Interaction between IRF6 and TGFA Genes Contribute to the Risk of Nonsyndromic Cleft Lip/Palate

Previous evidence from tooth agenesis studies suggested IRF6 and TGFA interact. Since tooth agenesis is commonly found in individuals with cleft lip/palate (CL/P), we used four large cohorts to evaluate if IRF6 and TGFA interaction contributes to CL/P. Markers within and flanking IRF6 and TGFA genes were tested using Taqman or SYBR green chemistries for case-control analyses in 1,000 Brazilian individuals. We looked for evidence of gene-gene interaction between IRF6 and TGFA by testing if markers associated with CL/P were overtransmitted together in the case-control Brazilian dataset and in the additional family datasets. Genotypes for an additional 142 case-parent trios from South America drawn from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), 154 cases from Latvia, and 8,717 individuals from several cohorts were available for replication of tests for interaction. Tgfa and Irf6 expression at critical stages during palatogenesis was analyzed in wild type and Irf6 knockout mice. Markers in and near IRF6 and TGFA were associated with CL/P in the Brazilian cohort (p<10⁻⁶). IRF6 was also associated with cleft palate (CP) with impaction of permanent teeth (p<10⁻⁶). Statistical evidence of interaction between IRF6 and TGFA was found in all data sets (p = 0.013 for Brazilians; p = 0.046 for ECLAMC; p = 10⁻⁶ for Latvians, and p = 0.003 for the 8,717 individuals). Tgfa was not expressed in the palatal tissues of Irf6 knockout mice. IRF6 and TGFA contribute to subsets of CL/P with specific dental anomalies. Moreover, this potential IRF6-TGFA interaction may account for as much as 1% to 10% of CL/P cases. The Irf6-knockout model further supports the evidence of IRF6-TGFA interaction found in humans.     

Targeting Tissular Immune Response Improves Diagnostic Performance of Anti-Saccharomyces cerevisiae Antibodies (ASCA) in Crohn’s Disease

Antibodies against Saccharomyces cerevisiae (ASCA) and Escherichia coli outer membrane porin C (anti-OmpC) are known to be detectable in the serum of patients with Crohn’s disease (CD) but display a very poor sensitivity for the disease especially in forms with isolated colonic involvement. In this study we aimed at evaluating performances of these markers in supernatant of cultured colonic biopsies.Patients with colonic CD (n =  67), ulcerative colitis (UC) (n = 35) and control individuals (n = 37) were prospectively recruited for colonoscopy pinch biopsies and blood sampling. Serum and supernatant of culture tissues were analyzed for ASCA and anti-OmpC. Direct immunofluorescence was also performed on colonic tissues for total IgA detection.We detected for the first time ASCA IgA/IgG and anti-OmpC IgA in cultured colonic tissue supernatants. For both markers, sensitivities for diagnosing CD were better in supernatants (ASCA: 53.7%, anti-OmpC: 28.4%) than in serum (ASCA: 31.3%, anti-OmpC: 22.4%). Combination of results from a panel of these tests gave the greatest sensitivity ever described for CD diagnosis in colonic forms (70.2%).In this study, we described, for the first time, ASCA in supernatant of colonic tissue cultures. This assaying approach in CD diagnosis should be taken into consideration in the future especially in CD forms with isolated colonic involvement.     

Marked defects in the expression and glycosylation of alpha2-HS glycoprotein/fetuin-A in plasma from neonates with intrauterine growth restriction: proteomics screening and potential clinical implications

Intrauterine growth restriction (IUGR) has been associated with increased perinatal morbidity and mortality and increased morbidity and metabolic abnormalities later in life. IUGR is characterized as the failure of a fetus to achieve his or her genetic growth potential in utero. Altered protein expression profiles associated with IUGR may be informative on the pathologic mechanisms of this condition and might reveal potential markers for postnatal complications. The aim of this study was to compare protein profiles of umbilical cord plasma from IUGR and appropriate for gestational age full-term neonates. Blood samples from doubly clamped umbilical cord at delivery from 10 IUGR and 10 appropriate for gestational age full-term neonates were analyzed by two-dimensional electrophoresis and MS. Prominent changes of the alpha2-HS glycoprotein/fetuin-A were observed in IUGR cases. Specifically we showed that these changes occur primarily at the level of post-translational modifications of the protein. Using a combination of mass spectrometry and classical biochemical assays, single and heavy chain forms of fetuin-A were found to lack the normally present O-linked sialic acids in IUGR neonates. Fetuin A is a glycoprotein that has been associated with promotion of in vitro cell replication, fetal growth and osteogenesis, and protection from Gram-negative bacterial endotoxins. Prominent defects in glycosylation/sialylation of fetuin-A revealed by our study might be responsible for impaired function of fetuin-A, leading to deficient fetal growth, especially osteogenesis, and/or to the development of complications frequently seen later in the lives of IUGR neonates.     

Een carrière als specialist ouderengeneeskunde; iets voor de huidige geneeskundestudent?

Background

Due to the rise of older patients with multi-morbidity, we need more elderly care physicians. However, not all available training slots for the elderly care medicine specialty have been fully utilized in recent years. To assess medical student interest in this specialty as well as potential causes for this interest we explored the interest of medical students in the profession of elderly care physician, as well their perception of this profession, both in the ‘old curriculum’ and in a ‘new curriculum’, where the new curriculum had a mandatory elderly care medicine clerkship and more competency-related learning.

Method

At VUmc 120 final year medical students were asked to complete a questionnaire in 2014 about professional preferences and professional characteristics. The same questionnaire had been presented five years earlier, in 2009, to 150 medical students at the end of their final year.

Results

The response rates were 100% and 85% respectively. Of the students in the new curriculum 16,7% considered a career in elderly care medicine. This percentage was 9,4% for students in the old curriculum (p?=?0,087). The characteristics of the profession that appealed most to the students, but were not considered applicable to elderly care medicine were: diagnostics skills, acute complaints, visible results. The professional characteristics that students found to be very much applicable to this specialty, but less attractive for their future profession were: psychosocial, chronic and terminal conditions.

Discussion

We observe a trend that students in the new curriculum are more interested in the profession of elderly care physician, even though this interest remains limited. We recommend that the basic medical training, both in the bachelor phase and in a mandatory elderly care medicine clerkship, focus more on demonstrating that the characteristics students find appealing in the medical profession are indeed present in this speciality. Also, the basic training should concentrate more on guidance and treatment of patients with chronic and terminal conditions.

     

Taxonomic investigation using DNA fingerprinting in Geitlerinema species (Oscillatoriales, Cyanobacteria)

The taxonomic study of 14 strains of Geitlerinema amphibium (Ag. ex Gom.) Anagnostidis and Geitlerinema unigranulatum (R.N. Singh) Komárek and Azevedo, coming from several localities was undertaken. Use was made of morphological data and molecular data were obtained by means of the DNA fingerprinting technique using highly iterated palindrome (HIP1) sequences. The employed morphological characteristics were those used for species taxonomic identification belonging to the Geitlerinema genus, namely, cell dimensions, shape of the apical cell, motility, number and localization of cyanophycin granules in the cell. The two species revealed as polymorphic were discriminated only by means of the average cellular diameters. In spite of this, minima and maxima values of the cellular diameters overlapped. It was found from molecular analysis that a high genetic diversity and the formation of two clusters consisted of G. amphibium and G. unigranulatum, plus a sole strain keeping itself isolated from the remaining. Also, these clusters were not related to the geographic location; they encompassed strains from water bodies distant from each other by as much as 3500 km, or Brazilian and Spanish strains. Molecular and morphological data support the possibility that G. unigranulatum could be considered a synonym for G. amphibium. HIP1 fingerprinting is a powerful tool for the study of genetic of cyanobacteria closely related taxa. This study points to the necessity of using other than morphological data in the taxonomic revision of cyanobacteria, as well as in the proposition of new taxons.     

Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution: production of an efficient enzyme for bioconversion of fine chemicals

The P450 cytochromes constitute a large family of hemoproteins that catalyze the monooxygenation of a diversity of hydrophobic substrates. CYP102A2 is a catalytically self-sufficient cytoplasmic enzyme from Bacillus subtilis, containing both a monooxygenase domain and a reductase domain on a single polypeptide chain. CYP102A2 was subjected to error-prone PCR to generate mutants with enhanced activity with fatty acids and other aromatic substrates. The library of CYP102A2 mutants was expressed in BL21(DE3) Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. After a single round of error-prone PCR, the variant Pro15Ser exhibiting modified substrate specificity was generated. This variant showed approximately 6- to 9-fold increased activity with SDS, lauric acid and 1,4-naphthoquinone, and enhanced activity for other substrates such as ethacrynic acid and epsilon-amino-n-caproic acid. Molecular modeling of the CYP102A2 monooxygenase domain suggested that Pro15 is located in a short helical segment and is involved in extensive interactions between the N-terminal domain and the beta2 sheet, which contribute to the formation of the substrate binding site. Thus, Pro15 appears to affect substrate binding and catalysis indirectly. These results clearly demonstrate the importance of remote residues, not readily predicted by rational design, for the determination of substrate specificity. In addition, we report here that the Pro15Ser variant of CYP102A2 can be efficiently immobilized on epoxy-activated Sepharose at pH 8.5 and 4 degrees C. The immobilized variant of CYP102A2 retains most of its activity (81%) and shows improved stability at 37 degrees C. The approach offers the possibility of designing a P450 bioreactor that can be operated over a long period of time with high efficiency and which can be used in fine chemical synthesis.