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Machado-Joseph disease (MJD/SCA3) is the most common form of dominantly inherited ataxia worldwide. The disorder is caused by an expanded CAG repeat in the ATXN3 gene. Past studies have revealed that the length of the expansion partly explains the disease age at onset (AO) variability of MJD, which is confirmed in this study (Pearson's correlation coefficient R2 = 0.62). Using a total of 786 MJD patients from five different geographical origins, a genome-wide association study (GWAS) was conducted to identify additional AO modifying factors that could explain some of the residual AO variability. We identified nine suggestively associated loci (P < 1 × 10-5). These loci were enriched for genes involved in vesicle transport, olfactory signaling, and synaptic pathways. Furthermore, associations between AO and the TRIM29 and RAG genes suggests that DNA repair mechanisms might be implicated in MJD pathogenesis. Our study demonstrates the existence of several additional genetic factors, along with CAG expansion, that may lead to a better understanding of the genotype-phenotype correlation in MJD. FA and CL were funded by the Fonds de Recherche du Québec–Santé. SM is funded by FCT (CEECIND/00684/2017) and by NORTE-01-0145- FEDER-000008, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). FM and LI are funded by Fundaçao de Amparo a Pesquisa do Estado de São Paulo (FAPESP, 2013/07559-3). MLSP and LBJ were funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq) and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). GAR holds a Canada Research Chair in Genetics of the Nervous System and the Wilder Penfield Chair in Neurosciences.

The use of anti-hail nets on orchards changes the microclimate underneath the net. This might be of great importance in apple growing regions characterized by high radiation levels and hot and dry climates during the summer season. But, depending on the net colour and on the local climatic conditions, the shade promoted triggers different responses by the trees. Grey and black anti-hail nets were applied in an apple orchard (cv. ‘Golden Delicious’) located in Northeast Portugal. Under the nets a lower concentration of glomalin related-soil proteins was observed, along with an improvement on trees water status, stomatal conductance, net photosynthetic rate, total chlorophylls, N, Mg, Fe and Cu concentrations, as well as an increase in mean fruit weight. The major difference between nets was on the photosynthetic efficiency, being higher on black net in sunny days, while grey net performed better under cloudy conditions. The use of netting systems proved to be effective in improving “Golden Delicious” apple trees performance under a Mediterranean climate, mainly when the radiation reaching the plants surpass the tree saturation point for photosynthesis. Therefore, these findings anticipate solutions for current and forecasted negative effects of climate change. This research was funded by the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UIDB/00690/2020) and CITAB (UIDB/04033/2020), as well by the project “Introdução de telas anti-granizo em macieira: Avaliação de efeitos colaterais e impacte económico” PA 54824, funded by the European Agricultural Fund for Rural Development and the Portuguese state under the measure 4.1- Cooperation for the Innovation-Rural Development Program (PRODER). info:eu-repo/semantics/publishedVersion

Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis and limited treatment strategies. Determining the role of cell-extrinsic regulators of leukemic cells is vital to gain clinical insights into the biology of AML. Iron is a key extrinsic regulator of cancer, but its systemic regulation remains poorly explored in AML. To address this question, we studied iron metabolism in patients with AML at diagnosis and explored the mechanisms involved using the syngeneic MLL-AF9–induced AML mouse model. We found that AML is a disorder with a unique iron profile, not associated with inflammation or transfusion, characterized by high ferritin, low transferrin, high transferrin saturation (TSAT), and high hepcidin. The increased TSAT in particular, contrasts with observations in other cancer types and in anemia of inflammation. Using the MLL-AF9 mouse model of AML, we demonstrated that the AML-induced loss of erythroblasts is responsible for iron redistribution and increased TSAT. We also show that AML progression is delayed in mouse models of systemic iron overload and that elevated TSAT at diagnosis is independently associated with increased overall survival in AML. We suggest that TSAT may be a relevant prognostic marker in AML.

Background:The prevalence of thyroid dysfunction and autoimmunity in the Portuguese population has not yet been estimated. However, the national prevalence of the metabolic syndrome remains high. The association of thyroid pathology with cardiovascular risk has been addressed but is still unclear. Our study aimed to evaluate the prevalence of thyroid dysfunction and autoimmunity and to assess the associations of thyroid-stimulating hormone and thyroid hormones and antibodies with metabolic syndrome, its components, and other possible determinants in a national sample.Material and Methods:The present study included a subsample of 486 randomly selected participants from a nationwide cross-sectional study sample of 4095 adults. A structured questionnaire was administered on past medical history and socio-demographic and behavioural characteristics. Blood pressure and anthropometric measurements were collected, and the serum lipid profile, glucose, insulin, hs- CRP, TSH, FT4, FT3 and thyroid antibodies were measured.Results:In our sample, the prevalence of hypothyroidism, hyperthyroidism and undiagnosed dysfunction was 4.9%, 2.5% and 72.2%, respectively. Overall, the prevalence of positivity for the thyroid peroxidase and thyroglobulin antibodies was 11.9% and 15.0%, respectively. A positive association was found between free triiodothyronine and metabolic syndrome (OR: 2.019; 95% CI: 1.196, 3.410). Additionally, thyroid peroxidase antibodies had a negative association with metabolic syndrome (OR: 0.465; 95% CI: 0.236, 0.917) and its triglyceride component (OR: 0.321; 95% CI: 0.124, 0.836).Conclusion:The prevalence of undiagnosed thyroid dysfunction and autoimmunity was high. Thyroid peroxidase antibodies were negatively associated with metabolic syndrome and its triglyceride component, whereas the free triiodothyronine level was positively associated with metabolic syndrome.

Solanum lycopersicum L. is among the healthiest fruits/vegetables due to its richness in bioactive compounds. However, its fruits from off-season (usually obtained in greenhouses that block UV-rays) have lower reputation than the ones of in-season/field productions. We hypothesise that moderate UV-A/UV-B irradiation during fruit development is able to improve its bioactive compounds and sensorial attributes, increasing its healthy properties. We supplemented for 30 days ‘MicroTom’ fruiting plants with two daily doses of UV-A (1 or 4 h) and UV-B (2 or 5 min). Irradiated plants showed higher ripening synchronization and produced more and smaller fruits. UV-A irradiation stimulated the fruit’s antioxidant capacity, and the antiradical activity by the accumulation of phenolic compounds including the flavonoids. Only the UV-A1 h condition promoted the accumulation of ortho-diphenols in tomato fruits. Regarding the consumers’ preference for aroma/taste, a consumers’ panel test ranked the tomatoes as UV-A 1 h, UVA 4 h, Control, UV-B 5 min and UV-B 2 min. We conclude that the supplementation of UV-A during pre-harvest is particularly effective in increasing ripening synchronization and fruit’s nutritional properties, potentially making these fruits more appealing to consumers.

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.

The primary genetic risk factor for late onset Alzheimer&rsquo, s disease (LOAD) is the APOE4 allele of Apolipoprotein E (APOE) gene. The three most common variants of APOE are determined by single nucleotide polymorphisms (SNPs) rs429358 and rs7412. Our aim was to estimate allele and genotype frequencies of APOE variants in an Iberian cohort, thus helping to understand differences in APOE-related LOAD risk observed across populations. We analyzed saliva or buccal swab samples from 229 LOAD patients and 89 healthy elderly controls (&ge, 68 years old) from Northern Portugal and Castile and Le&oacute, n region, Spain. The genotyping was performed by Sanger sequencing, optimized to overcome GC content drawbacks. Results obtained in our Iberian LOAD and control cohorts are in line with previous large meta-analyses on APOE frequencies in Caucasian populations, however, we found differences in allele frequencies between our Portuguese and Spanish subgroups of AD patients. Moreover, when comparing studies from Iberian and other Caucasian cohorts, differences in APOE2 and APOE4 frequencies and subsequent different APOE-related LOAD risks must be clarified. These results show the importance of studying genetic variation at the APOE gene in different populations (including analyses at a regional level) to increase our knowledge about its clinical significance.

Background The Zika virus (ZIKV) is a flavivirus and the human disease caused by this virus has been described in the Americas in 2015. ZIKV has been identified as an etiological agent of acute exanthematous disease in Brazil. In the same year, an epidemic of microcephaly with images suggestive of congenital infection raised the suspicion of a relationship between these alterations and ZIKV infection. Epidemiological and histopathological studies point to a strong relationship between prenatal Zika virus infection and microcephaly. Newborns with microcephaly, may also present auditory and visual changes, seizures and severe neurodevelopmental impairment. In 2016, the World Health Organization (WHO) declared Zika virus (ZIKV) infection a public health emergency of international concern. Aims Clinical observation in the ambulatory school of ABRAH (Brazilian Association of Recycling and Homeopathy Assistance) of patients with encephalopathy of various origins, using the medicine Helleborus niger as equalizer of the NS (Nervous System), as described in the Complex Systems of Carillo, shows improvement in motor, cognitive and seizure disorders. Based on this observation, we propose to use this method in patients with ZIKV congenital infection. Methodology To evaluate 15 patients in follow-up at the AFR (Fluminense Rehabilitation Association), through homeopathic anamnesis, clinical, diathesic, biotypological and temperamental classification. The Gross Motor Function Classification System (GMFCS) will be used. All patients will receive Helleborus niger 6 Ch daily for 6 months. Results and discussion This study was forwarded to the research ethics committee and will begin in July 2019. Will be evaluated the use of Helleborus niger in patients with congenital infection by ZIKV, as equalizer of NS. The expected result is overall neurological improvement. Conclusion Helleborus niger, probably stimulating self-regulation through uninjured neurological pathways, will promote autopoiesis and construction of new programs of action, and will enable the improvement of patients with congenital ZIKV infection.

Brain waves, measured by electroencephalography (EEG), are a powerful tool in the investigation of neurophysiological traits and a noninvasive and cost-effective alternative in the diagnostic of some neurological diseases. In order to identify novel Quantitative Trait Loci (QTLs) for brain wave relative power (RP), we collected resting state EEG data in five frequency bands (&delta, &theta, &alpha, &beta, 1, and &beta, 2) and genome-wide data in a cohort of 105 patients with late onset Alzheimer&rsquo, s disease (LOAD), 41 individuals with mild cognitive impairment and 45 controls from Iberia, correcting for disease status. One novel association was found with an interesting candidate for a role in brain wave biology, CLEC16A (C-type lectin domain family 16), with a variant at this locus passing the adjusted genome-wide significance threshold after Bonferroni correction. This finding reinforces the importance of immune regulation in brain function. Additionally, at a significance cutoff value of 5 &times, 10&minus, 6, 18 independent association signals were detected. These signals comprise brain expression Quantitative Loci (eQTLs) in caudate basal ganglia, spinal cord, anterior cingulate cortex and hypothalamus, as well as chromatin interactions in adult and fetal cortex, neural progenitor cells and hippocampus. Moreover, in the set of genes showing signals of association with brain wave RP in our dataset, there is an overrepresentation of loci previously associated with neurological traits and pathologies, evidencing the pleiotropy of the genetic variation modulating brain function.

Neck circumference (NC) has been proposed as a simple and practical tool, independently associated with cardiometabolic risk factors. However, the association of NC with inter-muscular adipose tissue (IMAT) is still to be determined. We aimed to examine the association of NC with thigh IMAT, and visceral adipose tissue (VAT) measured with computed tomography (CT) in overweight/obese women. 142 premenopausal overweight and obese Caucasian women participated in this cross-sectional study. NC was measured with an inextensible metallic tape above the thyroid cartilage according to International Society for Advancement of Kinanthropometry protocol. Thigh IMAT and VAT volumes were measured with a single cross-sectional CT. Regarding the covariates, fat mass (FM) was assessed with dual-energy x-ray absorptiometry and physical activity was objectively measured with accelerometry. NC was positively associated with thigh IMAT and VAT volumes (standardized β coefficient: β = 0.45, P-value = ≤0.001, β = 0.60, P = ≤ 0.001; respectively), which persisted after adjusting for age, height, overall FM or moderate-to-vigorous physical activity. Our findings show that NC is associated with thigh IMAT volume in overweight and obese premenopausal Caucasian women, regardless of the amount of lower-body fatness. These results suggest underscoring the relevance of NC as a marker of adipose tissue content in thigh skeletal muscle.

The zebrafish is a widely used animal model in biomedical research. Despite this, pathogens continue to be common in laboratory zebrafish. It is important to determine and describe the pathophysiology of cryptic infections on zebrafish to elucidate the impacts on experimental endpoints. Body condition is a basic measurement used experimentally and in health monitoring of animals. We exposed three wild-type zebrafish strains: AB, WIK, and 5D to Pseudoloma neurophilia. After 8 weeks postexposure, we individually imaged and processed fish for histology. Morphometric analysis was performed on images and an index of body condition was calculated based on the ratio of length/width from the dorsal aspect. Histological sections from each fish were examined to establish sex, severity of infection, and tissue distribution. We observed a significant decrease in body condition in female fish that was not observed in males. In addition, we observed a negative correlation between the total gonadal area of P. neurophilia exposed females and unexposed controls. These results illustrate the sex-specific impacts of a common chronic pathogen on zebrafish health and a commonly used experimental endpoint, further supporting the establishment of rigorous health monitoring programs in laboratory zebrafish colonies that include screening for chronic infectious diseases.

Hypertrophic cardiomyopathy (HCM), the most common inherited heart disease, is predominantly caused by mutations in genes that encode sarcomere-associated proteins. Effective gene-based diagnosis is critical for the accurate clinical management of patients and their family members. However, the introduction of high-throughput DNA sequencing approaches for clinical diagnostics has vastly expanded the number of variants of uncertain significance, leading to many inconclusive results that limit the clinical utility of genetic testing. More recently, developments in RNA analysis have been improving diagnostic outcomes by identifying new variants that interfere with splicing. This review summarizes recent discoveries of RNA mis-splicing in HCM and provides an overview of research that aims to apply the concept of RNA therapeutics to HCM.

AIM: Explore the use of transferrin-receptor peptide-functionalized nanoparticles (NPs) targeting blood-brain barrier (BBB) as siRNA carriers to silence P-glycoprotein (P-gp).MATERIALS & METHODS: Permeability experiments were assessed through a developed BBB cell-based model; P-gp mRNA expression was evaluated in vitro; rhodamine 123 permeability was assessed after cell monolayer treatment with siRNA NPs.RESULTS: Beyond their ability to improve siRNA permeability through the BBB by twofold, 96-h post-transfection, functionalized polymeric NPs successfully reduced P-gp mRNA expression up to 52%, compared with nonfunctionalized systems. Subsequently, the permeability of rhodamine 123 through the human BBB model increased up to 27%.CONCLUSION: Developed BBB-targeted NPs induced P-gp downregulation and consequent increase on P-gp substrate permeability, revealing their ability to modulate drug efflux at the BBB.

Machado-Joseph disease (MJD/SCA3) is the most common form of dominantly inherited ataxia worldwide. The disorder is caused by an expanded CAG repeat in the ATXN3 gene. Past studies have revealed that the length of the expansion partly explains the disease age at onset (AO) variability of MJD, which is confirmed in this study. Using a total of 786 MJD patients from five different geographical origins, a genome-wide association study (GWAS) was conducted to identify additional AO modifying factors that could explain some of the residual AO variability. We identified nine suggestively associated loci (P < 1 × 10−5). These loci were enriched for genes involved in vesicle transport, olfactory signaling, and synaptic pathways. Furthermore, associations between AO and the TRIM29 and RAG genes suggests that DNA repair mechanisms might be implicated in MJD pathogenesis. Our study demonstrates the existence of several additional genetic factors, along with CAG expansion, that may lead to a better understanding of the genotype-phenotype correlation in MJD.

At least 40 human diseases are associated with repeat expansions; yet, the mutational origin and instability mechanisms remain unknown for most of them. Previously, genetic epidemiology and predisposing backgrounds for the instability of some expanding loci have been studied in different populations through the analysis of diversity flanking the respective pathogenic repeats. Here, we aimed at developing a pipeline to assess disease-associated haplotypes at oligonucleotide repeat loci, combining analysis of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). Machado-Joseph disease (MJD/SCA3), the most frequent dominant ataxia worldwide, was used as an example of a detailed procedure. Thus, to identify genetic backgrounds that segregate with expanded/mutated alleles in MJD, we selected a set of 26 SNPs and 7 STRs flanking the causative CAG repeat. Key criteria and steps for this selection are described, and included (1) haplotype blocks minimizing the occurrence of recombination (for SNPs); and (2) match scores to increase potential for polymorphic information content of repetitive sequences found in Tandem Repeats Finder (for STRs). To directly assess SNP haplotypes in phase with MJD expansions, we optimized a strategy with preferential amplification of normal over expanded alleles, in addition to SNP allele-specific amplifications; this allowed the identification of disease-associated SNP haplotypes, even when only the proband is available in a given family. To infer STR haplotypes, we optimized a multiplex PCR, including 7 STRs plus the MJD_CAG repeat, followed by analysis of segregation or the use of the PHASE software. This protocol is a ready-to-use tool to assess MJD haplotypes in different populations. The pipeline designed can be used to assess disease-associated haplotypes in other repeat-expansion diseases. This should be of great utility to study (1) genetic epidemiology (population-of-origin, age and spreading routes of mutations) and (2) mechanisms responsible for de novo expansions, in these neurological diseases; (3) to detect predisposing haplotypes and (4) phenotype modifiers; (5) to help solving cases of apparent homoallelism (two same-size normal alleles) in diagnosis; and (6) to identify the best targets for the development of allele-specific therapies in ethnically diverse patient populations. This work was financed by FEDER-Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020-Operacional Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT-Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). SM is funded by FCT (IF/00930/2013). This article is a result of the project NORTE-01-0145-FEDER-000008, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).

Machado-Joseph disease (MJD, also known as spinocerebellar ataxia 3 or SCA3) is the most common dominant ataxia worldwide, with an overall average prevalence of 1-5/100,000. To this date, two major ancestral lineages have been found throughout the world. In China, the relative frequency of MJD among the SCAs reaches as high as 63%, however, little is known about its mutational origin in this country. We analyzed 50 families with MJD patients in two or more generations to study the hypothesis that new mutational events have occurred in this population. Haplotypes based on 20 SNPs have shown new genetic backgrounds segregating with MJD mutations in our cohort from China. We found the "Joseph-derived" lineage (Joseph lineage with a G variant in rs56268847) to be very common among Chinese MJD patients. Moreover, we estimated the time for the origin of this MJD SNP background based on STR diversity flanking the (CAG)n of ATXN3. It was surprising to find that the Chinese MJD population originated from 8,000 to 17,000 years ago, far earlier than the previous literature reports, which will be an important evidence to explain the origin, spread and founder effects of MJD. This work was supported by the National Key Research and Development Program of China (Nos. 2016YFC0905100 and 2016YFC0901504 to HJ), the National Natural Science Foundation of China (Nos. 81771231 and 81471156 to HJ), the Clinical Research Funds of Xiangya Hospital (2014L03 to HJ), the Clinical and rehabilitation fund of Peking University Weiming Biotech Group (No. xywm2015I10 to HJ), Youth Foundation of Xiangya Hospital (No. 2017Q03 to ZC), and Independent Exploration and Innovation Project of Graduate Students of Central South University (No. 1053320170177 to TL). Moreover, SM is funded by the project IF/00930/2013 from FCT. This article is a result of the project NORTE-01-0145-FEDER-000008, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, and through the European Regional Development Fund (ERDF).

Zebrafish (Danio rerio) is one of the top model organisms used in biomedical research. Therefore, it is fundamental that zebrafish facilities continuously improve husbandry methods to provide fish with the best physiological and welfare conditions that suit each experimental purpose. Nutrition is a husbandry aspect that needs further optimization, as it greatly affects growth, reproduction, health and behaviour. Here, we have compared the impact of different feeding regimens on zebrafish survival, growth and reproductive performance. Mutant and wild-type zebrafish were raised using several combinations of two cold-extruded processed feeds&mdash, Skretting&reg, GemmaMicro and Sparos&reg, Zebrafeed&mdash, and one live feed (rotifers). Zebrafeed&reg, outperformed GemmaMicro&reg, in terms of survival rate, and embryo viability was also higher when the spawners were fed with Zebrafeed&reg, either from larval stage or upon sexual maturation. In contrast, GemmaMicro&reg, favoured growth, both in size and weight. The use of rotifers until 60 days post-fertilization improved survival of fish co-fed with GemmaMicro&reg, while delaying their growth. Zebrafeed&reg, performance was not affected by co-feeding rotifers. Overall, we showed that different nutritional formulas affect physiological parameters, allowing for the establishment of feeding protocols adapted to the objectives of each facility. At the same time, we validated Skretting&reg, Zebrafeed as two commercially available feeds that are well suited for zebrafish nutrition in a laboratory environment.

Background Cropping practices focusing on agronomic water use efficiency and their impact on quality parameters must be investigated to overcome constraints affecting olive groves. We evaluated the response of olive trees (Olea europaea, cv. 'Cobrancosa') to different water regimes: full irrigation (FI, 100% crop evapotranspiration (ETc )), and three deficit irrigation strategies (DIS) (regulated (RDI, irrigated with 80% of crop evapotranspiration (ETc ) in phases I and III of fruit growth and 10% of ETc in the pit hardening stage), and two continuous sustained strategies (SDI) - a conventional SDI (27.5% of ETc ), and low-frequency irrigation adopted by the farmer (SDIAF, 21.2% of ETc ). Results The effects of water regimes on the plant water status, photosynthetic performance, metabolite fluctuations and fruit quality parameters were evaluated. All DIS treatments enhanced leaf tissue density; RDI and SDI generally did not affect leaf water status and maintained photosynthetic machinery working properly, and the SDIAF treatment impaired olive tree physiological indicators. The DIS treatments maintained the levels of primary metabolites in leaves, but SDIAF plants showed signs of oxidative stress. Moreover, DIS treatments led to changes in the secondary metabolism, both in leaves and in fruits, with increased total phenolic compounds, ortho-diphenols, and flavonoid concentration, and higher total antioxidant capacity, as well higher oil content. Phenolic profiles showed the relevance of an early harvest in order to obtain higher oleuropein levels with associated higher health benefits. Conclusion Adequate DIS are essential for sustainable olive growing, as they enhance the competitiveness of the sector in terms of olive production and associated quality parameters. © 2019 Society of Chemical Industry.

Regarding the foreseeing climate change is reasonable to expect harmful consequences to olive tree (Olea europaea L.), an iconic species of Mediterranean basin. In large areas of the Region, the sustained deficit irrigation (SDI) accounts with a reduced amount of water that might not be enough to keep the plant functioning at desirable levels. In this study the use of a reflective clay, kaolin, was evaluated in order to alleviate the negative summer stress effects on olive trees (cv. Cobrançosa) under an SDI system. Kaolin reduced the stomatal and the non-stomatal limitations to photosynthesis at high light intensity, improved plant water status, and decreased the oxidative damages. The amelioration of physiological and metabolic functions and the lower allocation of resources to summer stress tolerance mechanisms, allowed the KL-sprayed trees to increase crop yield, the oil yield and its oxidative stability, with the reduction of peroxide index and K232. Thus, the application of kaolin had a positive synergistic effect with SDI, especially important for semi-arid areas where water available for irrigation do not cover all the necessities. This work was financed by project Interact: Integrative Research in Environment, Agro-Chain, and Technology, operation NORTE-01-0145- FEDER-000017, research line ISAC, co-funded by European Regional Development Fund (FEDER) through NORTE 2020 (Programa Operacional Regional do Norte 2014/2020), and supported by the project “Novas pr´aticas em olivais de sequeiro: estrat´egias de mitigaç˜ao e adaptaç˜ao `as alteraç˜oes clim´aticas”, PDR2020-101-032119, financed by the European Agricultural Fund for Rural Development (EAFRD) and the Portuguese State under Aç˜ao 1.1 “Grupos Operacionais”, integrada na Medida 1. “Inovaç˜ao” do PDR 2020: Programa de Desenvolvimento Rural do Continente. Institution. This work is supported by National Funds by FCT—Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020. The authors are also grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020). info:eu-repo/semantics/publishedVersion

Machado-Joseph disease (MJD) is the most common autosomal dominant spinocerebellar ataxia reported worldwide, but it shows marked geographic differences in prevalence. The study of ancestral origins and spreading routes of MJD mutational events has contributed to explain such differences. During human evolution, at least two independent de novo MJD expansions occurred in distinct haplotype backgrounds: TTACAC and GTGGCA (named Joseph and Machado lineages). The most ancient Joseph lineage, probably of Asian origin, has been introduced recently in Europe, where founder effects are responsible for the high MJD prevalence, as occurs in the Portuguese/Azorean island of Flores and Northeastern mainland. The Machado lineage is geographically more restricted, with most known families in Portugal (island of Sao Miguel and along the Tagus valley). The hypothesis of other mutational origins has been raised, namely to explain the disease among Australian aborigines; however, a comprehensive haplotype study suggested the introduction of the Joseph lineage in that community via Asia. Also, additional SNP-based haplotypes (TTAGAC, TTGGAC and GTGCCA) were observed in other MJD families, but phylogenetic analysis with more polymorphic flanking markers did not point to independent mutational events, reinforcing the hypothesis of a very low mutation rate underlying this repeat expansion locus.

Current estimates indicate that approximately one-third of all disease-causing mutations are expected to disrupt splicing. Abnormal splicing often leads to disruption of the reading frame with introduction of a premature termination codon (PTC) that targets the mRNA for degradation in the cytoplasm by nonsense mediated decay (NMD). In addition to NMD there are RNA surveillance mechanisms that act in the nucleus while transcripts are still associated with the chromatin template. However, the significance of nuclear RNA quality control in the context of human genetic diseases is unknown. Here we used patient-derived lymphoblastoid cell lines as disease models to address how biogenesis of mRNAs is affected by splice site mutations. We observed that most of the mutations analyzed introduce PTCs and trigger mRNA degradation in the cytoplasm. However, for some mutant transcripts, RNA levels associated with chromatin were found down-regulated. Quantification of nascent transcripts further revealed that a subset of genes containing splicing mutations (SM) have reduced transcriptional activity. Following treatment with the translation inhibitor cycloheximide the cytoplasmic levels of mutant RNAs increased, while the levels of chromatin-associated transcripts remained unaltered. These results suggest that transcription-coupled surveillance mechanisms operate independently from NMD to reduce cellular levels of abnormal RNAs caused by SM.

© 2017. Published by The Company of Biologists Ltd., Spliceostatin A, meayamycin, and pladienolide B are small molecules that target the SF3b subunit of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP). These compounds are attracting much attention as tools to manipulate splicing and for use as potential anti-cancer drugs. We investigated the effects of these inhibitors on mRNA transport and stability in human cells. Upon splicing inhibition, unspliced pre-mRNAs accumulated in the nucleus, particularly within enlarged nuclear speckles. However, a small fraction of the pre-mRNA molecules were exported to the cytoplasm. We identified the export adaptor ALYREF as being associated with intron-containing transcripts and show its requirement for the nucleo-cytoplasmic transport of unspliced pre-mRNA. In contrast, the exon junction complex (EJC) core protein eIF4AIII failed to form a stable complex with intron-containing transcripts. Despite the absence of EJC, unspliced transcripts in the cytoplasm were degraded by nonsense-mediated decay (NMD), suggesting that unspliced transcripts are degraded by an EJC-independent NMD pathway. Collectively, our results indicate that although blocking the function of SF3b elicits a massive accumulation of unspliced pre-mRNAs in the nucleus, intron-containing transcripts can still bind the ALYREF export factor and be transported to the cytoplasm, where they trigger an alternative NMD pathway., This work was supported by Fundação para a Ciência e a Tecnologia (PTDC/BEX-BCM/5899/2014)

Fragile X syndrome (FXS), the most common cause of inherited intellectual disability, is due to the expansion over 200 CGGs and methylation of this polymorphic region, in the 5'-UTR (untranslated region) of FMR1 (Xq27.3). We have identified four FXS mosaic males: M1-(CGG)35/(CGG)>200; M2-(CGG)26/(CGG)>200; M3-(CGG)39/(CGG)>200; and M4-(CGG)18/(CGG)125/(CGG)>200. After genotyping their respective mothers, we suggested that normal alleles of these patients resulted from post-zygotic contractions of full expansions. The detection of these four rare independent cases led us to hypothesize the existence of a large-contraction predisposing haplotype in our population. Next, we questioned whether other normal pure CGGs would have arisen through similar contractions from fully expanded alleles. To address these questions, we identified stable single-nucleotide polymorphism (SNP) lineages and related short tandem repeat (STR) haplotypes (DXS998-DXS548-FRAXAC1-FRAXAC2) of the four mosaics, 123 unrelated FXS patients and 212 controls. An extended flanking haplotype (34-44-38-336) shared by mosaics from lineage A suggested a risk lineage-specific haplotype more prone to large contractions. Other normal pure FMR1 alleles from this SNP background also shared phylogenetically close STR haplotypes, although a single (CGG)exp>(CGG)24 contraction or the loss of AGG interruptions may explain their origin. In both scenarios, multistep FMR1 mutations involving the gain or loss of several CGGs seem to underlie the evolution of the repeat. We acknowledge all patients and their families for participating in this study, and medical geneticists Ana Maria Fortuna, Mariana Soeiro e Sa Oana Moldovan and Ana Berta Sousa for referring the patients. UMIB is funded by National Funds through FCT Foundation for Science and Technology, under the Pest-OE/SAU/UI0215/2014. SM is funded by the project IF/00930/2013/CP1184/CT0002 from FCT. IM received a research grant 2014 DEFI-CHP, E.P.E.

© 2017 Mendes de Almeida et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., High throughput sequencing technologies have revolutionized the identification of mutations responsible for genetic diseases such as hypertrophic cardiomyopathy (HCM). However, approximately 50% of individuals with a clinical diagnosis of HCM have no causal mutation identified. This may be due to the presence of pathogenic mutations located deep within the introns, which are not detected by conventional sequencing analysis restricted to exons and exon-intron boundaries., This work was supported by Fundação para a Ciência e Tecnologia and FEDER, Portugal (Scholarship BD/82023/2011 to R.M.A, grant EXPL/BIM-MEC/0201/2013 to S.M., grant Programa de Actividades Conjuntas n.º 016394 to M.C.-F, and LISBOA-01-0145-FEDER-007391), and an unrestricted grant from Merck Sharp & Dohme to M.C.-F. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The androgen receptor (AR) gene encodes a type of nuclear receptor that functions as a steroid-hormone activated transcription factor. In its coding region, AR includes a CAG repeat, which has been intensely studied due to the inverse correlation between repeat size and AR transcriptional activity. Several studies have reported different (CAG)n sizes associated with the risk of androgen-linked diseases. We aimed at clarifying the mechanisms on the origin of newly CAG sized alleles through a strategy involving the analysis of the associated haplotype diversity. We genotyped 374 control individuals of European and Asian ancestry, and reconstructed the haplotypes associated with the CAG repeat, defined by 10 SNPs and 6 flanking STRs. The most powerful SNPs to tag AR lineages are rs7061037-rs12012620 and rs34191540-rs6625187-rs2768578 in Europeans and Asians, respectively. In the most frequent AR lineage, (CAG)18 alleles seem to have been generated by a multistep mutation mechanism, most probably from longer alleles. We further noticed that the DXS1194-DXS1111 haplotype, in linkage disequilibrium with AR-(CAG)n expanded alleles responsible for spinal bulbar muscular atrophy (SBMA), is rare among our controls; however, the haplotype strategy here described may be used to clarify the origin of expansions in other populations, as in future association studies.

Thymidylate synthase (TYMS) has three polymorphisms that may modulate thymidylate synthase (TS) expression levels: (1) 28 base pairs (bp) variable number tandem repeat (VNTR) (rs34743033); (2) single nucleotide polymorphism (SNP) C>G at the twelfth nucleotide of the second repeat of 3R allele (rs2853542); and (3) 6 bp sequence deletion (1494del6, rs34489327). This study was conducted to evaluate the influence of TYMS polymorphisms on the survival of Portuguese patients with advanced non-small cell lung cancer (NSCLC) undergoing platinum-based chemotherapy. Our results showed no statistically significant differences between VNTR genotypes; although, considering the SNP C>G, homozygotes 3RG presented a better prognostic at 36 months (p = 0.004) and overall survival (p = 0.003) when compared to 2R3RG patients. Patients with “median/high expression genotypes” demonstrated a better survival at 12 months (p = 0.041) when compared to “low expression genotypes”. Furthermore, 6 bp− carriers (p = 0.006) showed a better survival at 12 months when compared to 6 bp+ homozygotes patients. When analyzing TYMS haplotypes, better survival at 12 months was observed for patients carrying haplotypes with the 6 bp− allele (2R6 bp−; p = 0.026 and 3RG6 bp−; p = 0.045). This is the first report that evaluates the three major TYMS polymorphisms in the therapeutic outcome of NSCLC in Portugal. According to our results, the TYMS polymorphisms may be useful tools to predict which advanced NSCLC patients could benefit more from platinum-based chemotherapy regimens.

The genus Corylus, a member of the birch family Betulaceae, includes several species that are widely distributed throughout temperate regions of the Northern Hemisphere. This study assesses the genetic diversity in 26 international cultivars and 32 accessions of Corylus avellana L. from Portugal: 13 wild genotypes and 19 landraces. The genetic relationships among the 58 hazelnuts (Corylus avellana L.) were analyzed using inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) markers. Eighteen ISSR primers and seven AFLP primer pairs generated a total of 570 unambiguous and repeatable bands, respectively, from which 541 (95.03 %) were polymorphic for both markers. Genetic similarity index values ranged from 0.239 for wild types and cultivars to 0.143 for landraces and wild types. The genetic relationships were presented as a Neighbor-Joining method dendrogram and a two-dimensional principal coordinate analysis (PCoA) plot. The Neighbor-Joining dendrogram showed three main clusters, and the PCoA analysis has shown to be congruent with the hierarchical analysis. Bayesian analysis clustered all individuals into three groups showing a good separation among wild genotypes, landraces and cultivars. The genetic diversity found on wild genotypes and Portuguese landraces may provide relevant information for the diversity conservation and it will be useful in breeding programs and to identify local selections for preservation.

Worldwide incidence of central nervous system (CNS) disorders is rising along with the age of population, making it an increasingly relevant subject. Protecting the CNS is the blood-brain barrier (BBB) which, consequently, hampers the success of current therapies by blocking drugs access to brain. Thus, developing innovative CNS treatments and improving the ability to cross BBB is urgent. Therefore, a BBB model where permeability experiments could be performed in a simple and fast way is crucially necessary to obtain high-throughput screening tools. This is where phospholipid vesicle-based permeability assay (PVPA) method opportunity arises. Through this technique, a non-cellular based BBB model able to assess passive transcellular-like permeability used as a tool for in vivo permeability prediction was developed based on phospholipid vesicles that mimic cellular membranes. Simultaneously, use of small interfering RNA (siRNA) to specifically silence efflux proteins present at BBB surface could be a useful advantage to circumvent BBB blockage. To improve siRNA delivery to brain and increase its ability to permeate BBB, lipid and polymeric nanoparticles were tested and their effect was observed by measuring siRNA permeability through the BBB-PVPA model. The permeability of siRNA significantly increased from 3.7 × 10−6±1.7 × 10−7 cm/s (free siRNA) to 5.5×10−6±3.1 × 10−7 cm/s and 6.9 × 10−6±4.5 × 10−7 cm/s after encapsulation into polymeric and lipidic nanoparticles, respectively.

The number of central nervous system (CNS) disorders has increased over the years as a result of a continuously aged population. Because current therapeutics frequently do not succeed well, there is an increasing need to find successful ways to reach the CNS to achieve successful treatment. The blood–brain barrier (BBB) is a unique membrane that creates this restrictive CNS environment. Therefore, regarding CNS disorders, failed therapeutics, and BBB responsiveness, BBB in vitro models that mimic in vivo BBB are a key factor in this promising research field. This chapter aims to describe different cell-based in vitro BBB models, static and dynamic, with a focus on which type of cells are commonly used, and their monoculture and co-culture, with or without fluid flow and shear stress exposure. Moreover, permeability through these models is discussed, centering on procedures to assess this crucial feature. Finally, the in vitro models are compared.

The genus Corylus, a member of the birch family Betulaceae, includes several species that are widely distributed throughout temperate regions of the Northern Hemisphere. The development of microsatellites or simple sequence repeats (SSRs) for non-coding regions of the chloroplast genome and their higher sequence variation compared with coding regions has provided a higher resolution tool for the study of cultivars and closely related taxa. Chloroplast polymorphisms provide a marker system to evaluate the genetic structure of plant populations. This study investigated genetic diversity in three cultivars and 32 genotypes of Corylus avellana L. from Portugal: 13 wild genotypes and 19 Portuguese landraces. Four of ten cpSSR loci were polymorphic, with diversity indices ranging from 0.111 to 0.244. Eleven chlorotypes were detected, and their relationships were analyzed using a network model. Haplotype A was most frequent in landraces and cultivars. Four chlorotypes (H, I, J and L) were found only in wild hazelnuts. The diversity of chlorotypes in the wild hazels, and the limited number reported in cultivars, suggests that northern Portugal was a refuge for hazel during the last ice age.

In the nucleus of higher eukaryotes, maturation of mRNA precursors involves an orderly sequence of transcription-coupled interdependent steps. Transcription is well known to influence splicing, but how splicing may affect transcription remains unclear. Here we show that a splicing mutation that prevents recruitment of spliceosomal snRNPs to nascent transcripts causes co-transcriptional retention of unprocessed RNAs that remain associated with polymerases stalled predominantly at the 3' end of the gene. In contrast, treatment with spliceostatin A, which allows early spliceosome formation but destabilizes subsequent assembly of the catalytic complex, abolishes 3' end pausing of polymerases and induces leakage of unspliced transcripts to the nucleoplasm. Taken together, the data suggest that recruitment of splicing factors and correct assembly of the spliceosome are coupled to transcription termination, and this might ensure a proofreading mechanism that slows down release of unprocessed transcripts from the transcription site.

Woad (Isatis tinctoria L.) was introduced in Europe in ancient times to produce indigo, a natural blue pigment used mainly for dyestuff. This species was cultivated in Portugal until the beginning of the 20th century, especially in the inner North and South. A set of nine inter-simple sequence repeat (ISSR) markers generated 177 reproducible fragments, of which 171 were polymorphic. The mean number of fragments/accession was 111, ranging between 100 (Portugal-Coimbra) and 124 (Poland). The total polymorphism observed was 0.3272, the average polymorphism was 0.1784 and the gene differentiation between accessions was 0.4546. Polymorphism ranged between 53.8% (Austria) and 73.1% (Belgium). The genetic relationship among woad accessions was obtained with unweighted pair group method with arithmetic mean dendrogram based on a molecular marker, clearly clustering the woad accessions according to their geographic origin. The genetic diversity observed in this collection shows that there is a considerable potential for its improvement and that ISSR could be used to evaluate intra- and inter-accession similarities in I. tinctoria species.

Woad (Isatis tinctoria L.) was the most important source of natural blue indigo, a pigment used mainly for dyestuff until the beginning of the 20th century, when the increased use of synthetic dyes by the industry lead to a decrease in the interest of natural dyes and to the abandonment of dye crops. The aim of this study was to characterize, according to morphological and agronomical traits, 11 woad accessions from different countries and to quantify the indigo precursor content by high-performance liquid chromatograph-diode array detection (HPLC-DAD). Qualitative traits revealed a low variability and a great variation was observed in quantitative traits. Principal component analysis (PCA) diagrams divided the accessions into four groups, primarily according to their geographic origin. The first three components of the PCA accounted for 76.8% of the total variation. Reciprocal interactions between indigo precursors and fresh leaf weight were compared through genetic diversity, with significant differences in isatan B (0.5–5.1 g/kg), indoxyl (0.3–2.0 g/kg) and residual indigo (0.3–0.5 g/kg). This information, together with genetic data, can be used to assist local farmers to re-introduce Isatis species in the European agricultural system, not only indicating the higher indigo yielding genotypes, but also the most suitable harvest time.

Machado-Joseph disease (MJD) has been described in Africans, but no cases have been reported from Nigeria. Current MJD global distribution results from both the ancestral populations-of-origin and the founder effects of mutations, some as a consequence of the Portuguese sea travels in the 15th to 16th century. Two main ancestral haplotypes have been identified: the Machado lineage, which is more recent, predominant in families of Portuguese extraction, and the Joseph lineage, which is much older and worldwide spread, postulated to have an Asian origin. We report a Nigerian family with MJD from Calabar, once settled by Portuguese slave traders, and assessed its mutational origin. The proband was a 33-year-old man with progressive unsteady gait, weakness of all limbs, dysphagia, dysarthria, urinary frequency and diaphoresis. He had end-of-gaze nystagmus, spastic quadriparesis and atrophic small muscles of the hand. He showed fibrillation potentials on EMG, and nerve conduction studies suggested a central axonopathy without demyelination. This family bears the Joseph haplotype, which has a founder effect in the island of Flores, in the Azores (and their descendants in North-America), but is also the most common in non-Portuguese populations worldwide, with an estimated mutation age of around 7000 years. SM is the recipient of a scholarship (SFRH/BPD/77969/2011) from the Portuguese Foundation for Science and Technology (FCT); IPATIMUP is partially supported by FCT. This work was co-financed by the European Social Fund (Human Potential Thematic Operational Programme).

The spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disease characterized by gait and limb ataxia. This disease is caused by the expansion of a (CAG)(n) located in the ATXN2, that encodes a polyglutamine tract of more than 34 repeats. Lately, alleles with 32-33 CAGs have been associated to late-onset disease cases. Repeat interruptions by CAA triplets are common in normal alleles, while expanded alleles usually contain a pure repeat tract. To investigate the mutational origin and the instability associated to the ATXN2 repeat, we performed an extensive haplotype study and sequencing of the CAG/CAA repeat, in a cohort of families of different geographic origins and phenotypes. Our results showed (1) CAA interruptions also in expanded ATXN2 alleles; (2) that pathological CAA interrupted alleles shared an ancestral haplotype with pure expanded alleles; and (3) higher genetic diversity in European SCA2 families, suggesting an older European ancestry of SCA2. In conclusion, we found instability towards expansion in interrupted ATXN2 alleles and a shared ancestral ATXN2 haplotype for pure and interrupted expanded alleles; this finding has strong implications in mutation diagnosis and counseling. Our results indicate that interrupted alleles, below the pathological threshold, may be a reservoir of mutable alleles, prone to expansion in subsequent generations, leading to full disease mutation.

Recent progress in global sequence and microarray data analysis has revealed the increasing complexity of the human transcriptome. Alternative splicing generates a huge diversity of transcript variants and disruption of splicing regulatory networks is emerging as an important contributor to various diseases, including cancer. Current efforts to establish the dynamic repertoire of transcripts that are generated in health and disease are showing that many cancer-associated alternative-splicing events occur in the absence of mutations in the affected genes. A growing body of evidence reveals changes in splicing-factor expression that correlate with cancer development, progression and response to therapy. Here, we discuss how recent links between cancer and altered expression of proteins implicated in splicing regulation are bringing the splicing machinery to the fore as a potential target for anticancer treatment.

Saccharomyces cerevisiae cells (strain W303-1A) treated with 5-fluorouracil and grown in 2% (fermentative conditions) or in 0.1 % glucose (oxidative conditions) accumulated two types of 5-fluoro-UDP-sugars (FUDP-sugars): FUDP-N-acetylglucosamine and FUDP-glucose. No difference was observed in both conditions of culture. The viability of yeast cells on treatment with 5-fluorouracil was also followed. Both FUDP-sugars were partially purified by column chromatography (on Hypersil ODS and Mono Q columns) and characterized by: (i) treatment with alkaline phosphatase (EC 3.1.3.1), snake venom phosphodiesterase (EC 3.1.4.1) and UDP-glucose dehydrogenase (EC 1.1.1.22); (ii) UV spectra; and (iii) matrix-assisted laser desorption/ionization-time of flight mass analysis and 1H-nuclear magnetic resonance spectrometry. The syntheses of both FUDP-sugars were inversely related to the concentration of uracil and directly related to the concentration of 5-fluorouracil in the culture medium. The strain W303-1A, requiring uracil for growth, was useful as a tool to analyze the effect of 5-fluorouracil on nucleotide metabolism. © 2007 Federation of European Microbiological Societies., This investigation was supported by grants from Dirección General de Investigación Científica y Técnica (BFU2006-04307/BMC 2002-00866).

In repeat expansion disorders, the size of pathological alleles is the most relevant factor accounting for the disease severity and age-at-onset, emphasizing the clinical significance of their underlying intergenerational instability. In one of these diseases, Machado-Joseph disease (MJD), the sex of transmitting progenitor and the C987GG/G987GG polymorphism are the best studied factors acting on intergenerational instability of expanded alleles. Here, we assessed the influence of other cis and inter-allelic acting factors, at the ATXN3 locus, through the analysis of MJD lineages, flanking STR-based haplotypes, the initial repeat size and parental age. A total of 100 transmissions of the expanded MJD allele were analyzed according to the sex of the transmitting parent. We have shown that independent origin mutations (identified by intragenic SNP-based haplotypes) behave differently, as the status of instability (contraction, no change or further expansion) is concerned. Indeed, 72% of expansions were associated to the worldwide spread TTACAC lineage, whereas the GTGGCA displayed 75% of all contractions observed. The analysis of flanking recombinant haplotypes did not suggest any further distant cis elements acting up- or downstream the ATXN3 locus. Considering the increased amplitude of expansions seen in older transmitting fathers, a repair-based mechanism may be suggested for the meiotic instability at this locus; furthermore, the lack of correlation between the initial repeat size and degree of instability did not support a replication-based mechanism. In summary, our findings point to different mechanisms of instability underlying male and female meioses, as well as contraction and expansion processes in MJD. © 2007 Wiley-Liss, Inc.

The blood–brain barrier plays an important role in protecting the brain from injury and diseases, but also restrains the delivery of potential therapeutic drugs for the treatment of brain illnesses, such as tumors. Glioma is most common cancer type of central nervous system in adults and the most lethal in children. The treatment is normally poor and ineffective. To better understand the ability of drug delivery systems to permeate this barrier, a blood–brain barrier model using human brain endothelial cells and a glioma cell line is herein proposed. The consistent trans-endothelial electrical values, immunofluorescence and scanning electronic microscopy showed a confluent endothelial cell monolayer with high restrictiveness. Upon inclusion of glioma cell line, the trans-endothelial electrical resistance decreased, with consequent increase of apparent permeability of fluorescein isothiocyanate dextran used as model drug, revealing a reduction of the barrier robustness. In addition, it was demonstrated a cell shape modification in the co-culture, with loss of tight junctions. The microenvironment of co-cultured model presented significant increase of of CCL2/MCP-1 and IL-6 production, correlating with the modulation of permeation. The results encourage the use of the proposed in vitro model as a screening tool when performing drugs permeability for the treatment of disorders among the central nervous system.

As the population ages, brain pathologies such as neurodegenerative diseases and brain cancer increase their incidence, being the need to find successful treatments of upmost importance. Drug delivery to the central nervous system (CNS) is required in order to reach diseases causes and treat them. However, biological barriers, mainly blood-brain barrier (BBB), are the key obstacles that prevent the effectiveness of possible treatments due to their ability to strongly limit the perfusion of compounds into the brain. Over the past decades, new approaches towards overcoming BBB and its efflux transporters had been proposed. One of these approaches here reviewed is through small interfering RNA (siRNA), which is capable to specifically target one gene and silence it in a post-transcriptional way. There are different possible functional proteins at the BBB, as the ones responsible for transport or just for its tightness, which could be a siRNA target. As important as the effective silence is the way to delivery siRNA to its anatomical site of action. This is where nanotechnology-based systems may help, by protecting siRNA circulation and providing cell/tissue-targeting and intracellular siRNA delivery. After an initial overview on incidence of brain diseases and basic features of the CNS, BBB and its efflux pumps, this review focuses on recent strategies to reach brain based on siRNA, and how to specifically target these approaches in order to treat brain diseases.

Hazelnut (Corylus avellana L.), a member of the family Betulaceae and the order Fagales, is native to Europe, Turkey, and the Caucasus. It has been cultivated for more than 5000 years. The majority of cultivars were selected from local wild populations over the past few centuries. Hazelnut is native to northern Portugal, with local production from landrace clonal selections. In recent years, hazelnut production has declined, and landraces are in danger of being lost. In this study, we used 16 microsatellite markers to investigate genetic diversity in wild populations, Portuguese landraces, and reference cultivars. A high level of polymorphism was found, as indicated by mean values for expected heterozygosity (0.74), observed heterozygosity (0.71), and polymorphism information content (0.78). A neighbor-joining dendrogram showed a clear separation of the wild genotypes from the landraces and reference cultivars. Bayesian analysis placed individuals in three groups, showing differentiation of wild populations, landraces, and reference cultivars. Our studies provide new insights on the origin of Portuguese landraces.

Despite the intense debate around the repeat instability reported on the large group of neurological disorders caused by trinucleotide repeat expansions, little is known about the mutation process underlying alleles in the normal range that, ultimately, expand to pathological size. In this study, we assessed the mutation mechanisms by which wild-type Machado-Joseph disease (MJD) alleles have been generated throughout human evolution. Haplotypes including the CAG repeat, six intragenic SNPs and four flanking microsatellites were analysed in 431 normal chromosomes of European, Asian and African origin. A bimodal CAG repeat length frequency distribution was found in the four most frequent wild-type lineages (H1-GCGGCA; H2-GTGGCA; H3-TTAGAC and H4-TTACAC). Based on flanking microsatellite haplotypes, the variance calculated by analysis of molecular variance between modal (CAG)n alleles was little or null in lineages H1, H2 and H4, as were the pairwise differences. Moreover, genetic distances among all the alleles from each lineage did not reflect the allele sizes differences, as expected if a stepwise mutation model was the main process of evolution. On the contrary, when exposed in maximum parsimonious phylogenetic trees, a large number of mutation steps separated same-size alleles, whereas several microsatellite haplotypes were shared by modal CAGs. In conclusion, our results suggest that the main mutation mechanism occurring in the evolution of the polymorphic CAG region at MJD/SCA3 locus is a multistep one, either by gene conversion or DNA slippage; repeats with 14, 21, 23 and 27 CAGs are the main alleles involved in this process.

Seventeen landraces of common beans ( Phaseolus vulgaris L.) sampled in the North and Centre of Portugal were analyzed at population level for 689 RAPD loci amplified by using forty random primers. The two different parameters used to estimate the genetic variability in and between samples indicate that the inter-population component of the genetic variability is mainly responsible for the diversity found, since only about a 10% would be of an intra-population nature. In addition, the gametic disequilibrium was estimated and reached an average value of 40% for the different combinations of pairs in the 689 loci studied taking the 17 samples as a whole population. Self-pollination, genetic drift and adaptation would thus be favouring the formation of multilocus associations. In addition, the fingerprinting study suggests that each landrace produced unique amplification products allowing it to be distinguished from the other tested genotypes, but, nevertheless, the landraces show a considerable genetic similarity (56% and 70.5% using two different methods). The Neighbour-Joining dendrogram did not show any relation between the geographical distribution of landraces and genetic distance. The results suggest that these Portuguese landraces conserved an important genetic diversity that can be useful to widen the genetic base of currently cultivated beans.

Dentatorubropallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by a variable combination of progressive ataxia, epilepsy, myoclonus, choreoathetosis and dementia. This disease is caused by a (CAG)(n) expansion in the DRPLA gene, on chromosome 12p13. DRPLA is prevalent in Japan, but several families of non-Japanese ancestry have already been published. To identify the origin of expanded alleles in Portuguese families with DRPLA, we studied two previously reported intragenic SNPs in introns 1 and 3, in addition to the CAG repeat of the DRPLA gene. The results showed that all four Portuguese DRPLA families shared the same haplotype, which is also common to that reported for Japanese DRPLA chromosomes. This haplotype is also the most frequent in Japanese normal alleles, whereas it was rare in Portuguese control chromosomes. Thus, our findings support that a founder DRPLA haplotype of Asian origin was introduced in Portugal, being responsible for the frequency of the disease in this country.

Twelve neurological disorders are caused by gene-specific CAG/CTG repeat expansions that are highly unstable upon transmission to offspring. This intergenerational repeat instability is clinically relevant since disease onset, progression and severity are associated with repeat size. Studies of model organisms revealed the involvement of some DNA replication and repair genes in the process of repeat instability, however, little is known about their role in patients. Here, we used an association study to search for genetic modifiers of (CAG)n instability in 137 parent-child transmissions in Machado-Joseph disease (MJD/SCA3). With the hypothesis that variants in genes involved in DNA replication, repair or recombination might alter the MJD CAG instability patterns, we screened 768 SNPs from 93 of these genes. We found a variant in ERCC6 (rs2228528) associated with an expansion bias of MJD alleles. When using a gene-gene interaction model, the allele combination G-A (rs4140804-rs2972388) of RPA3-CDK7 is also associated with MJD instability in a direction-dependent manner. Interestingly, the transcription-coupled repair factor ERCC6 (aka CSB), the single-strand binding protein RPA, and the CDK7 kinase part of the TFIIH transcription repair complex, have all been linked to transcription-coupled repair. This is the first study performed in patient samples to implicate specific modifiers of CAG instability in humans. In summary, we found variants in three transcription-coupled repair genes associated with the MJD mutation that points to distinct mechanisms of (CAG)n instability. This work was supported by the Canadian Institutes of Health Research; and the Levesque Chair for research in Neurogenetics [to G. A. R.]; and the Portuguese Foundation for Science and Technology [SFRH/BPD/77969/2011 to S.M].

Background Several strategies have been developed to counteract liver injury as a consequence of nonsteroid anti-inflammatory drugs toxicity. Here, we aimed to determine whether physical exercise results in liver mitochondrial protection against in vitro diclofenac toxicity. Material and methods Male adult Sprague-Dawley rats were divided into sedentary, 12-week endurance training (ET) and voluntary activity (VPA). In vitro liver mitochondrial function as assessed by oxygen consumption, transmembrane electric potential (ΔΨ) and susceptibility to the mitochondrial permeability transition pore (MPTP) was evaluated in the absence and presence of diclofenac. Mitochondrial oxidative stress markers [MnSOD, aconitase, -SH and MDA, SIRT3, p66shc(Ser36)/p66shc ratio] and apoptotic signalling (caspases 3, 8 and 9, Bax, Bcl-2 and CypD) were assessed. Content of OXPHOS components and qualitative liver morphological evaluation were assessed. Results Despite no effects of ET and VPA on basal liver mitochondrial oxygen consumption or ΔΨ endpoints, exercised animals showed lower susceptibility to MPTP. Diclofenac-induced decrease in ΔΨ, increased state 4 respiration and susceptibility to MPTP opening were all prevented by exercise. Under untreated conditions, VPA group showed higher aconitase activity, while ET decreased MDA and increased Bax content. VPA decreased p66shc(Ser36), complex III and V OXPHOS subunits. Both ET and VPA increased complex IV OXPHOS subunit, and SIRT3 and Bcl-2 content and decreased caspase 9 activity. Unexpectedly, ET and VPA decreased ANT. Conclusions Both chronic physical exercise models augmented the resistance to in vitro diclofenac-induced mitochondrial alterations, including increased MPTP susceptibility, possibly by modulating oxidative stress and MPTP regulators.

Agriculture can only be considered as sustainable if it includes a suitable system of plant genetic resources conservation. Crop genetic resources conservation is an insurance against risks to food security. The landraces stand important genetic resources for plant breeders due to its genotypic variability. In Portugal, the production of Cucurbita is based in old local cultivars and is mainly for self-consumption (human food or feed) and is sale on local markets. To evaluate the diversity of 54C. pepo, 32C. maxima and 21C. moschata populations collected in Northern and inner center of Portugal, 20 morphological traits from the Minimum Descriptors for Cucurbita spp. and six microsatellites were used. The traits that registered the highest mean values were the fruit length, the skin thickness and the seed weight, in C. pepo, the fruit width and 100 seed weight in C. maxima and the fruit weight and flesh thickness in C. moschata populations. The ANOVA revealed significant differences (P

Zebrafish is already one of the most used model organisms in biomedical sciences and other research fields. It is therefore becoming increasingly important to assure that zebrafish maintained in laboratory aquaculture conditions are raised and housed under rigorous standards that promote health and welfare to guarantee the required quality and reproducibility of research data. Specifying the programs each facility is adopting would be the first step to achieve this by allowing other facilities to compare, improve, and discuss their protocols and fish performance. We provide in this article a detailed description of an integrated facility health management program, with protocols and readouts, fully designed and aimed at maximizing fish health, welfare, and performance for research.