Molecular characterization of Portuguese patients with hereditary cerebellar ataxia

© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Hereditary cerebellar ataxia (HCA) comprises a clinical and genetic heterogeneous group of neurodegenerative disorders characterized by incoordination of movement, speech, and unsteady gait. In this study, we performed whole-exome sequencing (WES) in 19 families with HCA and presumed autosomal recessive (AR) inheritance, to identify the causal genes. A phenotypic classification was performed, considering the main clinical syndromes: spastic ataxia, ataxia and neuropathy, ataxia and oculomotor apraxia (AOA), ataxia and dystonia, and ataxia with cognitive impairment. The most frequent causal genes were associated with spastic ataxia (SACS and KIF1C) and with ataxia and neuropathy or AOA (PNKP). We also identified three families with autosomal dominant (AD) forms arising from de novo variants in KIF1A, CACNA1A, or ATP1A3, reinforcing the importance of differential diagnosis (AR vs. AD forms) in families with only one affected member. Moreover, 10 novel causal-variants were identified, and the detrimental effect of two splice-site variants confirmed through functional assays. Finally, by reviewing the molecular mechanisms, we speculated that regulation of cytoskeleton function might be impaired in spastic ataxia, whereas DNA repair is clearly associated with AOA. In conclusion, our study provided a genetic diagnosis for HCA families and proposed common molecular pathways underlying cerebellar neurodegeneration.This work was funded by National Funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., under the project UIDB/04293/2020. It was also supported in part by the FCT grant FCT-ANR/BEX-GMG/0008/2013; the Porto Neurosciences and Neurologic Disease Research Initiative at the i3S (Norte-01-0145-FEDER-000008), supported by Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement, also through FEDER; and by GenomePT (POCI-01-0145-FEDER-022184). M.S. was the recipient of a fellowship (SFRH/BPD/116046/2016) and acknowledges funding from FCT through program DL 57/2016—Norma Transitória.info:eu-repo/semantics/publishedVersio

pH and Reactive Oxygen Species-Sequential Responsive Nano-in-Micro Composite For Targeted Therapy of Inflammatory Bowel Disease

Oxidative stress and abnormally high levels of reactive oxygen species (ROS) play an essential role in the pathogenesis and progression of inflammatory bowel disease (IBD). Oxidation‐responsive nanoparticles (NPs) are formulated from a phenylboronic esters‐modified dextran (OxiDEX) that degrades selectively in response to hydrogen peroxide (H2O2). OxiDEX NPs are coated with chitosan and encapsulated in a pH‐sensitive polymer to produce nano‐in‐micro composites. The microparticles are spherical with homogeneous particle size (53 ± 3 µm) and maintain integrity at acidic pH, preventing the premature release of the NPs in gastric conditions. The degradation of NPs is highly responsive to the level of H2O2, and the release of the drug is sustained in the presence of physiologically relevant H2O2 concentrations. The presence of chitosan on the particles surface significantly enhances NPs stability in intestinal pH and their adhesion on the intestinal mucosa. Compared to a traditional enteric formulation, this formulation shows tenfold decreased drug permeability across C2BBe1/HT29‐MTX cell monolayer, implying that lower amount of drug would be absorbed to the blood stream and, therefore, limiting the undesired systemic side effects. Based on these results, a successful nano‐in‐micro composite for targeted therapy of IBD is obtained by combination of the responsiveness to pH and ROS.Peer reviewe

Interaction of [(VO)-O-IV(acac)(2)] with Human Serum Transferrin and Albumin

VO(acac)(2)] is a remarkable vanadium compound and has potential as a therapeutic drug. It is important to clarify how it is transported in blood, but the reports addressing its binding to serum proteins have been contradictory. We use several spectroscopic and mass spectrometric techniques (ESI and MALDI-TOF), small-angle X-ray scattering and size exclusion chromatography (SEC) to characterize solutions containing [VO(acac)(2)] and either human serum apotransferrin (apoHTF) or albumin (HSA). DFT and modeling protein calculations are carried out to disclose the type of binding to apoHTF. The measured circular dichroism spectra, SEC and MALDI-TOF data clearly prove that at least two VOacac moieties may bind to apoHTF, most probably forming [(VO)-O-IV(acac)(apoHTF)] complexes with residues of the HTF binding sites. No indication of binding of [VO(acac)(2)] to HSA is obtained. We conclude that (VO)-O-IV-acac species may be transported in blood by transferrin. At very low complex concentrations speciation calculations suggest that [(VO)(apoHTF)] species form.Fundacao para a Ciencia e Tecnologia (FCT), Portugal [ RECI/QEQMED/0330/2012, PTDC/QEQ-MED/1902/2014]FCT [IF/00100/2013, IF/00007/2015]PROTEOMASS Scientific SocietyUCIBIO, Unidade de Ciencias Biomoleculares Aplicadas [UID/Multi/04378/2013]ERDF [POCI-01-0145-FEDER-007728, POCI-01-0145-FEDER-007265]info:eu-repo/semantics/publishedVersio

O ERRO E A AVALIAÇÃO DA APRENDIZAGEM: AS CONCEPÇÕES DE PROFESSORES E ALUNOS

A prática avaliativa classicatória, no interior das escolas, vem cedendo lugar para outra, exercida com sentido mais formativo. Nesse contexto, espera-se que o erro passe a ser concebido como indicador diagnóstico para orientar professores e alunos. Todavia, cumpre questionar: como professores e alunos concebem o erro no processo de ensino e aprendizagem? Como professores e alunos sentem-se quando erram em situações avaliativas, no contexto escolar? Este trabalho, assim, tem por objetivo geral o de confrontar e analisar as concepções de erro manifestas por professores e alunos. A pesquisa qualitativa configurou-se como a opção mais pertinente. A coleta de dados teve por fonte a representação pictórica do sentimento frente ao erro cometido em situações de avaliação escolar, bem como pelo registro das explicações manifestas por 47 alunos e 29 professores do ensino fundamental II. A análise das imagens, aliada à análise das explicações proferidas possibilitou determinar as unidades temáticas. A análise dos dados revelou não haver discrepância entre as concepções discente e docente no concernente ao erro. Todos temem e se entristecem frente a ele e poucos reagem favoravelmente, tracejando alternativas para a sua superação. A predominância de sentimentos negativos, como: ansiedade, medo, frustração, raiva, desespero, desesperança faz-se presente nas imagens traçadas sobre o papel, bem como nas palavras escritas no verso das folhas. A evitação é uma das consequências e, pari passu, outra é o abandono do desejo de tentar

Individual common variants exert weak effects on the risk for autism spectrum disorders.

While it is apparent that rare variation can play an important role in the genetic architecture of autism spectrum disorders (ASDs), the contribution of common variation to the risk of developing ASD is less clear. To produce a more comprehensive picture, we report Stage 2 of the Autism Genome Project genome-wide association study, adding 1301 ASD families and bringing the total to 2705 families analysed (Stages 1 and 2). In addition to evaluating the association of individual single nucleotide polymorphisms (SNPs), we also sought evidence that common variants, en masse, might affect the risk. Despite genotyping over a million SNPs covering the genome, no single SNP shows significant association with ASD or selected phenotypes at a genome-wide level. The SNP that achieves the smallest P-value from secondary analyses is rs1718101. It falls in CNTNAP2, a gene previously implicated in susceptibility for ASD. This SNP also shows modest association with age of word/phrase acquisition in ASD subjects, of interest because features of language development are also associated with other variation in CNTNAP2. In contrast, allele scores derived from the transmission of common alleles to Stage 1 cases significantly predict case status in the independent Stage 2 sample. Despite being significant, the variance explained by these allele scores was small (Vm< 1%). Based on results from individual SNPs and their en masse effect on risk, as inferred from the allele score results, it is reasonable to conclude that common variants affect the risk for ASD but their individual effects are modest

ST3Gal.I sialyltransferase relevance in bladder cancer tissues and cell lines

<p>Abstract</p> <p>Background</p> <p>The T antigen is a tumor-associated structure whose sialylated form (the sialyl-T antigen) involves the altered expression of sialyltransferases and has been related with worse prognosis. Since little or no information is available on this subject, we investigated the regulation of the sialyltransferases, able to sialylate the T antigen, in bladder cancer progression.</p> <p>Methods</p> <p>Matched samples of urothelium and tumor tissue, and four bladder cancer cell lines were screened for: <it>ST3Gal.I</it>, <it>ST3Gal.II </it>and <it>ST3Gal.IV </it>mRNA level by real-time PCR. Sialyl-T antigen was detected by dot blot and flow cytometry using peanut lectin. Sialyltransferase activity was measured against the T antigen in the cell lines.</p> <p>Results</p> <p>In nonmuscle-invasive bladder cancers, <it>ST3Gal.I </it>mRNA levels were significantly higher than corresponding urothelium (p < 0.001) and this increase was twice more pronounced in cancers with tendency for recurrence. In muscle-invasive cancers and matching urothelium, <it>ST3Gal.I </it>mRNA levels were as elevated as nonmuscle-invasive cancers. Both non-malignant bladder tumors and corresponding urothelium showed <it>ST3Gal.I </it>mRNA levels lower than all the other specimen groups. A good correlation was observed in bladder cancer cell lines between the <it>ST3Gal.I </it>mRNA level, the ST activity (r = 0.99; p = 0.001) and sialyl-T antigen expression, demonstrating that sialylation of T antigen is attributable to ST3Gal.I. The expression of sialyl-T antigens was found in patients' bladder tumors and urothelium, although without a marked relationship with mRNA level. The two <it>ST3Gal.I </it>transcript variants were also equally expressed, independently of cell phenotype or malignancy.</p> <p>Conclusion</p> <p>ST3Gal.I plays the major role in the sialylation of the T antigen in bladder cancer. The overexpression of <it>ST3Gal.I </it>seems to be part of the initial oncogenic transformation of bladder and can be considered when predicting cancer progression and recurrence.</p

The First Cellular Models Based on Frataxin Missense Mutations That Reproduce Spontaneously the Defects Associated with Friedreich Ataxia

BACKGROUND:Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)(n) expansion within the first intron of the frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation. METHODOLOGY:We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN(G130V) and hFXN(I154F)) frataxin. Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN(I154F) mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients. CONCLUSIONS:These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating frataxin deficiency

Identification of metabolic pathways influenced by the G-protein coupled receptors GprB and GprD in Aspergillus nidulans

Heterotrimeric G-protein-mediated signaling pathways play a pivotal role in transmembrane signaling in eukaryotes. Our main aim was to identify signaling pathways regulated by A. nidulans GprB and GprD G-protein coupled receptors (GPCRs). When these two null mutant strains were compared to the wild-type strain, the DeltagprB mutant showed an increased protein kinase A (PKA) activity while growing in glucose 1% and during starvation. In contrast, the DeltagprD has a much lower PKA activity upon starvation. Transcriptomics and (1)H NMR-based metabolomics were performed on two single null mutants grown on glucose. We noted modulation in the expression of 11 secondary metabolism gene clusters when the DeltagprB and DeltagprD mutant strains were grown in 1% glucose. Several members of the sterigmatocystin-aflatoxin gene cluster presented down-regulation in both mutant strains. The genes of the NR-PKS monodictyphenone biosynthesis cluster had overall increased mRNA accumulation in DeltagprB, while in the DeltagprD mutant strain the genes had decreased mRNA accumulation. Principal component analysis of the metabolomic data demonstrated that there was a significant metabolite shift in the DeltagprD strain. The (1)H NMR analysis revealed significant expression of essential amino acids with elevated levels in the DeltagprD strain, compared to the wild-type and DeltagprB strains. With the results, we demonstrated the differential expression of a variety of genes related mainly to secondary metabolism, sexual development, stress signaling, and amino acid metabolism. We propose that the absence of GPCRs triggered stress responses at the genetic level. The data suggested an intimate relationship among different G-protein coupled receptors, fine-tune regulation of secondary and amino acid metabolisms, and fungal development