciparum have been selected. The inhibition of haemozoin formation in vitro, an indirect test that does not require P. falciparum cultures, has been described and this test is believed to improve antimalarial drug discovery. Clinical trials conducted with new funds from international agencies and the participation of several industries committed to the eradication of malaria should accelerate the discovery of drugs that are as effective as artemisinin derivatives, thus providing new hope for the control of malaria.

A male with unilateral microphthalmia reveals a role for TMX3 in eye development.

Anophthalmia and microphthalmia are important birth defects, but their pathogenesis remains incompletely understood. We studied a patient with severe unilateral microphthalmia who had a 2.7 Mb deletion at chromosome 18q22.1 that was inherited from his mother. In-situ hybridization showed that one of the deleted genes, TMX3, was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TMX3 in 162 patients with anophthalmia or microphthalmia, and found two missense substitutions in unrelated patients: c.116G>A, predicting p.Arg39Gln, in a male with unilateral microphthalmia and retinal coloboma, and c.322G>A, predicting p.Asp108Asn, in a female with unilateral microphthalmia and severe micrognathia. We used two antisense morpholinos targeted against the zebrafish TMX3 orthologue, zgc:110025, to examine the effects of reduced gene expression in eye development. We noted that the morphant larvae resulting from both morpholinos had significantly smaller eye sizes and reduced labeling with islet-1 antibody directed against retinal ganglion cells at 2 days post fertilization. Co-injection of human wild type TMX3 mRNA rescued the small eye phenotype obtained with both morpholinos, whereas co-injection of human TMX3(p.Arg39Gln) mutant mRNA, analogous to the mutation in the patient with microphthalmia and coloboma, did not rescue the small eye phenotype. Our results show that haploinsufficiency for TMX3 results in a small eye phenotype and represents a novel genetic cause of microphthalmia and coloboma. Future experiments to determine if other thioredoxins are important in eye morphogenesis and to clarify the mechanism of function of TMX3 in eye development are warranted.

Effect of iodine prophylaxis during pregnancy on neurocognitive development of children during the first two years of life.

CONTEXT The association between thyroid function during pregnancy and the later mental and psychomotor development of the child is supported by numerous experimental, clinical, and epidemiological studies. OBJECTIVE The aim of the study was to evaluate the psychological development of infants aged 3 to 18 months whose mothers had received 300 microg of potassium iodide during the first trimester of their pregnancy and compare with infants whose mothers had received no iodine supplements. DESIGN AND STUDY SUBJECTS: The study included 133 women who had received 300 microg of potassium iodine and 61 women who had received no iodine supplements. MAIN OUTCOME MEASURES The neuropsychological status of the children was evaluated with the Bayley Scales of Infant Development, and measurements were made of TSH, free T(3), free T(4), and urinary iodine. RESULTS Those children whose mothers had received an iodine supplement of 300 microg had a more favorable psychometric assessment than those of the other group of mothers. They had higher scores on the Psychomotor Development Index (P = 0.02) and the Behavior Rating Scale. CONCLUSIONS Dietary iodine supplements not only have no harmful effect on the neurodevelopment of the children, they may even be beneficial. Given the possible presence of confounding variables not controlled for in this study, these findings should be considered as preliminary.

Novel (60%) and recurrent (40%) androgen receptor gene mutations in a series of 59 patients with a 46,XY disorder of sex development.

BACKGROUND Androgen receptor (AR) gene mutations are the most frequent cause of 46,XY disorders of sex development (DSD) and are associated with a variety of phenotypes, ranging from phenotypic women [complete androgen insensitivity syndrome (CAIS)] to milder degrees of undervirilization (partial form or PAIS) or men with only infertility (mild form or MAIS). OBJECTIVE The aim of the study was to characterize the contribution of the AR gene to the molecular cause of 46,XY DSD in a series of Spanish patients. SETTING We studied a series of 133 index patients with 46,XY DSD in whom gonads were differentiated as testes, with phenotypes including varying degrees of undervirilization, and in whom the AR gene was the first candidate for a molecular analysis. METHODS The AR gene was sequenced (exons 1 to 8 with intronic flanking regions) in all patients and in family members of 61% of AR-mutated gene patients. RESULTS AR gene mutations were found in 59 individuals (44.4% of index patients), of whom 46 (78%) were CAIS and 13 (22%) PAIS. Fifty-seven different mutations were found: 21.0% located in exon 1, 15.8% in exons 2 and 3, 57.9% in exons 4-8, and 5.3% intronic. Twenty-three mutations (40.4%) had been previously described and 34 (59.6%) were novel. CONCLUSIONS AR gene mutation is the most frequent cause of 46,XY DSD, with a clearly higher frequency in the complete phenotype. Mutations spread along the whole coding sequence, including exon 1. This series shows that 60% of mutations detected during the period 2002-2009 were novel.

New human kallikrein 14: enzymatic characterization and inhibitors development.

RESUME DESTINE A UN LARGE PUBLIC En biologie, si une d��couverte permet de r��pondre �� quelques questions, en g��n��ral elle en engendre beaucoup d'autres. C'est ce qui s'est produit r��cemment dans le monde des kallicr��ines. De la famille des prot��ases, prot��ines ayant la facult�� de couper plus ou moins sp��cifiquement d'autres prot��ines pour exercer un r��le biologique, la famille des kallicr��ines humaines n'��tait compos��e que de 3 membres lors du si��cle dernier. Parmi eux, une kallicr��ine mondialement utilis��e pour d��tecter le cancer de la prostate, le PSA. En 2000, un chercheur de l'h��pital universitaire Mont Sina�� �� Toronto, le Professeur Eleftherios Diamandis, a d��couvert la pr��sence de 12 nouveaux g��nes appartenant �� cette famille, situ��s sur le m��me chromosome que les 3 premi��res kallicr��ines. Cette d��couverte majeure a plac�� les sp��cialistes des kallicr��ines face �� une montagne d'interrogations car les fonctions de ces nouvelles prot��ases ��taient totalement inconnues. La kallicr��ine humaine 14 (hK14) pr��sente un int��r��t particulier, car elle se retrouve associ��e �� diff��rents cancers, notamment les carcinomes ovariens et mammaires. Cette association ne r��pond cependant pas �� la fonction de cette prot��ase. L'objectif de ce travail de th��se ��tait donc de d��couvrir, dans un premier temps, la sp��cificit�� de cette nouvelle kallicr��ine, c'est-��-dire le type de coupure qu'elle engendre au niveau des prot��ines qu'elle cible. Utilisant une technologie de pointe qui exploite la propri��t�� des bact��riophages �� se r��pliquer dans les bact��ries �� l'infini, des dizaines de millions de combinaisons prot��iques al��atoires ont ��t�� pr��sent��es �� hK14, qui a pu s��lectionner celles qui lui ��taient favorables pour la coupure. Cette technique qualitative porte le nom de Phage Display Substrate. Une fois la s��lection r��alis��e, il fallait transf��rer ces s��quences coup��es ou substrats dans un syst��me permettant de donner une valeur quantitative �� l'efficacit�� de coupure. Pour cela nous avons d��velopp�� une technologie qui permet d'��valuer cette efficacit�� en utilisant des prot��ines fluorescentes de m��duse, modifi��es g��n��tiquement, dont l'excitation de la premi��re (CFP : cyan fluorescent protein) par la lumi��re �� une certaine longue d'onde permet le transfert d'��nergie �� la seconde (YFP : yellow fluorescent protein), via un substrat qui les lie. Pour que ce transfert d'��nergie se produise, il faut que les deux prot��ines fluorescentes soient proches, comme c'est le cas lorsqu'elles sont li��es par un substrat. La coupure de ce lien provoque un changement de transfert d'��nergie qui est quantifiable en utilisant un spectrofluorom��tre. Cette technologie permet donc de suivre la r��action d'hydrolyse (coupure) des prot��ases. Afin de poursuivre certaines exp��riences permettant de mieux comprendre la fonction biologique d'hK14 ainsi que son ��ventuelle implication dans le cancer, nous avons d��velopp�� des inhibiteurs sp��cifiques d'hK14. Les s��quences qui on ��t�� le plus efficacement coup��es par hK14 ont ��t�� utilis��es pour transformer deux types d'inhibiteurs classiques, qui circulent dans notre sang, en inhibiteurs d'hK14 hautement efficaces et sp��cifiques. Selon les r��sultats obtenus in vitro, ils pourront ��tre ��valu��s in vivo en tant que traitement potentiel contre le cancer. RESUME Les prot��ases sont des enzymes impliqu��es dans des processus physiologiques mais aussi parfois pathologiques. La famille des kallicr��ines tissulaires humaines repr��sente le plus grand groupe de prot��ases humaines, dont plusieurs pourraient participer au d��veloppement de certaines maladies. D'autre part, ces prot��ases sont apparues comme des marqueurs de pathog��nicit�� potentiels, notamment dans les cas de cancers hormono-d��pendants. La kallicr��ine humaine 14 a ��t�� r��cemment d��couverte et son implication dans quelques maladies, particuli��rement dans le cas de tumeurs, semble probable. En effet, son expression g��nique est augment��e au niveau des tissus canc��reux de la prostate et du sein et son expression prot��ique s'est r��v��l��e plus ��lev��e dans le s��rum de patientes atteintes d'un cancer du sein ou des ovaires. Cependant, comme c'est le cas pour la plupart des kallicr��ines, sa fonction est encore inconnue. Afin de mieux conna��tre son r��le biologique et/ou pathologique, nous avons d��cid�� de caract��riser son activit�� enzymatique. Nous avons tout d'abord mis au point un syst��me de substrats enti��rement biologique permettant d'��tudier in vitro l'activit�� des prot��ases. Ce syst��me est bas�� sur le ph��nom��ne de FRET, �� savoir le transfert d'��nergie de r��sonance fluorescente qui intervient entre deux mol��cules fluorescentes voisines si le spectre d'��mission de la prot��ine donneuse chevauche le spectre d'excitation de la prot��ine receveuse. Nous avons fusionn�� de mani��re covalente une prot��ine fluorescente bleue (CFP) et une jaune (YFP) en les liant avec diverses s��quences. Par clivage de la s��quence de liaison, une perte du transfert d'��nergie peut ��tre mesur��e par un spectrofluorom��tre. Cette technologie repr��sente un moyen facile de suivre la r��action d'hydrolyse des prot��ases. Les conditions optimales de production de ces substrats CFP-YFP ont ��t�� d��termin��es, de m��me que les param��tres pouvant ��ventuellement influencer le FRET. Ce syst��me poss��de une grande r��sistance �� la prot��olyse non sp��cifique et est applicable �� un grand nombre de prot��ase. Contrairement aux substrats fluorog��niques, il permet d'��tudier les acides amin��s se trouvant des deux c��t��s du site de clivage. Ce syst��me ��tant enti��rement biologique, il est le reflet des interactions prot��ine-prot��ine et repr��sente un outil biologique facile, bon march�� et rapide pour caract��riser les prot��ases. Dans un premier temps, hK14 a ��t�� mise en pr��sence d' une banque de haute diversit�� de pentapeptides al��atoires pr��sent��e �� la surface de phages afin d'identifier des substrats sp��cifiques. Ensuite, le syst��me CFP-YFP a ��t�� employ�� pour trier les peptides s��lectionn��s afin d'identifier les s��quences de substrats les plus sensibles et sp��cifiques pour hK14. Nous avons montr��, qu'en plus de sa pr��visible activit�� de type trypsine, hK14 poss��de aussi une tr��s surprenante activit�� de type chymotrypsine. Les s��quences les plus sensibles ont ��t�� choisies pour cribler la banque de donn��e Swissprot, permettant ainsi l'identification de 6 substrats prot��iques humains potentiels pour hK14. Trois d'entre eux, la laminine α-5, le collag��ne IV et la matriline-4, qui sont des composants de la matrice extracellulaire, ont d��montr�� une grande susceptibilit�� �� l'hydrolyse par hK14. De plus, la s��paration ��l��ctrophor��tique a montr�� que la d��gradation de la laminine α-5 et de la matriline-4 par hK14 devait se produire aux sites identifi��s par la technologie du phage display. Pour terminer, nous avons transform��, par mutagen��se dirig��e, deux serpines (inhibiteurs de prot��ases de type s��rine) connues, AAT et ACT (alpha anti-trypsine et alpha anti-chymotrypsine), qui inhibent un vaste ��ventail d'enzymes humaines en inhibiteurs d'hK14 hautement efficaces et sp��cifiques. Ces inhibiteurs pourront ��tre utilis��s d'une part pour poursuivre certaines exp��riences permettant de mieux comprendre l'implication d'hK14 dans des voies physiologiques ou dans le cancer et d'autre part pour les ��valuer in vivo en tant que traitement potentiel contre le cancer. SUMMARY Proteases consist of enzymes involved in physiological events, but also, in case of dysregulation, in pathogenicity. The human tissue kallikrein family represents the largest human protease cluster and includes several members that either could participate in the course of certain diseases or emerged as potential biological markers, especially in hormone dependent cancers. The human kallikrein 14 has been recently discovered and suggested implications in some disorders, particularly in tumors since its gene expression is up-regulated in prostate and breast cancer tissues and its protein expression increased in the serum of patients with breast and ovarian cancers. However, like most kallikreins, its function remains unknown. To better understand hK14 biological and/or pathological role, we decided to characterize its enzymatic activity. First of all, we developped a biological system suitable for in vitro study of protease activity. This system is based on the so-called FRET phenomenon, that is the Fluorescence Resonance Energy Transfer that occurs between two nearby fluorescent proteins if the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. We fused covalently a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) with diverses sequences. Upon cleavage of the linker sequence by protease, the loss of energy transfer can be measured by a spectrofluorometer allowing an easy following of hydrolysis reaction. The optimal conditions to produce in bacterial system these CFP-YFP substrates were determined as well as the parameters that could eventually influence the FRET. This system demonstrated a high degree of resistance to non-specific proteolysis and applicability to various conditions corresponding to a great number of existing proteases. Other avantages are the possibility to study the amino acids located both sides of the cleavage site as well as the interest to work in a full biological system reflecting protein-protein interaction. A phage substrate library with exhaustive diversity was used prior to CFP-substrate-YFP system to isolate specific human kallikrein 14 substrates. After that the CFP-YFP system was used to sort peptides and identify highly sensitive and specific substrate sequences for hK14. We showed that besides its predictable trypsin-like activity, hK14 also possesses a surprising chymotrypsin-like activity. The screening of the Swissprot database was achieved with the most sensitive sequences and allowed the identification of 6 potential human protein substrates for hK14. Three of them, laminin α-5, collagen IV and matrilin-4, which are components of the extracellular matrix were incubated with hK14, by which they were efficiently hydrolyzed. Moreover, electrophoretic separation revealed that degradation of laminin α-5 and matrilin-4 by hK14 generated fragments with identical molecular size than the predicted N-terminal fragments that would result from hK14 specific cleavage, proving the value of phage display substrate to identify potential substrates. Finally, with site-directed mutagenesis, we transformed two well-known serpins (serine protease inhibitors), AAT and ACT (alpha anti-trypsin and alpha anti-chymotrypsin), which inhibit a vast spectrum of human enzymes into highly efficient and specific hK14 inhibitors. These inhibitors will be used to pursue experiments that could help understand hK14 implication in physiological pathways as well as in cancer biology and also to perform their in vivo evalution as potential cancer treatment.

Paper de l��� Haemophilus influenzae en les exacerbacions de la Malaltia Pulmonar Obstructiva Cr��nica (MPOC): tipificaci�� molecular i factors de virul��ncia

Objetivos: 1.-Identificar los factores cl��nicos y microbiol��gicos que ayuden a predecir la aparici��n de exacerbaciones en la EPOC. 2.-Diagn��stico y cuantificaci��n de las especies bacterianas aisladas en esputo (fase de exacerbaci��n y estable) .3.- Tipificaci��n genot��pica secuencial de las cepas de H. influenzae y P. aeruginosa. 4.- Impacto del tratamiento antibi��tico en la aparici��n de resistencias en estos pat��genos. 5.- Dise��o: Estudio prospectivo (3 a��os). ��mbito del estudio: Hospital Universitario de tercer nivel. Pacientes con EPOC grave atendidos en la Consulta Monogr��fica de EPOC del Servicio de Neumolog��a. M��todos microbiol��gicos: Cuantificaci��n de la carga bacteriana en muestras respiratorias en fase estable y en exacerbaci��n. Estudio de la sensibilidad ���in vitro���. Tipificaci��n molecular (PFGE y MLST) de H. influenzae y P. aeruginosa. Estudio de los genes de virulencia de H. influenzae mediante PCR. Resultados: Desde Febrero de 2010 a Julio de 2011 se han incluido 77 pacientes. Los microorganismos m��s frecuentemente aislados en fase de exacerbaci��n fueron: P. aeruginosa (29.3%), H. influenzae (15.92%), M. catarrhalis (12.74%), S. pneumoniae (10.19%) y S. aureus (5.10%). En los 88 episodios por P. aeruginosa se detectaron 38 genotipos diferentes. En los 41 episodios por H. influenzae se detectaron 39 genotipos diferentes. El 10% de los episodios fueron polimicrobianos. Los episodios de EAEPOC y de fase estable tuvieron una distribuci��n de microorganismos similar. Sin embargo, cuando se cuantificaron las cargas bacterianas fueron mayores en EAEPOC (intervalo 4x107 -2x108) que en fase estable (intervalo 2x105 -4x107). Conclusiones: El genotipo de las cepas de P. aeruginosa y H. influenzae aisladas en EAEPOC difieren de un paciente a otro, sin embargo la mayor��a de los episodios de cada paciente est��n causados por un genotipo ��nico.

Quelques m��canismes mol��culaires impliqu��s dans la pathogen��se du diab��te de type II et leur importance th��rapeutique potentielle [Various molecular mechanisms involved in the pathogenesis of type II diabetes and their potential therapeutic importance]

The pancreatic beta cell presents functional abnormalities in the early stages of development of non-insulin dependent diabetes mellitus (NIDDM). The disappearance of the first phase of insulin secretion induced by a glucose load is a early marker of NIDDM. This abnormality could be secondary to the low expression of the pancreatic glucose transporter GLUT2. Together with the glucokinase enzyme, GLUT2 is responsible for proper beta cell sensing of the extracellular glucose levels. In NIDDM, the GLUT2 mRNA levels are low, a fact which suggests a transcriptional defect of the GLUT2 gene. The first phase of glucose-induced insulin secretion by the beta pancreatic cell can be partly restored by the administration of a peptide discovered by a molecular approach, the glucagon-like peptide 1 (GLP-1). The gene encoding for the glucagon is expressed in a cell-specific manner in the A cells of the pancreatic islet and the L cells of the intestinal tract. The maturation process of the propeptide encoded by the glucagon gene is different in the two cells: the glucagon is the main hormone produced by the A cells whereas the glucagon-like peptide 1 (GLP-1) is the major peptide synthesized by the L cells of the intestine. GLP-1 is an incretin hormone and is at present the most potent insulinotropic peptide. The first results of the administration of GLP-1 to normal volunteers and diabetic patients are promising and may be a new therapeutic approach to treating diabetic patients.

Molecular characterization of signalling complexes involved in G-protein coupled receptor-induced cardiac hypertrophy

In response to pathological stresses, the heart undergoes a remodelling process associated with cardiac hypertrophy. Since sustained hypertrophy can progress to heart failure, there is an intense investigation about the intracellular signalling pathways that control cardiomyocyte growth. Accumulating evidence has demonstrated that most stimuli known to initiate pathological changes associated with the development of cardiac hypertrophy activate G protein-coupled receptors (GPCRs) including the αl-adrenergic- (αl-AR), Angiotensin II- (AT-R) and endothelin-1- (ET-R) receptors. In this context, we have previously identified a cardiac scaffolding protein, called AKAP-Lbc (Α-kinase anchoring protein), with an intrinsic Rho specific guanine nucleotide exchange factor activity, that plays a key role in integrating and transducing hypertrophic signals initiated by these GPCRs (Appert-Collin, Cotecchia et al. 2007). Activated RhoA controls the transcriptional activation of genes involved in cardiomyocyte hypertrophy through signalling pathways that remain to be characterized. Here, we identified the nuclear factor-Kappa Β (NF-κΒ) activating kinase ΙΚΚβ as a novel AKAP-Lbc interacting protein. This raises the hypothesis that AKAP-Lbc might promote cardiomyocyte growth by maintaining a signalling complex that promotes the activation of the pro-hypertrophic transcription factor NF-κΒ. In fact, the activation of NF- κΒ-dependent transcription has been detected in numerous disease contexts, including hypertrophy, ischemia/reperfusion injury, myocardial infarction, allograft rejection, myocarditis, apoptosis, and more (Hall, Hasday et al. 2006). While it is known by more than a decade that NF-κΒ is a critical mediator of cardiac hypertrophy, it is currently poorly understood how pro-hypertrophic signals controlling NF-κΒ transcriptional activity are integrated and coordinated within cardiomyocytes. In this study, we show that AKAP-Lbc and ΙΚΚβ form a transduction complex in cardiomyocytes that couples activation of αl-ARs to NF-κB-mediated transcriptional reprogramming events associated with cardiomyocyte hypertrophy. In particular, we can show that activation of ΙΚΚβ within the AKAP-Lbc complex promotes NF-κB-dependent production of interleukine-6 (IL-6), which, in turn, enhances foetal gene expression. These findings indicate that the AKAP-Lbc/ΙΚΚβ complex is critical for selectively directing catecholamine signals to the induction of cardiomyocyte hypertrophy.

Functional and molecular evolutionary analysis of the "Brevix Radix" gene family in mono-and dicotyledonous plants

ABSTRACTIn contrast to animals, plants cannot move from their place of birth and, therefore, need to adapt to their particular habitat in order to survive. Thus, plant development is remarkably plastic, making plants an ideal system for the isolation of genes that account for intraspecific natural variation and possibly environmental adaptation. However, to date, this approach mostly identified null alleles and missed mutations with subtle effects. For instance, BREVIS RADIX (BRX) has been isolated as a key regulator of root growth through a naturally occurring loss-of-function allele in the Arabidopsis thaliana accession Uk-1 and is the founding member of a highly-conserved plant-specific gene family.In this work, we show that a strong selective pressure is acting on the BRX gene family and dates back before the monocot-dicot divergence. However, functional diversification is observed mainly in dicotyledon BRX family genes and is correlated with acceleration in the evolutionary rates in the N-terminal regions. Population genetic data revealed that BRX is highly conserved across Arabidopsis accessions and presents signatures of adaptation. Interestingly, a seven amino acid deletion polymorphism in BRX sequence was found in a few accessions, which seems to be responsible for their enhanced primary root growth. Nevertheless, BRX might not only be active in the root, as suggested by its expression in the shoot. Indeed, leaves and cotyledons of brx mutants are significantly smaller than wild- type. This phenotype is a direct consequence of the absence of BRX function in the shoot rather than an indirect effect of an altered root system growth. Interestingly, cotyledons of brx plants reflect the same physiological defects as the root. Moreover, phenotypes in BRX gain-of-function plants, such as epinastic leaves and increased epidermal cell size, could be associated with an increase in leaf brassinosteroid content.Collectively, these results indicate that BRX contributes to local adaptation by ubiquitously regulating plant growth, probably through the modulation of brassinosteroid biosynthesis.R��SUM��Contrairement �� la plupart des animaux, les plantes ne peuvent se mouvoir et doivent ainsi s'adapter �� leur environnement pour survivre. Pour cette raison, elles repr��sentent un syst��me id��al pour l'identification de g��nes contribuant �� la variation naturelle intra- sp��cifique, ainsi qu'�� l'adaptation. Cependant, cette approche a, jusqu'�� pr��sent, surtout permis d'isoler des all��les nuls et non des mutations conf��rant des effets plus subtiles. C'est le cas du g��ne  REVIS RADIX (BRX), un r��gulateur cl�� de la croissance racinaire, qui a ��t�� identifi�� gr��ce �� un all��le non-fonctionnel pr��sent dans l'accession naturelle d'Arabidopsis thaliana Uk-1. BRX et ses homologues des plantes mono- et dicotyl��dones forment une famille tr��s conserv��e et sp��cifique aux plantes.Dans ce travail, nous d��montrons que la famille de g��nes BRX est soumise �� une forte pression de s��lection qui remonte avant la divergence entre mono- et dicotyl��dones. Cependant, une diversification fonctionnelle a ��t�� observ��e chez les g��nes des dicotyl��dones et corr��le avec une acc��l��ration de la vitesse d'��volution dans leur r��gion N- terminale. Une analyse g��n��tique de diff��rentes accessions naturelles d'Arabidopsis a r��v��l�� que BRX est hautement conserv�� et pr��sente des signatures d'adaptation. Remarquablement, un polymorphisme de d��l��tion de sept acides amin��s a ��t�� d��tect�� dans quelques accessions et a pour cons��quence une plus forte croissance de la racine primaire. N��anmoins, il semble que le r��le de BRX ne se limite pas qu'�� la racine, comme indiqu�� par son expression dans les parties a��riennes de la plante. En effet, les mutants brx pr��sentent des cotyl��dons et des feuilles significativement plus petits que le type sauvage, une cons��quence directe de l'absence d'activit�� de BRX dans ces organes. Nous avons aussi not�� que les cotyl��dons des mutants brx, �� l'instar des racines, ont une perception alt��r��e de l'auxine et peuvent ��tre compl��ment��s par l'application exog��ne de brassinost��ro��des. De plus, dans des plantes pr��sentant un gain de fonction BRX, les feuilles sont ��pinastiques et les cellules de leur ��piderme plus grandes. Ces ph��notypes sont accompagn��s d'une augmentation de la concentration de brassinost��ro��des dans les feuilles. Conjointement, ces r��sultats d��montrent que BRX contribue �� une adaptation locale de la plante par la r��gulation g��n��rale de sa croissance, probablement en modulant la biosynth��se des brassinost��ro��des.

Molecular and immunological evaluation of the expression of cancer/testis gene products in human colorectal cancer.

Tumor-specific gene products, such as cancer/testis (CT) antigens, constitute promising targets for the development of T cell vaccines. Whereas CT antigens are frequently expressed in melanoma, their expression in colorectal cancers (CRC) remains poorly characterized. Here, we have studied the expression of the CT antigens MAGE-A3, MAGE-A4, MAGE-A10, NY-ESO-1 and SSX2 in CRC because of the presence of well-described HLA-A2-restricted epitopes in their sequences. Our analyses of 41 primary CRC and 14 metastatic liver lesions confirmed the low frequency of expression of these CT antigens. No increased expression frequencies were observed in metastatic tumors compared to primary tumors. Histological analyses of CRC samples revealed heterogeneous expression of individual CT antigens. Finally, evidence of a naturally acquired CT antigen-specific CD8(+) T cell response could be demonstrated. These results show that the expression of CT antigens in a subset of CRC patients induces readily detectable T cell responses.

First insights into the transcriptome and development of new genomic tools of a widespread circum-Mediterranean tree species, Pinus halepensis Mill.

Aleppo pine (Pinus halepensis Mill.) is a relevant conifer species for studying adaptive responses to drought and fire regimes in the Mediterranean region. In this study, we performed Illumina next-generation sequencing of two phenotypically divergent Aleppo pine accessions with the aims of (i) characterizing the transcriptome through Illumina RNA-Seq on trees phenotypically divergent for adaptive traits linked to fire adaptation and drought, (ii) performing a functional annotation of the assembled transcriptome, (iii) identifying genes with accelerated evolutionary rates, (iv) studying the expression levels of the annotated genes and (v) developing gene-based markers for population genomic and association genetic studies. The assembled transcriptome consisted of 48,629 contigs and covered about 54.6 Mbp. The comparison of Aleppo pine transcripts to Picea sitchensis protein-coding sequences resulted in the detection of 34,014 SNPs across species, with a Ka /Ks average value of 0.216, suggesting that the majority of the assembled genes are under negative selection. Several genes were differentially expressed across the two pine accessions with contrasted phenotypes, including a glutathione-s-transferase, a cellulose synthase and a cobra-like protein. A large number of new markers (3334 amplifiable SSRs and 28,236 SNPs) have been identified which should facilitate future population genomics and association genetics in this species. A 384-SNP Oligo Pool Assay for genotyping with the Illumina VeraCode technology has been designed which showed an high overall SNP conversion rate (76.6%). Our results showed that Illumina next-generation sequencing is a valuable technology to obtain an extensive overview on whole transcriptomes of nonmodel species with large genomes.

Cell autonomous lipin 1 function is essential for development and maintenance of white and brown adipose tissue.

Through analysis of mice with spatially and temporally restricted inactivation of Lpin1, we characterized its cell autonomous function in both white (WAT) and brown (BAT) adipocyte development and maintenance. We observed that the lipin 1 inactivation in adipocytes of aP2(Cre/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice resulted in lipodystrophy and the presence of adipocytes with multilocular lipid droplets. We further showed that time-specific loss of lipin 1 in mature adipocytes in aP2(Cre-ERT2/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice led to their replacement by newly formed Lpin1-positive adipocytes, thus establishing a role for lipin 1 in mature adipocyte maintenance. Importantly, we observed that the presence of newly formed Lpin1-positive adipocytes in aP2(Cre-ERT2/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice protected these animals against WAT inflammation and hepatic steatosis induced by a high-fat diet. Loss of lipin 1 also affected BAT development and function, as revealed by histological changes, defects in the expression of peroxisome proliferator-activated receptor alpha (PPARα), PGC-1α, and UCP1, and functionally by altered cold sensitivity. Finally, our data indicate that phosphatidic acid, which accumulates in WAT of animals lacking lipin 1 function, specifically inhibits differentiation of preadipocytes. Together, these observations firmly demonstrate a cell autonomous role of lipin 1 in WAT and BAT biology and indicate its potential as a therapeutical target for the treatment of obesity.

Light-regulated plant growth and development.

Plants are sessile and photo-autotrophic; their entire life cycle is thus strongly influenced by the ever-changing light environment. In order to sense and respond to those fluctuating conditions higher plants possess several families of photoreceptors that can monitor light from UV-B to the near infrared (far-red). The molecular nature of UV-B sensors remains unknown, red (R) and far-red (FR) light is sensed by the phytochromes (phyA-phyE in Arabidopsis) while three classes of UV-A/blue photoreceptors have been identified: cryptochromes, phototropins, and members of the Zeitlupe family (cry1, cry2, phot1, phot2, ZTL, FKF1, and LKP2 in Arabidopsis). Functional specialization within photoreceptor families gave rise to members optimized for a wide range of light intensities. Genetic and photobiological studies performed in Arabidopsis have shown that these light sensors mediate numerous adaptive responses (e.g., phototropism and shade avoidance) and developmental transitions (e.g., germination and flowering). Some physiological responses are specifically triggered by a single photoreceptor but in many cases multiple light sensors ensure a coordinated response. Recent studies also provide examples of crosstalk between the responses of Arabidopsis to different external factors, in particular among light, temperature, and pathogens. Although the different photoreceptors are unrelated in structure, in many cases they trigger similar signaling mechanisms including light-regulated protein-protein interactions or light-regulated stability of several transcription factors. The breath and complexity of this topic forced us to concentrate on specific aspects of photomorphogenesis and we point the readers to recent reviews for some aspects of light-mediated signaling (e.g., transition to flowering).