Amniotic membrane stimulates cell migration by modulating transforming growth factor‐β signalling

Keratinocyte migration is a mandatory aspect of wound healing. We have previously shown that amniotic membrane (AM) applied to chronic wounds assists healing through a process resulting in the overexpression of c‐Jun at the wound's leading edge. We have also demonstrated that AM modifies the genetic programme induced by transforming growth factor‐ß (TGF‐ß) in chronic wounds. Here we used a scratch assay of mink lung epithelial cells (Mv1Lu) and a spontaneously immortalized human keratinocyte cell line (HaCaT) cells to examine the influence of AM application on the underlying signalling during scratch closure. AM application induced c‐Jun phosphorylation at the leading edge of scratch wounds in a process dependent on MAPK and JNK signalling. Strikingly, when the TGF‐ß‐dependent Smad‐activation inhibitor SB431542 was used together with AM, migration improvement was partially restrained, whereas the addition of TGF‐ß had a synergistic effect on the AM‐induced cell migration. Moreover, antagonizing TGF‐ß with specific antibodies in both cell lines or knocking out TGF‐ß receptors in Mv1Lu cells had similar effects on cell migration as using SB431542. Furthermore, we found that AM was able to attenuate TGF‐ß‐Smad signalling specifically at the migrating edge; AM treatment abated Smad2 and Smad3 nuclear localization in response to TGF‐ß in a process dependent on mitogen‐activated protein kinase kinase 1 (MEK1) activation but independent of EGF receptor or JNK activation. The involvement of Smad signalling on AM effects on HaCaT keratinocytes was further corroborated by overexpression of either Smad2 or Smad3 and the use of Smad phosphorylation‐specific inhibitors, revealing a differential influence on AM‐induced migration for each Smad. Thus, AM TGF‐ß‐Smad signalling abating is essential for optimal cell migration and wound closure.     

Gap phenomenon in the bundle of his conduction]

His bundle electrograms recorded during atrial stimulation with the extrastimulus technique permitted the analysis of the so called gaps in bundle branch conduction. Three different types of gaps were identified each with a distinct electrophysiological mechanism. In type I gap a CLBBB pattern disappeared at shorter coupling (A1-A2) intervals because the more premature impulses encountered a greater degree of the delay at the A-V node therefore reaching the left bundle branch after its effective refractory period had ended. During this part of the cycle the H1-H2 were longer and H2-V2 intervals shorter than when CRBBB was present. In type II gap a CRBBB pattern disappeared at shorter coupling intervals because the premature impulses met a greater delay in the proximal portions of the His-Purkinje system. In consequence, they arrived at the right bundle branch when its effective refractory period had expired. When this occurred the H1-H2 interval were shorter and the H2-V2 longer than those at which CRBBB had been present. The patient with type III gap had CLBBB. A-V conduction was possible through the right bundle branch very late in the cycle. However, it could not occur at shorter coupling intervals finally reappearing at even shorter coupling intervals. True supernormal conduction or vagal pulsatile discharges could have been present. Yet, we favor, as the most likely possibility, the existence of 2:1 phase 4 block with latent or abortive escapes not recorded in the surface leads.     

The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.      

Elastin point mutations cause an obstructive vascular disease, supravalvular aortic stenosis

Supravalvular aortic stenosis (SVAS) is an inherited obstructive vascular disease that affects the aorta, carotid, coronary and pulmonary arteries. Previous molecular genetic data have led to the hypothesis that SVAS results from mutations in the elastin gene, ELN. In these studies, the disease phenotype was linked to gross DNA rearrangements (35 and 85 kb deletions and a translocation) in three SVAS families. However, gross rearrangements of ELN have not been identified in most cases of autosomal dominant SVAS. To define the spectrum of ELN mutations responsible for this disorder, we refined the genomic structure of human ELN and used this information in mutational analyses. ELN point mutations co-segregate with the disease in four familial cases and are associated with SVAS in three sporadic cases. Two of the mutations are nonsense, one is a single base pair deletion and four are splice site mutations. In one sporadic case, the mutation arose de novo. These data demonstrate that point mutations of ELN cause autosomal dominant SVAS.      

Avian mycobacteriosis in free-living raptors in Majorca Island,Spain

Avian mycobacteriosis is a chronic, infectious disease caused by different species of mycobacteria, usually belonging to the Mycobacterium avium complex. From 2004 to 2007, 589 raptors brought dead or sick to a wildlife rehabilitation centre in Majorca (Balearic Islands, Spain) were necropsied. The birds belonged to 12 different species, chiefly common kestrel (Falco tinnunculus) (n=297), scops owl (Otus scops) (n=109), barn owl (Tyto alba) (n=75), long-eared owl (Asio otus) (n=58), peregrine falcon (Falco peregrinus) (n=27), and booted eagle (Hieraaetus pennatus) (n=13). Gross lesions compatible with mycobacteriosis were observed in 14 birds (2.4%) found in several locations in Majorca. They were 12 kestrels (prevalence in this species, 4.0%), one long-eared owl (1.7%) and one scops owl (0.9%), all the birds presenting white–yellowish nodules from pinpoint size to 1 cm in diameter in diverse organs, mainly in the liver, spleen and intestine. Affected organs were subjected to bacteriology and molecular identification by polymerase chain reaction and, in all cases, infection with M. avium subspecies avium was confirmed. The observed prevalences are similar to those previously observed in Holland, although the actual prevalence detected in this study is likely to be higher than reported because only birds with gross lesions were subjected to culture. Further molecular characterization with a set of six mycobacterial interspersed repetitive unit–variable number tandem repeat loci was used to sub-type the isolates in order to show the existence of possible epidemiological links. Six different genotypes were found, which points to infection from multiple foci. No temporal or geographical aggregation of the cases was observed to be associated with the presence of positive birds or with the different variable number tandem repeat allelic profiles. The most feasible origin might be water or food sources, although the reservoir of mycobacteria remains unknown.     

A founder mutation in BBS2 is responsible for Bardet‐Biedl syndrome in the Hutterite population: utility of SNP arrays in genetically heterogeneous disorders

Innes AM, Boycott KM, Puffenberger EG, Redl D, MacDonald IM, Chudley AE, Beaulieu C, Perrier R, Gillan T, Wade A, Parboosingh JS. A founder mutation in BBS2 is responsible for Bardet‐Biedl syndrome in the Hutterite population: utility of SNP arrays in genetically heterogeneous disorders. Bardet‐Biedl syndrome (BBS) is a multisystem genetically heterogeneous disorder, the clinical features of which are largely the consequence of ciliary dysfunction. BBS is typically inherited in an autosomal recessive fashion, and mutations in at least 14 genes have been identified. Here, we report the identification of a founder mutation in the BBS2 gene as the cause for the increased incidence of this developmental disorder in the Hutterite population. To ascertain the Hutterite BBS locus, we performed a genome‐wide single nucleotide polymorphism (SNP) analysis on a single patient and his three unaffected siblings from a Hutterite family. The analysis identified two large SNP blocks that were homozygous in the patient but not in his unaffected siblings, one of these regions contained the BBS2 gene. Sequence analysis and subsequent RNA studies identified and confirmed a novel splice site mutation, c.472‐2A>G, in BBS2. This mutation was also found in homozygous form in three subsequently studied Hutterite BBS patients from two different leuts, confirming that this is a founder mutation in the Hutterite population. Further studies are required to determine the frequency of this mutation and its role, if any, in the expression of other ciliopathies in this population.     

Stepwise functional connectivity reveals altered sensory‐multimodal integration in medication‐naïve adults with attention deficit hyperactivity disorder

Neuroimaging studies indicate that children with attention‐deficit/hyperactivity disorder (ADHD) present alterations in several functional networks of the sensation‐to‐cognition spectrum. These alterations include functional overconnectivity within sensory regions and underconnectivity between sensory regions and neural hubs supporting higher order cognitive functions. Today, it is unknown whether this same pattern of alterations persists in adult patients with ADHD who had never been medicated for their condition. The aim of the present study was to assess whether medication‐naïve adults with ADHD presented alterations in functional networks of the sensation‐to‐cognition spectrum. Thirty‐one medication‐naïve adults with ADHD and twenty‐two healthy adults underwent resting‐state functional magnetic resonance imaging (rs‐fMRI). Stepwise functional connectivity (SFC) was used to characterize the pattern of functional connectivity between sensory seed regions and the rest of the brain at direct, short, intermediate, and long functional connectivity distances, thus covering the continuum from the sensory input to the neural hubs supporting higher order cognitive functions. As compared to controls, adults with ADHD presented increased SFC degree within primary sensory regions and decreased SFC degree between sensory seeds and higher order integration nodes. In addition, they exhibited decreased connectivity degree between sensory seeds and regions of the default‐mode network. Consistently, the higher the score in clinical severity scales the lower connectivity degree between seed regions and the default mode network.