Mutational mechanism for DAB1 (ATTTC) n insertion in SCA37: ATTTT repeat lengthening and nucleotide substitution

Dynamic mutations by microsatellite instability are the molecular basis of a growing number of neuromuscular and neurodegenerative diseases. Repetitive stretches in the human genome may drive pathogenicity, either by expansion above a given threshold, or by insertion of abnormal tracts in nonpathogenic polymorphic repetitive regions, as is the case in spinocerebellar ataxia type 37 (SCA37). We have recently established that this neurodegenerative disease is caused by an (ATTTC)n insertion within an (ATTTT)n in a noncoding region of DAB1. We now investigated the mutational mechanism that originated the (ATTTC)n insertion within an ancestral (ATTTT)n . Approximately 3% of nonpathogenic (ATTTT)n alleles are interspersed by AT-rich motifs, contrarily to mutant alleles that are composed of pure (ATTTT)n and (ATTTC)n stretches. Haplotype studies in unaffected chromosomes suggested that the primary mutational mechanism, leading to the (ATTTC)n insertion, was likely one or more T>C substitutions in an (ATTTT)n pure allele of approximately 200 repeats. Then, the (ATTTC)n expanded in size, originating a deleterious allele in DAB1 that leads to SCA37. This is likely the mutational mechanism in three similar (TTTCA)n insertions responsible for familial myoclonic epilepsy. Because (ATTTT)n tracts are frequent in the human genome, many loci could be at risk for this mutational process.We are grateful to the families and individuals who participated in this work. We thank Patricia Ribeiro for technical assistance. This study was financed by Fundo Europeu de Desenvolvimento Regional (FEDER), through the COMPETE 2020 Operational Pro- gram for Competitiveness and Internationalization (POCI) of Portugal 2020, and by the Fundacão para a Ciência e a Tecnologia (FCT) and Ministério da Ciência, Tecnologia e Ensino Superior (Portugal), in the framework of the project POCI-01-0145-FEDER-029255; (PTDC/MED-GEN/29255/2017) to I.S. J.R.L. and C.L.O. were sup- ported by scholarships from PEst-C/SAU/LA0002/2013. S.M. is funded by the project IF/00930/2013/ CP1184/CT0002 from FCT. This work was also funded by the Porto Neurosciences and Neurologic Disease Research Initiative at the Instituto de Investigação e Inovação em Saúde (Norte-01-0145-FEDER- 000008), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTU- GAL 2020 Partnership Agreement with FEDER

El ácido valproico tiene efecto neuroprotector en un modelo de estrés oxidativo agudo en ratas

The excess of the free radical production at has been implied has a common factor in pathogenesis of a big number of neurodegenerative processes, including Alzheimer’s and Parkinson’s disease and in ischemia/reperfusion induced brain damage. Valproic Acid (VPA), which traditionally has been used as an antiepileptic drug, now is proposed as a neuroprotective agent, but is not clear it’s mechanisms of action. In this paper, its effect was investigated in an acute oxidative stress model with Sprague Dawley rats, in which iron sulfate was used as injury and lipid peroxidation and carbonylated proteins were quantified at cerebral cortex, showing that it blocked formation of free radicals. Levels of MDA atcerebral cortex that received oxidative injury in the group of animals treated with VPA were 57% lower in comparison to control group, also the amount of carbonylated proteins was 65% lower in the cerebral cortex of experimental animals in contrast to control group. Furthermore, it was found that the antioxidant effect showed by the VPA, was equally significant than showed by vitamin E.El exceso de producción de radicales libres en el cerebro ha sido implicada como un factor común en la patogénesis de un gran número de procesos neurodegenerativos, incluyendo la enfermedad de Alzheimer, la enfermedad de Parkinson, y en la isquemia/reperfusión cerebral. El ácido valproico (VPA) que tradicionalmente ha sido utilizado como fármaco antiepiléptico, actualmente se propone como agente neuroprotector, pero aún no está claro cual es su mecanismo de acción. En esta investigación se probó su efecto en un modelo de estrés oxidativo agudo en ratas Sprague Dawley para lo cual se usó el sulfato de hierro como agente inductor de daño y se cuantificó la peroxidación lipídica y proteínas carboniladas en corteza cerebral, demostrándose que bloqueó la formación de radicales libres. Los niveles de malondialdehído en la corteza cerebral que recibió la agresión oxidativa en el grupo de animales tratados con VPA fueron 57% inferiores en relación al grupo control que no recibió VPA, mientras que la cantidad de proteínas carboniladas fue un 65% inferior en la corteza cerebral de los animales experimentales en comparación al grupo control. Adicionalmente se encontró que el efecto antioxidante exhibido por el VPA, fue igual de significativo al presentado por la vitamina E

Importance of web-based intervention in minimizing depressive symptoms and associated stigma in depressed medical students

[Excerpt] There is a considerable prevalence of depression in medical students, and those who are depressed more frequently endorse feeling stigmatized than non-depressed students.1 Because concerns about confidentiality are often cited by depressed medical students as a major barrier in seeking help, there is a significant need to develop an innovative way to provide medical students with safe and confidential access to services to improve prevention, detection, and intervention in depression and its associated stigma.1 A web-based approach could be potentially useful for addressing this issue and has already been used for delivering intervention in various health conditions, with benefits such as low cost, user convenience, timely information, privacy and confidentiality, reduced levels of stigmatization, and increased user and supplier control.2 Moreover, although current evidence is limited, a recent meta-review3 points to the efficacy of web-based cognitive behavioral interventions in treating/improving depression symptoms in adults. In relation to depression stigma, there is evidence that web-based interventions (e.g., MoodGYM) can reduce personal stigmatizing attitudes toward depression. The web-based approach assumes even greater importance when we consider that stigmatization increases with either the use of prescription medication or mental health counseling, and that only a small percentage of depressed medical students seek mental health counseling services, due to lack of time, confidentiality, stigma, and fear of documentation in academic records.4 However, unintended negative effects could easily arise with web-based approaches as well (e.g. decline in seeking support from family and friends, avoidance of in-person mental health services, inadequate assessment and diagnosis). [...]- (undefined

Mutations in SPG11, encoding spatacsin, are a major cause of spastic paraplegia with thin corpus callosum.

Autosomal recessive hereditary spastic paraplegia (ARHSP) with thin corpus callosum (TCC) is a common and clinically distinct form of familial spastic paraplegia that is linked to the SPG11 locus on chromosome 15 in most affected families. We analyzed 12 ARHSP-TCC families, refined the SPG11 candidate interval and identified ten mutations in a previously unidentified gene expressed ubiquitously in the nervous system but most prominently in the cerebellum, cerebral cortex, hippocampus and pineal gland. The mutations were either nonsense or insertions and deletions leading to a frameshift, suggesting a loss-of-function mechanism. The identification of the function of the gene will provide insight into the mechanisms leading to the degeneration of the corticospinal tract and other brain structures in this frequent form of ARHSP

A pentanucleotide ATTTC repeat insertion in the non-coding region of DAB1, mapping to SCA37, causes spinocerebellar ataxia.

Advances in human genetics in recent years have largely been driven by next-generation sequencing (NGS); however, the discovery of disease-related gene mutations has been biased toward the exome because the large and very repetitive regions that characterize the non-coding genome remain difficult to reach by that technology. For autosomal-dominant spinocerebellar ataxias (SCAs), 28 genes have been identified, but only five SCAs originate from non-coding mutations. Over half of SCA-affected families, however, remain without a genetic diagnosis. We used genome-wide linkage analysis, NGS, and repeat analysis to identify an (ATTTC)n insertion in a polymorphic ATTTT repeat in DAB1 in chromosomal region 1p32.2 as the cause of autosomal-dominant SCA; this region has been previously linked to SCA37. The non-pathogenic and pathogenic alleles have the configurations [(ATTTT)7-400] and [(ATTTT)60-79(ATTTC)31-75(ATTTT)58-90], respectively. (ATTTC)n insertions are present on a distinct haplotype and show an inverse correlation between size and age of onset. In the DAB1-oriented strand, (ATTTC)n is located in 5' UTR introns of cerebellar-specific transcripts arising mostly during human fetal brain development from the usage of alternative promoters, but it is maintained in the adult cerebellum. Overexpression of the transfected (ATTTC)58 insertion, but not (ATTTT)n, leads to abnormal nuclear RNA accumulation. Zebrafish embryos injected with RNA of the (AUUUC)58 insertion, but not (AUUUU)n, showed lethal developmental malformations. Together, these results establish an unstable repeat insertion in DAB1 as a cause of cerebellar degeneration; on the basis of the genetic and phenotypic evidence, we propose this mutation as the molecular basis for SCA37.We thank the families who participated in this study. We are grateful to Goncalo Abecasis, Miguel Costa, Tito Vieira, and Andre Torres for help with MERLIN analysis; Beatriz Sobrino, Jorge Amigo, and Pilar Cacheiro for next-generation sequencing analysis, performed at the Santiago de Compostela node of the Spanish National Genotyping Center; Nuno Santarem and Anabela Cordeiro-da-Silva for assistance with cloning; Antonio Amorim, Laura Vilarinho, and Paula Jorge for samples from the Portuguese population; and Paula Magalhaes from the Institute for Molecular and Cell Biology Cell Culture and Genotyping Core for DNA extraction. This work was financed by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operational Program for Competitiveness and Internationalization (POCI) of Portugal 2020 and by Portuguese funds through the Fundacao para a Ciencia e a Tecnologia (FCT) and Ministerio da Ciencia, Tecnologia, e Inovacao in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-007274); and by FCT grant PTDC/SAU-GMG/098305/2008 to I.S. A. I.S. was the recipient of an FCT scholarship (SFRH/BD/30702/2006). J.R.L. was supported by scholarships from PEst-C/SAU/LA0002/2013 and the European Molecular Biology Organization (ASTF494-2015). C.L.O. was supported by a scholarship from PEst-C/SAU/LA0002/2013. This work was also financed by the Porto Neurosciences and Neurologic Disease Research Initiative at the Instituto de Investigacao e Inovacao em Saude (Norte-01-0145-FEDER-000008), supported by Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through FEDER, and by the Fondo de Investigacion Sanitaria of the Instituto de Salud Carlos III (grant PI12/00742)

Performance adaptive training control strategy for recovering wrist movements in stroke patients: a preliminary, feasibility study

<p>Abstract</p> <p>Background</p> <p>In the last two decades robot training in neuromotor rehabilitation was mainly focused on shoulder-elbow movements. Few devices were designed and clinically tested for training coordinated movements of the wrist, which are crucial for achieving even the basic level of motor competence that is necessary for carrying out ADLs (activities of daily life). Moreover, most systems of robot therapy use point-to-point reaching movements which tend to emphasize the pathological tendency of stroke patients to break down goal-directed movements into a number of jerky sub-movements. For this reason we designed a wrist robot with a range of motion comparable to that of normal subjects and implemented a self-adapting training protocol for tracking smoothly moving targets in order to facilitate the emergence of smoothness in the motor control patterns and maximize the recovery of the normal RoM (range of motion) of the different DoFs (degrees of Freedom).</p> <p>Methods</p> <p>The IIT-wrist robot is a 3 DoFs light exoskeleton device, with direct-drive of each DoF and a human-like range of motion for Flexion/Extension (FE), Abduction/Adduction (AA) and Pronation/Supination (PS). Subjects were asked to track a variable-frequency oscillating target using only one wrist DoF at time, in such a way to carry out a progressive splinting therapy. The RoM of each DoF was angularly scanned in a staircase-like fashion, from the "easier" to the "more difficult" angular position. An Adaptive Controller evaluated online performance parameters and modulated both the assistance and the difficulty of the task in order to facilitate smoother and more precise motor command patterns.</p> <p>Results</p> <p>Three stroke subjects volunteered to participate in a preliminary test session aimed at verify the acceptability of the device and the feasibility of the designed protocol. All of them were able to perform the required task. The wrist active RoM of motion was evaluated for each patient at the beginning and at the end of the test therapy session and the results suggest a positive trend.</p> <p>Conclusion</p> <p>The positive outcomes of the preliminary tests motivate the planning of a clinical trial and provide experimental evidence for defining appropriate inclusion/exclusion criteria.</p

The usefulness of contrast during exercise echocardiography for the assessment of systolic pulmonary pressure

<p>Abstract</p> <p>Background</p> <p>The systolic pulmonary artery pressure (PAPs) can be accurately estimated, non-invasively, using continuous-wave Doppler (CWD) ultrasound measurement of the peak velocity of a tricuspid regurgitant (TR) jet.</p> <p>However, it is often difficult to obtain adequate tricuspid regurgitation signals for measurement of PAPs, what could lead to its underestimation. Therefore, utilization of air-blood-saline contrast has been implemented for the improvement of Doppler signal in several clinical contexts.</p> <p>It is now recommended in the evaluation of patients with pulmonary hypertension. Physical activity is severely restricted in patients with PAH, being exertional dypnea the most typical symptom. Exercise stress echo-Doppler imaging allows assessment of the response to exercise. It is an excellent screening test for patients with suspected PAH. Our purpose was to evaluate the value and accuracy of agitated saline with blood contrast echocardiography, in the improvement of the Doppler signal, to quantify PAPs during treadmill exercise-echocardiography.</p> <p>Purpose</p> <p>To evaluate the value of contrast echocardiography, using agitated saline with blood, in the improvement of the Doppler signal used to quantify the pulmonary artery systolic pressure during exercise.</p> <p>Methods</p> <p>From a total of 41 patients (pts), we studied 38 pts (93%), 35 women, aged 54 ± 12 years-old. 27 with the diagnosis of systemic sclerosis, 10 with history of pulmonary embolism and one patient with a suspected idiopathic PAH, who were referred to the Unity of Heart Failure and Pulmonary Hypertension for screening of PAH. According to the Unity protocol, a transthoracic echocardiogram was made, in left decubitus (LD), with evaluation of right ventricle-right atria gradient (RV/RAg). A peripheral venous access was obtained, with a 3-way stopcock and the patients were placed in orthostatism (O), with a new evaluation of RV/RAg. Exercise echocardiography (EE) was begun, with evaluation of RV/RAg at peak exercise (P) and afterwards agitated saline (8 cc with 1 cc of air and 1 cc of blood) was injected, followed by a new evaluation of RV/RAg (PC) and then the interruption of the EE. Pulmonary Hypertension was diagnosed when RV/RAg at the end of the exercise was superior to 40 mmHg.</p> <p>Results</p> <p>The quality of Doppler signal was deteriorated in 5 pts, maintained in 6 pts and improved in 26 pts, with the use of contrast. In one patient, an interventricular septal defect was diagnosed. In 6 pts, a Doppler signal was only obtained with the use of contrast. In 15 pts, a RV/RAg superior to 40 mmHg was only obtained with the use of contrast. Of these, 9 have already been submitted to right heart cathetherism, that confirmed the diagnosis of pulmonary hypertension in 5 of them (56%). RV/RAg (P) was 44 ± 11 mmHg and RV/RAg (PC) was 54 ± 11 mmHg, p < 0,001.</p> <p>Conclusion</p> <p>1. The method is applicable in a large number of patients. 2. RV/RA gradients obtained at peak exercise are higher with the use of contrast, and the clinical meaning of this difference should be evaluated in a larger number of pts submitted to right heart cathetherism. The high number of false positives should lead to a higher diagnostic threshold. 3. This method seems to have relevant clinical value in the diagnosis of pulmonary arterial hypertension.</p

Oenological characterisation of indigenous strains of S. cerevisiae isolated in a biodynamic winery in the Cortona DOC area

Genotypic and technological characterisation of the S. cerevisiae population isolated in a biodynamic winery in the Cortona DOC area was performed to gain better knowledge of the variables that influence winemaking. The oenological performance of 11 S. cerevisiae strains was evaluated with physiological tests; strain typing was performed through analysis of interdelta sequences and 26S rDNA sequencing. The analysis revealed a remarkable variability in terms of S. cerevisiae strains, despite the homogeneity of wine features, underlining the high levels of biodiversity characterising biodynamic agriculture. Some strains were found in wines of different vintages, suggesting the presence of an established microbiota in the winery. Oenological tests demonstrated that while some yeasts provided reliable oenological performance, other strains were not able to accomplish prompt and effective alcoholic fermentation, or were characterised by spoilage characteristics, such as excessive production of volatile phenols or acetic acid. Indigenous strains of S. cerevisiae could be a useful instrument for reliable winemaking without altering the native microbiota of each oenological environment. However, characterisation of their oenological suitability, and the application of practices able to drive the evolution of microbiota, must be employed to reduce the risk of wine spoilage

Impact of Continuous Axenic Cultivation in Leishmania infantum Virulence

Experimental infections with visceral Leishmania spp. are frequently performed referring to stationary parasite cultures that are comprised of a mixture of metacyclic and non-metacyclic parasites often with little regard to time of culture and metacyclic purification. This may lead to misleading or irreproducible experimental data. It is known that the maintenance of Leishmania spp. in vitro results in a progressive loss of virulence that can be reverted by passage in a mammalian host. In the present study, we aimed to characterize the loss of virulence in culture comparing the in vitro and in vivo infection and immunological profile of L. infantum stationary promastigotes submitted to successive periods of in vitro cultivation. To evaluate the effect of axenic in vitro culture in parasite virulence, we submitted L. infantum promastigotes to 4, 21 or 31 successive in vitro passages. Our results demonstrated a rapid and significant loss of parasite virulence when parasites are sustained in axenic culture. Strikingly, the parasite capacity to modulate macrophage activation decreased significantly with the augmentation of the number of in vitro passages. We validated these in vitro observations using an experimental murine model of infection. A significant correlation was found between higher parasite burdens and lower number of in vitro passages in infected Balb/c mice. Furthermore, we have demonstrated that the virulence deficit caused by successive in vitro passages results from an inadequate capacity to differentiate into amastigote forms. In conclusion, our data demonstrated that the use of parasites with distinct periods of axenic in vitro culture induce distinct infection rates and immunological responses and correlated this phenotype with a rapid loss of promastigote differentiation capacity. These results highlight the need for a standard operating protocol (SOP) when studying Leishmania species