Fusarium verticillioides NAT1 (FDB2) N-malonyltransferase is structurally, functionally and phylogenetically distinct from its N-acetyltransferase (NAT) homologues.

Fusarium endophytes damage cereal crops and contaminate produce with mycotoxins. Those fungi overcome the main chemical defence of host via detoxification by a malonyl-CoA-dependent enzyme homologous to xenobiotic metabolizing arylamine N-acetyltransferase (NAT). In Fusarium verticillioides (teleomorph Gibberella moniliformis, GIBMO), this N-malonyltransferase activity is attributed to (GIBMO)NAT1, and the fungus has two additional isoenzymes, (GIBMO)NAT3 (N-acetyltransferase) and (GIBMO)NAT2 (unknown function). We present the crystallographic structure of (GIBMO)NAT1, also modelling other fungal NAT homologues. Monomeric (GIBMO)NAT1 is distinctive, with access to the catalytic core through two "tunnel-like" entries separated by a "bridge-like" helix. In the quaternary arrangement, (GIBMO)NAT1 monomers interact in pairs along an extensive interface whereby one entry of each monomer is covered by the N-terminus of the other monomer. Although monomeric (GIBMO)NAT1 apparently accommodates acetyl-CoA better than malonyl-CoA, dimerization changes the active site to allow malonyl-CoA to reach the catalytic triad (Cys110, His158 and Asp173) via the single uncovered entry, and anchor its terminal carboxyl-group via hydrogen bonds to Arg109, Asn157 and Thr261. Lacking a terminal carboxyl-group, acetyl-CoA cannot form such stabilizing interactions, while longer acyl-CoAs enter the active site but cannot reach catalytic Cys. Other NAT isoenzymes lack such structural features, with (GIBMO)NAT3 resembling bacterial NATs and (GIBMO)NAT2 adopting a structure intermediate between (GIBMO)NAT1 and (GIBMO)NAT3. Biochemical assays confirmed differential donor substrate preference of (GIBMO)NAT isoenzymes, with phylogenetic analysis demonstrating evolutionary separation. Given the role of (GIBMO)NAT1 in enhancing Fusarium pathogenicity, unravelling the structure and function of this enzyme may benefit research into more targeted strategies for pathogen control.

Communication between infant boys and their mothers with ADHD symptoms.

AIM: This preliminary longitudinal study examined timing features and type of interaction between infant boys and their mothers with attention deficit hyperactivity disorder (ADHD) symptoms METHOD: Ten infants and their mothers with ADHD symptoms and 10 control dyads were video recorded at home during free play interactions when infants were 2-, 4-, 6-, and 9-month old. Microanalysis of the video recordings was carried out to assess synchronization, turn-taking, and type of interaction. Infants' temperament was also assessed RESULTS: ADHD dyads showed shorter synchronization at 2 months and shorter duration of Joint Attention. Partial least squares regression analysis revealed that infant's ability for Joint Attention is predicted mainly by duration of maternal behavior as well as by earlier forms of communication, that is, protoconversations. CONCLUSION: The data from our preliminary study suggest that mothers with ADHD symptoms may have difficulties maintaining their behavior for enough time possibly due to the core symptoms of the disorder, that is, inattention, hyperactivity, and impulsivity. This maternal deficit seems to affect temporal coordination with their infants and maybe the development of more complex forms of interaction. Clinical implications of these findings are also discussed.

Propósito: Este preliminar estudio longitudinal examinó las características de tiempo y el tipo de interacción entre infantes varones y sus madres con síntomas de Trastorno con Déficit de Atención con Hiperactividad (TDAH). Método: Diez infantes y sus madres con síntomas de TDAH y 10 díadas de control fueron grabados en video en casa durante interacciones de juego libre cuando los infantes tenían 2, 4, 6 y 9 meses de edad. Se llevó a cabo un microanálisis de las grabaciones de video para evaluar la sincronización, el turnarse y el tipo de interacción. También se evaluó el temperamento de los infantes. Resultados: Las díadas TDAH mostraron una más corta sincronización a los 2 meses y una duración más corta de la Atención Compartida. El análisis de Regresión de Cuadrados Mínimos Parciales reveló que la habilidad del infante para la Atención Compartida es afirmada principalmente por la duración de la conducta materna, así como también por formas más tempranas de comunicación, v.g. protoconversaciones. Conclusión: La información de nuestro estudio preliminar sugiere que las madres con síntomas de TDAH pudieran tener dificultades para mantener su conducta por suficiente tiempo posiblemente a causa de los síntomas centrales del trastorno, v.g. falta de atención, hiperactividad e impulsividad. Este déficit materno parece afectar la coordinación temporal con sus infantes y quizás el desarrollo de más complejas formas de interacción. También se discuten las implicaciones clínicas de estos resultados.

But: Cette étude longitudinale préliminaire a examiné les traits de timing et les types d'interaction entre des nourrissons garçons et leurs mères avec des symptômes de TDAH. Méthode: Dix bébés et leurs mères avec des symptômes de TDAH et 1- dyades de contrôle ont été filmés à la vidéo durant des interactions de jeu libre quand les bébés avaient 2, 4, 6, et 9 mois. Une microanalyse des enregistrements vidéo a été faite afin d'évaluer la synchronisation, les tours de role et le type d'interaction. Le tempérament des bébés a aussi été évalué. Résultats: Les dyades TDAH ont fait preuve d'une synchronisation plus courte à deux mois et d'une durée plus courte d'Attention Commune. Une analyse de régression partielle par les moindres carrés a révélé que la capacité du nourrisson à une Attention Commune est prédite essentiellement par la durée du comportement maternel ainsi que par les formes précédentes de communication, comme des protoconversations. Conclusion: Les données de notre étude préliminaire suggèrent que les mères avec des symptômes TDAH peuvent avoir des difficultés à maintenir leur comportement pour assez de temps, peut-être du fait des symptômes essentiels du trouble: inattention, hyperactivité et impulsivité. Ce déficit maternel semble affecter la coordination temporelle avec leurs bébés et peut-être le développement de formes d'interaction plus complexes. Les implications cliniques de ces résultats sont aussi discutées.

Homologues of xenobiotic metabolizing N-acetyltransferases in plant-associated fungi: Novel functions for an old enzyme family.

Plant-pathogenic fungi and their hosts engage in chemical warfare, attacking each other with toxic products of secondary metabolism and defending themselves via an arsenal of xenobiotic metabolizing enzymes. One such enzyme is homologous to arylamine N-acetyltransferase (NAT) and has been identified in Fusarium infecting cereal plants as responsible for detoxification of host defence compound 2-benzoxazolinone. Here we investigate functional diversification of NAT enzymes in crop-compromising species of Fusarium and Aspergillus, identifying three groups of homologues: Isoenzymes of the first group are found in all species and catalyse reactions with acetyl-CoA or propionyl-CoA. The second group is restricted to the plant pathogens and is active with malonyl-CoA in Fusarium species infecting cereals. The third group generates minimal activity with acyl-CoA compounds that bind non-selectively to the proteins. We propose that fungal NAT isoenzymes may have evolved to perform diverse functions, potentially relevant to pathogen fitness, acetyl-CoA/propionyl-CoA intracellular balance and secondary metabolism.

Distinct Wnt-driven primitive streak-like populations reflect in vivo lineage precursors.

During gastrulation, epiblast cells are pluripotent and their fate is thought to be constrained principally by their position. Cell fate is progressively restricted by localised signalling cues from areas including the primitive streak. However, it is unknown whether this restriction accompanies, at the individual cell level, a reduction in potency. Investigation of these early transition events in vitro is possible via the use of epiblast stem cells (EpiSCs), self-renewing pluripotent cell lines equivalent to the postimplantation epiblast. Strikingly, mouse EpiSCs express gastrulation stage regional markers in self-renewing conditions. Here, we examined the differentiation potential of cells expressing such lineage markers. We show that undifferentiated EpiSC cultures contain a major subfraction of cells with reversible early primitive streak characteristics, which is mutually exclusive to a neural-like fraction. Using in vitro differentiation assays and embryo grafting we demonstrate that primitive streak-like EpiSCs are biased towards mesoderm and endoderm fates while retaining pluripotency. The acquisition of primitive streak characteristics by self-renewing EpiSCs is mediated by endogenous Wnt signalling. Elevation of Wnt activity promotes restriction towards primitive streak-associated lineages with mesendodermal and neuromesodermal characteristics. Collectively, our data suggest that EpiSC pluripotency encompasses a range of reversible lineage-biased states reflecting the birth of pioneer lineage precursors from a pool of uncommitted EpiSCs similar to the earliest cell fate restriction events taking place in the gastrula stage epiblast.

Comparative genomic and phylogenetic investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family.

Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes characterized in several bacteria and eukaryotic organisms. We report a comprehensive phylogenetic analysis employing an exhaustive dataset of NAT-homologous sequences recovered through inspection of 2445 genomes. We describe the first NAT homologues in viruses, archaea, protists, many fungi and invertebrates, providing complete annotations in line with the consensus nomenclature. Contrary to the NAT genes of vertebrates, introns are commonly found within the homologous coding regions of lower eukaryotes. The NATs of fungi and higher animals are distinctly monophyletic, but evidence supports a mixed phylogeny of NATs among bacteria, protists and possibly some invertebrates.

Phenotypic mismatch repair hMSH2 and hMLH1 gene expression profiles in primary non-small cell lung carcinomas.

BACKGROUND: Defects in the human DNA mismatch repair genes (MMR) hMSH2 and hMLH1 are responsible for the development of sporadic and hereditary colorectal cancers. The role of MMR genes in the pathogenesis of lung cancer has not been elucidated. The aim of this study was to address the phenotypic mRNA expression profiles of mismatch DNA repair system in lung cancer. MATERIALS AND METHODS: We evaluated the mRNA levels of the hMSH2 and hMLH1 components of the mismatch DNA repair (MMR) system in 29 unselected frozen pairs of primary non-small cell lung carcinomas (NSCLCs) and their adjacent normal tissue (ANTs) specimens by quantitative real-time PCR analysis relative to housekeeping Porphobilinogen deaminase (hPBGD) mRNA. To simplify and potentially improve the analysis of data, we defined for each individual MMR mRNA two possible phenotypes: a regular (R(2): hMSH2/hPBGD mRNAs> or =1 and R(1): hMLH1/hPBGD mRNAs> or =1) and a reduced (r(2): hMSH2/hPBGD mRNAs<1 and r(1): hMLH1/hPBGD mRNAs<1). The presence of MMR gene expression was evaluated after conversion of the molecular mRNA levels into clinically distinct phenotypic entities by these working criteria, based on the hypothesis that reduced mRNA and protein levels result in lower or non-functional MMR. RESULTS: Phenotyping defined four distinct MMR system expression profiles, R(2)R(1), r(2)R(1), R(2)r(1) and r(2)r(1) by ascending tumor progression rate and identified a previously unrecognized disease-associated phenotypic entity (r(2)r(1)). The phenotype-based biological aspects of the MMR system suggested that its two components: (1) function independently and (2) are not directly involved in the onset of the transformation process, since healthy lung tissue was devoid of r(2)r(1) phenotypes. CONCLUSION: These findings link MMR mRNA levels of paired lung tissue specimens to patients' clinical condition and suggest that phenotypic translation of molecular MMR data refines the biology of the MMR system with consequent diagnostic implications in the clinical assessment of lung cancer patients.

Development of a single-tube polymerase chain reaction assay for the simultaneous detection of Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus spp. directly in clinical samples.

This study describes the development and evaluation of a multiplex single-tube polymerase chain reaction assay for the simultaneous detection of Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus spp. used as target species-specific or genus-specific genes. The assay enables the detection of 5 to 50 pg of bacterial DNA. The sensitivity of the assay was evaluated as 100% for P. aeruginosa, S. aureus, and Streptococcus spp., and 94.3% for H. influenzae; the specificity was 100% for all 4 microorganisms (positive predictive value, 100%; negative predictive value, 98.2%). The assay permits rapid and accurate detection of these 4 microorganisms in a wide range of clinical samples such as whole blood, cerebrospinal, ear, pleural and ophthalmic fluids, as well as bronchoalveolar lavage and bronchial secretions.

Microsatellite instability and loss of heterozygosity at the MEN1 locus in lung carcinoid tumors: a novel approach using real-time PCR with melting curve analysis in histopathologic material.

The possible causes and genetic mechanisms of pulmonary carcinoid tumor development are unclear. In this study, we examined genetic alterations at the MEN1 locus in archival material from 15 pulmonary carcinoids. We employed, for the first time in this setting, real-time PCR with melting curve analysis in order to identify loss of heterozygosity (LOH) or microsatellite instability (MI) in two polymorphic markers (PYGM, D11S449) at the MEN1 locus and one additional marker (D11S906) of a putative oncosuppressive region distal to the MEN1 gene. Sequencing data were available in a selected subset of tumors in order to verify the reliability of real-time PCR analysis. We observed LOH at PYGM in 38% of the cases and MI in 13.3% of the cases. Our data indicate that real-time PCR with melting curve analysis is a reliable technique for LOH and MI detection and indicate that genetic errors at the MEN1 locus but also distal to it may be involved in the development of sporadic pulmonary carcinoid tumors.