Identification of a novel mutation in MEF2C gene in an atypical patient with frontotemporal lobar degeneration.

The MEF2C gene encodes a transcription factor known to play a crucial role in molecular pathways affecting neuronal development. MEF2C mutations were described as a genetic cause of developmental disease (MRD20), and several reports sustain its involvement in dementia-related conditions, such as Alzheimer's disease and amyotrophic lateral sclerosis. These pathologies and frontotemporal degeneration (FTLD) are thought to share common physiopathological pathways. In this exploratory study, we searched for alterations in the DNA sequence of exons and boundaries, including 5'- and 3'-untranslated regions (5'UTR, 3'UTR), of MEF2C gene in 11 patients with clinical phenotypes related with MRD20 or FTLD. We identified a heterozygous deletion of 13 nucleotides in the 5'UTR region of a 69 years old FTLD patient. This alteration was absent in 200 healthy controls, suggesting a contribution to this patient's disease phenotype. In silico analysis of the mutated sequence indicated changes in mRNA secondary structure and stability, thus potentially affecting MEF2C protein levels. Furthermore, in vitro functional analysis of this mutation revealed that the presence of this deletion abolished the transcriptional activity of the gene in human embryonic cells and rat brain neurons, probably by modifying MEF2C expression. Altogether, our results provide evidence for the involvement of MEF2C in FTLD manifesting with seizures.

MEF2C orthologues from zebrafish: Evolution, expression and promoter regulation.

MEF2C is a crucial transcription factor for cranial neural crest cells development. An abnormal expression of this protein leads to severe abnormalities in craniofacial features. Recently, a human disease (MRD20) was described as a consequence of MEF2C haploinsufficiency. These patients show severe developmental delay, intellectual disability and dysmorphic features. Zebrafish presents two MEF2C orthologues, mef2ca and mef2cb. In this study we demonstrate a highly conserved pattern of chromosome localization for MEF2C between human and zebrafish, a similar protein sequence and tissue expression profile. We have focused our functional analysis on the zebrafish orthologue mef2cb. We identified three new exons through 5' RACE and described two new transcriptional start sites (TSS). These alternative TSS reflect the occurrence of two alternative promoters differentially regulated by nuclear factors related to craniofacial or neuronal development such as Sox9b, Sox10 and Runx2. We also predict that mef2cb gene may be post transcriptionally regulated by analysing the structure of its 5' UTR region, conserved throughout evolution. Our study provides new insights in MEF2C conservation and provides the first evidence of mef2cb regulation by both transcriptional and post transcriptional mechanisms, thus contributing to validate zebrafish as a good model for future studies concerning MEF2C dependent pathologies.

mef2ca and mef2cb Double Mutant Zebrafish Show Altered Craniofacial Phenotype and Motor Behaviour.

The transcription factor MEF2C is crucial in neuronal, cardiac, bone and cartilage molecular processes, as well as for craniofacial development. MEF2C was associated with the human disease MRD20, whose patients show abnormal neuronal and craniofacial development. Zebrafish mef2ca;mef2cb double mutants were analysed for abnormalities in craniofacial and behaviour development through phenotypic analysis. Quantitative PCR was performed to investigate the expression levels of neuronal marker genes in mutant larvae. The motor behaviour was analysed by the swimming activity of 6 dpf larvae. We found that mef2ca;mef2cb double mutants display several abnormal phenotypes during early development, including those already described in zebrafish carrying mutations in each paralog, but also (i) a severe craniofacial phenotype (comprising both cartilaginous and dermal bone structures), (ii) developmental arrest due to the disruption of cardiac oedema and (iii) clear alterations in behaviour. We demonstrate that the defects observed in zebrafish mef2ca;mef2cb double mutants are similar to those previously described in MEF2C-null mice and MRD20 patients, confirming the usefulness of these mutant lines as a model for studies concerning MRD20 disease, the identification of new therapeutic targets and screening for possible rescue strategies.

Listeria monocytogenes biofilm-associated protein (BapL) may contribute to surface attachment of L. monocytogenes but is absent from many field isolates.

Listeria monocytogenes is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the L. monocytogenes EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in L. monocytogenes to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all L. monocytogenes strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of L. monocytogenes in mice.

Occurrence and persistence of Listeria spp. in the environment of ewe and cow's milk cheese dairies in Portugal unveiled by an integrated analysis of identification, typing and spatial-temporal mapping along production cycle.

Eight dairies, located in two distant geographic regions of Portugal, were screened along the production cycle in order to evaluate the presence and distribution of Listeria spp. in their environment. Three dairies in each region were positive for the presence of listeriae and 213 isolates were obtained. Based on an integrated analysis of RAPD fingerprints with three primers, molecular identification and genomic typing of isolates was performed followed by spatial and temporal mapping on dairy plants. The occurrence of Listeria species by region was noticeable different. Listeria monocytogenes prevailed in South Portugal dairies and L. innocua presented the highest occurrence in Azores, whereas L. seeligeri and L. ivanovii were detected in distinct regions. Dairies were at risk of contamination, from more than one source, whatever the stage in the production cycle and the surface materials used. For the three prevalent species, most of the genomic types were dairy and sampling time specific. Nonetheless, more than one type could be found in each dairy at a particular site and, in a few cases, even for different species. Some dairies also shared types, mainly for L. innocua and usually at the same stage of the production cycle. For L. monocytogenes, PCR serotyping was applied and 52% of genomic types were serotype 4b. An equal frequency of genomic types (24%) was found for serotypes 1/2b or 3b and 1/2a or 3a. The global pattern of types within a dairy is not constant, suggesting cycles of elimination and recontamination along the production cycle.