👤 V M R Pires

Batten disease is characterized by early-onset blindness, juvenile dementia and death within the second decade of life. The most common genetic cause are mutations in CLN3, encoding a lysosomal protein. Currently, no therapies targeting disease progression are available, largely because its molecular mechanisms remain poorly understood. To understand how CLN3 loss affects cellular signaling, we generated human CLN3 knock-out cells (CLN3-KO) and performed RNA-seq analysis. Our multi-dimensional analysis reveals the transcriptional regulator YAP1 as a key factor in remodeling the transcriptome in CLN3-KO cells. YAP1-mediated pro-apoptotic signaling is also increased as a consequence of CLN3 functional loss in retinal pigment epithelia cells, and in the hippocampus and thalamus of Cln3 Show less

Mitochondrial dysfunction is one of the leading causes of disease development. Dysfunctional mitochondria limit energy production, increase reactive oxygen species generation, and trigger apoptotic signals. Photobiomodulation is a noninvasive, nonthermal technique involving the application of monochromatic light with low energy density, inducing non-thermal photochemical effects at the cellular level, and it has been used due to its therapeutic potential. This review focuses on the mitochondrial dynamic's role in various diseases, evaluating the possible therapeutic role of low-power lasers (LPL) and light-emitting diodes (LED). Studies increasingly support that mitochondrial dysfunction is correlated with severe neurodegenerative diseases such as Parkinson's, Huntington's, Alzheimer's, and Charcot-Marie-Tooth diseases. Furthermore, a disturbance in mitofusin activity is also associated with metabolic disorders, including obesity and type 2 diabetes. The effects of PBM on mitochondrial dynamics have been observed in cells using a human fibroblast cell line and in vivo models of brain injury, diabetes, spinal cord injury, Alzheimer's disease, and skin injury. Thus, new therapies aiming to improve mitochondrial dynamics are clinically relevant. Several studies have demonstrated that LPL and LED can be important therapies to improve health conditions when there is dysfunction in mitochondrial dynamics. Show less

The effects of feeding Cistus ladanifer (Cistus) and a blend of soybean and linseed oil (1 : 2 vol/vol) on fatty acid (FA) composition of lamb meat lipids and messenger RNA (mRNA) expression of desaturase enzymes was assessed. In total, 54 male lambs were randomly assigned to 18 pens and to nine diets, resulting from the combination of three inclusion levels of Cistus (50 v. 100 v. 200 g/kg of dry matter (DM)) and three inclusion levels of oil (0 v. 40 v. 80 g/kg of DM). The forage-to-concentrate ratio of the diets was 1 : 1. Longissimus muscle lipids were extracted, fractionated into neutral (NL) and polar lipid (PL) and FA methyl esters obtained and analyzed by GLC. The expression of genes encoding Δ5, Δ6 and Δ9 desaturases (fatty acid desaturase 1 (FADS1), fatty acid desaturase 2 (FADS2) and stearoyl CoA desaturase (SCD)) was determined. Intramuscular fat, NL and PL contents were not affected by oil or Cistus. Oil supplementation reduced (P<0.05) 16:0, c9-16:1, 17:0, c9-17:1 and c9-18:1 FA and increased (P<0.05) 18:2n-6, 18:3n-3 and the majority of biohydrogenation intermediates in NL. Cistus alone had few effects on FA of NL but interacted with oil (P<0.05) by increasing t10-18:1,t10,t12-18:2,t10,c12-18:2 and t7,c9-18:2. The t10-/t11-18:1 ratio increased with both Cistus and oil levels. The c9, t11-18:2 did not increase (P<0.05) with both oil and Cistus dietary inclusion. Oil reduced c9-16:1, 17:0, c9-17:1,c9-18:1, 20:4n-6, 22:4n-6 and 20:3n-9 proportions in PL, and increased 18:2n-6, 18:3n-3, 20:3n-3 and of most of the biohydrogenation intermediates. The Cistus had only minor effects on FA composition of PL. Cistus resulted in a reduction (P<0.05) of 20:5n-3 and 22:6n-3 in the meat PL. The expression level of SCD mRNA increased (P=0.015) with Cistus level, although a linear relationship with condensed tannins intake (P=0.11) could not be established. FADS1 mRNA expressed levels increased linearly (P=0.019) with condensed tannins intake. In summary, the inclusion of Cistus and oil in 1 : 1 forage-to-concentrate ratio diets resulted in a large increase in t10-18:1 and no increase in c9,t11-18:2 or n-3 long chain poor in polyunsaturated fatty acids in lamb meat. Show less

The influence of genotype (lean v. fatty) and dietary protein level (normal v. reduced) on plasma metabolites, hepatic fatty acid composition and mRNA levels of lipid-sensitive factors is reported for the first time, using the pig as an experimental model. The experiment was conducted on forty entire male pigs (twenty lean pigs of Large White×Landrace×Pietrain cross-breed and twenty fatty pigs of Alentejana purebreed) from 60 to 93 kg of live weight. Each pig genotype was divided into two subgroups, which were fed the following diets: a normal protein diet (NPD) equilibrated for lysine (17·5 % crude protein and 0·7 % lysine) and a reduced protein diet (RPD) not equilibrated for lysine (13·1 % crude protein and 0·4 % lysine). The majority of plasma metabolites were affected by genotype, with lean pigs having higher contents of lipids, whereas fatty pigs presented higher insulin, leptin and urea levels. RPD increased plasma TAG, free fatty acids and VLDL-cholesterol compared with NPD. Hepatic total lipids were higher in fatty pigs than in the lean genotype. RPD affected hepatic fatty acid composition but had a slight influence on gene expression levels in the liver. Sterol regulatory element-binding factor 1 was down-regulated by RPD, and fatty acid desaturase 1 (FADS1) and fatty acid binding protein 4 (FABP4) were affected by the interaction between genotype and diet. In pigs fed RPD, FADS1 was up-regulated in the lean genotype, whereas FABP4 increased in the fatty genotype. Although there is a genotype-specific effect of dietary protein restriction on hepatic lipid metabolism, lipogenesis is not promoted in the liver of lean or fatty pigs. Show less

Nitric oxide (NO) is a chemical messenger involved in the control of oocyte maturation. It stimulates guanylate cyclase to produce cyclic guanosine monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase (PKG) and some phosphodiesterases that may interfere with cAMP levels, a nucleotide also involved in meiosis resumption. The aim of this study was to determine the role played by NO on the cGMP/cAMP pathway during meiosis resumption in bovine oocytes. The effects of increasing NO generated by S-nitroso-N-acetylpenicillamine (SNAP; 10(-7)-10(-3) mol/L) and of other drugs that may affect the NO/cGMP pathway (proptoporfirin IX and 8-Br-cGMP) on meiosis resumption were investigated in bovine cumulus-oocyte complexes (COCs) matured for 9 hours in a semidefined medium (TCM199 + 3 mg/mL BSA). The COCs matured with 10(-7) mol/L SNAP associated or not with 100 μmol/L oxadiazole-one quinoxaline, a guanylate cyclase inhibitor, also had their cGMP and cAMP levels measured during the first hours of maturation (1, 3, and 6 hours). Quantitative polymerase chain reaction was performed by real-time polymerase chain reaction to determine the effects of NO on expression of genes encoding for enzymes of the NO/guanylate cyclase/cGMP and cAMP pathways during the first 9 hours of oocyte maturation. Increasing NO levels using 10(-7) mol/L SNAP resulted in lower rate of germinal vesicle breakdown (36% germinal vesicle breakdown; P < 0.05) at 9 hours IVM, whereas control group and the treatments with 10(-9) and 10(-8) mol/L SNAP showed about 70% germinal vesicle breakdown (P > 0.05). A temporary increase in cGMP levels was also observed with the same treatment (4.51 pmol/COC) at 1 hour IVM, which was superior to the control group (2.97 pmol/COC; P < 0.05) and was reversed by inhibiting guanylate cyclase activity with 100 μmol/L oxadiazole-one quinoxaline. Neither cAMP levels nor gene expression were affected by NO. These results suggest that NO acts via guanylate cyclase/cGMP and that even a temporary increase in cGMP levels leads to a delay in meiosis resumption, even when cAMP levels have declined. Nitric oxide does not act on oocyte maturation by affecting cAMP levels or the expression of genes related to the NO/guanylate cyclase/cGMP and cAMP pathways. Also, to our knowledge this is the first report to detect PKG1, PKG2, phosphodiesterase-5A, ADCY3, ADCY6, and ADCY9 transcripts in bovine oocytes. Show less

In ruminants, unsaturated dietary fatty acids are biohydrogenated in the rumen and are further metabolised in various tissues, including liver, which has an important role in lipid and lipoprotein metabolism. Therefore, manipulation of muscle fatty acid composition should take into account liver metabolism. In the present study, the influence of breed and diet on liver lipid composition and gene expression was investigated in order to clarify the role of this organ in the lipid metabolism of ruminants. Forty purebred young bulls from two phylogenetically distant autochthonous cattle breeds, Alentejana and Barrosã, were assigned to two different diets (low vs. high silage) and slaughtered at 18 months of age. Liver fatty acid composition, mRNA levels of enzymes and transcription factors involved in lipid metabolism, as well as the plasma lipid profile, were assessed. In spite of similar plasma non-esterified fatty acids levels, liver triacylglycerols content was higher in Barrosã than in Alentejana bulls. Moreover, the fatty acid composition of liver was clearly distinct from the remaining tissues involved in fatty acid metabolism of ruminants, as shown by Principal Components Analysis. The hepatic tissue is particularly rich in α-linolenic acid and their products of desaturation and elongation. Results indicate that DGAT1, ELOVL2, FADS1 and FADS2 genes influence the fatty acid composition of the liver the most. Moreover, genes such as DGAT1 and ELOVL2 appear to be more sensitive to genetic background than to dietary manipulation, whereas genes encoding for desaturases, such as FADS1, appear to be modulated by dietary silage level. Our results indicate that liver plays an important role in the biosynthesis of n-3 LC-PUFA. It is also suggested that dietary silage level influences the hepatic fatty acid metabolism in a breed-dependent manner, through changes in the expression of genes encoding for enzymes associated with the desaturation and elongation pathway. The importance of devising custom-made feeding strategies taking into account the genetic background is, therefore, stressed by the results from this experiment. Show less

Hypertrophic Cardiomyopathy (HCM) is a complex myocardial disorder with a recognized genetic heterogeneity. The elevated number of genes and mutations involved in HCM limits a gene-based diagnosis that should be considered of most importance for basic research and clinical medicine. In this report, we evaluated High Resolution Melting (HRM) robustness, regarding HCM genetic testing, by means of analyzing 28 HCM-associated genes, including the most frequent 4 HCM-associated sarcomere genes, as well as 24 genes with lower reported HCM-phenotype association. We analyzed 80 Portuguese individuals with clinical phenotype of HCM allowing simultaneously a better characterization of this disease in the Portuguese population. HRM technology allowed us to identify 60 mutated alleles in 72 HCM patients: 49 missense mutations, 3 nonsense mutations, one 1-bp deletion, one 5-bp deletion, one in frame 3-bp deletion, one insertion/deletion, 3 splice mutations, one 5'UTR mutation in MYH7, MYBPC3, TNNT2, TNNI3, CSRP3, MYH6 and MYL2 genes. Significantly 22 are novel gene mutations. HRM was proven to be a technique with high sensitivity and a low false positive ratio allowing a rapid, innovative and low cost genotyping of HCM. In a short return, HRM as a gene scanning technique could be a cost-effective gene-based diagnosis for an accurate HCM genetic diagnosis and hopefully providing new insights into genotype/phenotype correlations. Show less

Left ventricular noncompaction (LVNC) is a rare and potentially progressive cardiomyopathy, characterized by the persistence of multiple trabeculations and deep intratrabecular recesses in the ventricular myocardium. Although two-dimensional and color Doppler echocardiography are the most useful diagnostic modalities, cardiac magnetic resonance imaging has proved to have high sensitivity and specificity in the diagnosis of this anomaly. To characterize the clinical and imaging features of LVNC in a pediatric population and to assess their evolution. We performed a retrospective chart review of five pediatric patients with LVNC, followed at Coimbra Pediatric Hospital between January 1999 and December 2007. Median age at presentation was five months (ranging from one day to 13 years), and they were mainly male (1.5:1). Two of the children had a family history of sudden death. In one case the clinical presentation was cardiac arrest due to ventricular fibrillation and in three others, congestive cardiac failure. None of the five cases had associated congenital cardiac anomalies. Involvement of the ventricular apical region was found in all cases. Four children additionally had ventricular dysfunction which improved with diuretic and vasodilator therapy. Mean follow-up was 34 months, ranging from six months to seven years. In one case a change in the morphological phenotype was noted, from a dilated to a hypertrophic form. In this case and in the child's father a mutation in the MYBPC3 gene was identified, which is associated with hypertrophic cardiomyopathy. No thromboembolic phenomena or deaths occurred during the study period. In the pediatric population, congestive cardiac failure is the most common clinical presentation of LVNC, which can coexist with other cardiomyopathies, particularly dilated and hypertrophic forms. The sample presented in this analysis is statistically non-significant due to its limited size and the authors highlight the need for larger prospective studies in the pediatric population in order to clarify this disease and its diagnostic criteria. Show less