Metabolic Rewiring and Altered Glial Differentiation in an iPSC-Derived Astrocyte Model Derived from a Nonketotic Hyperglycinemia Patient.

The pathophysiology of nonketotic hyperglycinemia (NKH), a rare neuro-metabolic disorder associated with severe brain malformations and life-threatening neurological manifestations, remains incompletely understood. Therefore, a valid human neural model is essential. We aimed to investigate the impact of GLDC gene variants, which cause NKH, on cellular fitness during the differentiation process of human induced pluripotent stem cells (iPSCs) into iPSC-derived astrocytes and to identify sustainable mechanisms capable of overcoming GLDC deficiency. We developed the GLDC27-FiPS4F-1 line and performed metabolomic, mRNA abundance, and protein analyses. This study showed that although GLDC27-FiPS4F-1 maintained the parental genetic profile, it underwent a metabolic switch to an altered serine-glycine-one-carbon metabolism with a coordinated cell growth and cell cycle proliferation response. We then differentiated the iPSCs into neural progenitor cells (NPCs) and astrocyte-lineage cells. Our analysis showed that GLDC-deficient NPCs had shifted towards a more heterogeneous astrocyte lineage with increased expression of the radial glial markers GFAP and GLAST and the neuronal markers MAP2 and NeuN. In addition, we detected changes in other genes related to serine and glycine metabolism and transport, all consistent with the need to maintain glycine at physiological levels. These findings improve our understanding of the pathology of nonketotic hyperglycinemia and offer new perspectives for therapeutic options.

Ubiquitin-specific protease 7 (USP7): an emerging drug target for cancer treatment.

INTRODUCTION: Ubiquitin-specific protease 7 (USP7) also known as herpesvirus-associated ubiquitin-specific protease (HAUSP) is a well-characterized cysteine protease that belongs to the largest subfamily of deubiquitinating enzymes (DUBs). It is involved in multiple signaling pathways, some of them dysregulated in malignant tumors. USP7 inhibition can lead to cell growth arrest and apoptosis through inhibition of tumor promoters and stabilization of tumor suppressors, making it a promising druggable target for cancer therapy. AREAS COVERED: This review covers the structure of USP7, its function in multiple signaling pathways and relevance in cancer, as well as recent advances and future perspectives in the development of USP7 inhibitors for cancer therapy. EXPERT OPINION: Literature reports display the multiple antitumor activities of USP7 inhibitors, both in vitro and in vivo. Nonetheless, none have entered clinical trials so far, highlighting the need to delve into a deeper understanding of USP7 binding sites and the development of more accurate compound screening methods. Despite these challenges, further development of USP7 inhibitors is promising as a valuable new approach for cancer treatment, including the ability to address chemoresistance.

The Influence of (Poly)phenol Intake in Saliva Proteome: Short- and Medium-Term Effects of Apple.

The relationship between salivary proteome and dietary habits was studied in previous works, where a relationship between salivary proteins like cystatins and polyphenol/tannin levels in diet was observed. However, it remains to be elucidated if this association results from an effect of polyphenol-rich food ingestion on saliva composition. The aim of this work was to test the effects of apple intake on the saliva proteome, both in the short and medium term (after 4 days of continuous intake). By incubating saliva samples with apple phenolic-rich extract, protein bands containing α-amylase, S-type cystatins, and proline-rich proteins (PRPs) appeared in the fraction that precipitated, showing the potential of these (poly)phenols to precipitate salivary proteins. Among these, it was salivary cystatins that presented changes in their levels both in the saliva samples collected immediately after apple intake and in the ones collected after 4 days of intake of an extra amount of apple. These results support the thought that intake is reflected in the salivary proteome. The effect of a polyphenol-rich food, like the apple, on salivary cystatin levels is in line with results observed in animal models and, due to the involvement of these proteins in oral food perception, it would be interesting to explore in future studies the effect of these changes on sensory perception and acceptance of polyphenol-rich food.

Pathogenic variants of the coenzyme A biosynthesis-associated enzyme phosphopantothenoylcysteine decarboxylase cause autosomal-recessive dilated cardiomyopathy.

Coenzyme A (CoA) is an essential cofactor involved in a range of metabolic pathways including the activation of long-chain fatty acids for catabolism. Cells synthesize CoA de novo from vitamin B5 (pantothenate) via a pathway strongly conserved across prokaryotes and eukaryotes. In humans, it involves five enzymatic steps catalyzed by four enzymes: pantothenate kinase (PANK [isoforms 1-4]), 4'-phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC), and CoA synthase (COASY). To date, inborn errors of metabolism associated with all of these genes, except PPCDC, have been described, two related to neurodegeneration with brain iron accumulation (NBIA), and one associated with a cardiac phenotype. This paper reports another defect in this pathway (detected in two sisters), associated with a fatal cardiac phenotype, caused by biallelic variants (p.Thr53Pro and p.Ala95Val) of PPCDC. PPCDC enzyme (EC 4.1.1.36) catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine in CoA biosynthesis. The variants p.Thr53Pro and p.Ala95Val affect residues highly conserved across different species; p.Thr53Pro is involved in the binding of flavin mononucleotide, and p.Ala95Val is likely a destabilizing mutation. Patient-derived fibroblasts showed an absence of PPCDC protein, and nearly 50% reductions in CoA levels. The cells showed clear energy deficiency problems, with defects in mitochondrial respiration, and mostly glycolytic ATP synthesis. Functional studies performed in yeast suggest these mutations to be functionally relevant. In summary, this work describes a new, ultra-rare, severe inborn error of metabolism due to pathogenic variants of PPCDC.

Pathogenic variants in GCSH encoding the moonlighting H-protein cause combined nonketotic hyperglycinemia and lipoate deficiency.

Maintaining protein lipoylation is vital for cell metabolism. The H-protein encoded by GCSH has a dual role in protein lipoylation required for bioenergetic enzymes including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase, and in the one-carbon metabolism through its involvement in glycine cleavage enzyme system, intersecting two vital roles for cell survival. Here, we report six patients with biallelic pathogenic variants in GCSH and a broad clinical spectrum ranging from neonatal fatal glycine encephalopathy to an attenuated phenotype of developmental delay, behavioral problems, limited epilepsy and variable movement problems. The mutational spectrum includes one insertion c.293-2_293-1insT, one deletion c.122_(228 + 1_229-1) del, one duplication of exons 4 and 5, one nonsense variant p.Gln76*and four missense p.His57Arg, p.Pro115Leu and p.Thr148Pro and the previously described p.Met1?. Via functional studies in patient's fibroblasts, molecular modeling, expression analysis in GCSH knockdown COS7 cells and yeast, and in vitro protein studies, we demonstrate for the first time that most variants identified in our cohort produced a hypomorphic effect on both mitochondrial activities, protein lipoylation and glycine metabolism, causing combined deficiency, whereas some missense variants affect primarily one function only. The clinical features of the patients reflect the impact of the GCSH changes on any of the two functions analyzed. Our analysis illustrates the complex interplay of functional and clinical impact when pathogenic variants affect a multifunctional protein involved in two metabolic pathways and emphasizes the value of the functional assays to select the treatment and investigate new personalized options.

Effect of thermal and chemical treatments used for SARS-COV-2 inactivation in the measurement of saliva analytes.

The present study aims to assess the effects of thermal and chemical inactivating procedures, that can be used for SARS-CoV-2 inactivation, on different salivary analytes. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) protein profile and a panel of 25 specific biomarkers of oxidative status, stress, metabolism and tissue damage were evaluated in samples subjected to different treatments: thermal (65 °C or 92 °C) and chemical with detergents [sodium dodecyl sulphate (SDS), Triton X-100 or NP-40]. Salivary SDS-PAGE profile was most affected by heating at 92 °C, with three and two protein bands decreasing and increasing their expression levels, respectively. This treatment also affected the results of several enzymes, with some of them being also affected by heating at 65 °C and incubation with SDS. The use of Triton X-100 or NP-40 resulted in increased values of cortisol, triglycerides and glucose, not affecting the other tested biomarkers. The present results will help researchers and clinicians to select the best protocols to work in safe conditions with saliva, taking into account the target analyte planned to be measured.

Impact of gastroplasty on salivary characteristics, dental health status and oral sensory aspects: A controlled clinical study.

BACKGROUND: Saliva is a non-invasive source of biomarkers useful in the study of different pathophysiological conditions. The qualitative and quantitative study of saliva, as well as the assessment of oral health, can be particularly useful for a better understanding of obesity due to its importance in the food oral perception and ingestion. OBJECTIVES: To evaluate the effect of treatment of morbid obesity (dietary counselling versus gastroplasty) on salivary characteristics, oral sensory perception and dental health in a controlled study. METHODS: Seventy-three adults (60 females; 19-59 years) with morbid obesity were divided in: Control group (CG; n = 34) followed-up during a 6-months dietary programme, and Gastroplasty group (GG; n = 39) evaluated immediately before, 3 and 6 months after gastroplasty. Dietary habits, Oral Health Impact Profile and xerostomia complaints were investigated by interview. The clinical examination included anthropometric and caries experience evaluation. Salivary flow rate, buffering capacity, total protein and alpha-amylase levels, and sensitivity for the four basic tastes were assessed. Two-way mixed model and sign test were applied. RESULTS: Physical and clinical aspects did not differ between groups in the beginning of the study, and GG showed a rapid weight loss after surgery (p < .001). An improvement in most of the dietary aspects was observed, especially in the GG. A worsening in the dental health status was observed over time in both groups, with an increase in the number of filled and decayed teeth. CG showed a better oral health-related quality of life, while xerostomia complains increased in GG after gastroplasty. Salivary flow rate remained stable in both groups, but a decrease in buffering capacity, total protein and alpha-amylase activity was observed in GG after 6-months; taste sensitivity increased from baseline to 6-months in GG (p < .05). CONCLUSION: After 6-months of follow-up, patients undergoing gastroplasty presented an improvement in dietary habits and taste sensitivity. However, changes in saliva composition and a worsening in dental health status and xerostomia complaints were also observed.

Clinical Data on Canabinoids: Translational Research in the Treatment of Autism Spectrum Disorders.

Translational research made with Cannabis sativa L. and its biocompounds provides data for some targeted diseases, as also symptoms associated with Autism Spectrum Disorders (ASDs). The main compounds ∆9-tetrahydrocannabinol (THC) and cannabidiol (CBD), are capable of modulating the endocannabinoid system since its dysregulation interferes with the pathophysiology of ASDs there are clinical evidence for its potential use in the treatment of the disease. Conventional therapy still has limitations, as it does not always treat the central symptoms, and there are many patients who do not respond to treatment, which demands more research on new therapies. Through the analysis of published literature on this topic, it is verified that cannabinoids, in particular CBD, improves symptoms associated with common comorbidities in ASDs. Some studies also demonstrate the therapeutic potential of these compounds in the treatment of central symptoms of autism. In addition, cannabinoid therapy to ASDs is associated with low adverse effects and a reduction in concomitant medication. Although it appears to be promising, it is essential to do the translation of this data into clinical research and some of its potential and critical gaps are discussed in this review pointing to large-scale and long-term clinical trials that should include more patients and homogeneous samples.

Salivary Protein Profile and Food Intake: A Dietary Pattern Analysis.

Saliva research has gained interest due to its potential as a source of biomarkers. One of the factors inducing changes in saliva, in the short term, is food intake, and evidence exist about changes in salivary proteome induced by some food components. Since this topic of research is in its early stages, it was hypothesized that saliva protein composition could be associated with different levels of adherence to dietary patterns that contain higher amounts of plant products. The aim of the present study was to test this hypothesis, in adults, by comparing salivary protein electrophoretic profiles of individuals with different diet characteristics, particularly dietary patterns (DP) that exhibit different proportions of animal and plant-based products. Dietary habits were assessed in 122 adults (61 from each sex, with ages ranging from 20 to 59 years) using Food Frequency Questionnaires. To identify the dietary patterns, a principal component analysis was used. Individual's non-stimulated saliva was evaluated for flow rate, pH, protein concentration, α-amylase activity, and electrophoretic protein profiles. Seven dietary patterns (DP) were identified. Salivary amylase enzymatic activity was positively associated with animal-based and starchy foods DP, and with plant-based fatty foods without wine DP. At the same time, protein bands containing amylase and type S cystatins were positively associated with the cheese/yoghurt and wine DP. Our results support the association of salivary proteomics and different dietary patterns and highlight the need of considering food consumption habits in studies using saliva, since this is a factor associated with variations in the composition of this fluid.

Relationship between Mediterranean Diet Adherence and Saliva Composition.

Dietary polyphenol exposure is known to change protein saliva composition in rodents, but less is known in humans. The present study aimed to assess the relationship between saliva protein composition and adherence to Mediterranean Diet (MD) and polyphenol intake levels. Participants were assessed for their dietary habits, which were converted in Mediterranean adherence level, according to Mediterranean Diet Adherence Score (MEDAS) score. Total polyphenol and total flavanol intakes were extrapolated from dietary data, using Phenol explorer database. Whole saliva was collected, and proteins were separated by SDS-PAGE. Salivary S-type cystatins were highly expressed in the group with medium adherence to MD, being positively correlated with wine intake in overweight individuals. The association between salivary amylase and MD adherence also depended on Body Mass Index (BMI), with a positive association only in normal weight individuals. Polyphenol intake was positively associated with S-type cystatins levels, particularly when flavanols were considered separately. These results show that saliva relationship with MD adherence depend on BMI, suggesting that normal weight and overweight individuals may have different salivary responses to diet. Moreover, these results reinforce the link between saliva and dietary polyphenols (flavanols) levels, leading to the hypothesis that salivary proteome can have a role in polyphenol-rich foods acceptance.

How Different Snacks Produce a Distinct Effect in Salivary Protein Composition.

Saliva secretion changes in response to different stimulation. Studies performed in animals and humans suggest that dietary constituents may influence saliva composition, although the dynamics of these changes, and how they are specific for each type of food, are little known. The objective of the present study was to access the short-term effects of different foods in salivation and salivary protein composition. Twelve participants were tested for four snacks (yoghurt, bread, apple and walnuts). Non-stimulated saliva was collected before and at 0', 5' and 30' after each snack intake. Flow rate, total protein, alpha-amylase enzymatic activity and salivary protein profile were analyzed. Yoghurt and apple were the snacks resulting in higher salivary changes, with higher increases in flow rate and alpha-amylase activity immediately after intake. The expression levels of immunoglobulin chains decreased after the intake of all snacks, whereas cystatins and one pink band (proline-rich proteins-PRPs) increased only after yoghurt intake. Walnut's snack was the one resulting in lower changes, probably due to lower amounts eaten. Even so, it resulted in the increase in one PRPs band. In conclusion, changes in saliva composition varies with foods, with variable changes in proteins related to oral food processing and perception.

Changes in Salivary Proteome in Response to Bread Odour.

It is widely recognized that smelling food results in a mouth-watering feeling and influences appetite. However, besides changes in volume, little is known about the effects that food odours have on the composition of saliva. The aim of the present study was to access the effects that smelling bread has on saliva proteome and to compare such effects with those of chewing and ingesting it. Besides a significant increase in saliva flow rate, together with a decrease in total protein concentration, bread odour induced changes in the proportion of different salivary proteins. The expression levels of two spots of cystatins and two spots of amylase increased due to olfactory stimulation, similar to what happened with bread mastication, suggesting that odour can allow anticipation of the type of food eaten and consequently the physiological oral changes necessary to that ingestion. An interesting finding was that bread odour increased the expression levels of several protein spots of immunoglobulin chains, which were decreased by both bread or rice mastication. This may be of clinical relevance since food olfactory stimulation of salivary immunoglobulins can be used to potentiate the oral immune function of saliva. Moreover, the effects of bread odour in the levels of salivary proteins, previously observed to be involved in oral food processing led to the hypothesis of an influence of this odour in the sensory perception of foods further ingested. Further studies are needed to elucidate this point, as well as whether the changes observed for bread odour are specific, or if different food odours lead to similar salivary proteome responses.

Generation and characterization of a human iPSC line (UAMi005-A) from a patient with nonketotic hyperglycinemia due to mutations in the GLDC gene.

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with nonketotic hyperglycinemia bearing the biallelic changes c.1742C > G (p.Pro581Arg) and c.2368C > T (p.Arg790Trp) in the GLDC gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. This cellular model provides a good resource for disease modeling and drug discovery.

Generation and characterization of a human iPSC line (UAMi004-A) from a patient with propionic acidemia due to defects in the PCCB gene.

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with propionic acidemia that has a homozygous mutation (c.1218_1231del14ins12 (p.G407 fs)) in the PCCB gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. The generated iPSC line represents a useful tool to study the pathomechanisms underlying the deficiency.

The electrocardiogram of vertebrates: Evolutionary changes from ectothermy to endothermy.

The electrocardiogram (ECG) reveals that heart chamber activation and repolarization are much faster in mammals and birds compared to ectothermic vertebrates of similar size. Temperature, however, affects electrophysiology of the heart and most data from ectotherms are determined at body temperatures lower than those of mammals and birds. The present manuscript is a review of the effects of temperature on intervals in the ECG of ectothermic and endothermic vertebrates rather than a hypothesis-testing original research article. However, the conclusions are supported by the inclusion of original data (Iguana iguana, N = 4; Python regius, N = 5; Alligator mississippiensis, N = 4). Most comparisons were of animals of approximately 1 kg. Compared to mammals and birds, the reptiles at 35-37 °C had 4 fold lower heart rates, 2 fold slower atrial and ventricular conduction (longer P- and QRS-wave durations), and 4 fold longer PR intervals (atrioventricular delay) and QT intervals (total ventricular repolarization). We conclude that the faster chamber activation in endotherms cannot be explained by temperature alone. Based on histology, we show that endotherms have a more compact myocardial architecture. In mammals, disorganization of the compact wall by fibrosis associates with conduction slowing and we suggest the compact tissue architecture allows for faster chamber activation. The short cardiac cycle that characterizes mammals and birds, however, is predominantly accommodated by shortening of the atrioventricular delay and the QT interval, which is so long in a 1 kg iguana that it compares to that of an elephant.

Nonketotic hyperglycinemia: Functional assessment of missense variants in GLDC to understand phenotypes of the disease.

The rapid analysis of genomic data is providing effective mutational confirmation in patients with clinical and biochemical hallmarks of a specific disease. This is the case for nonketotic hyperglycinemia (NKH), a Mendelian disorder causing seizures in neonates and early-infants, primarily due to mutations in the GLDC gene. However, understanding the impact of missense variants identified in this gene is a major challenge for the application of genomics into clinical practice. Herein, a comprehensive functional and structural analysis of 19 GLDC missense variants identified in a cohort of 26 NKH patients was performed. Mutant cDNA constructs were expressed in COS7 cells followed by enzymatic assays and Western blot analysis of the GCS P-protein to assess the residual activity and mutant protein stability. Structural analysis, based on molecular modeling of the 3D structure of GCS P-protein, was also performed. We identify hypomorphic variants that produce attenuated phenotypes with improved prognosis of the disease. Structural analysis allows us to interpret the effects of mutations on protein stability and catalytic activity, providing molecular evidence for clinical outcome and disease severity. Moreover, we identify an important number of mutants whose loss-of-functionality is associated with instability and, thus, are potential targets for rescue using folding therapeutic approaches.

Economic aspects of production of Caiman crocodilus yacare.

The breeding of crocodilians is still a recent activity in Brazil. Its peak was in the 1990's, but it has gaps in its production, as there are no norms for the commercial breeding of these animals in captivity. However, its economic potential is great, and the search for ecological balance and viability of commercial production has become a challenge among farmers of this activity. Therefor, the objective of the study was to economically analyze the production of Caiman crocodilus yacare on a farm located in Caceres, state of Mato Grosso, identifying relevant items of costs in the activity, as well as the parameters related to the profitability and viability of the activity. The economic results for the breeding of this animal were positive, with profitability ratios higher than 70%.

The shoot-specific expression of gamma-glutamylcysteine synthetase directs the long-distance transport of thiol-peptides to roots conferring tolerance to mercury and arsenic.

Thiol-peptides synthesized as intermediates in phytochelatin (PC) biosynthesis confer cellular tolerance to toxic elements like arsenic, mercury, and cadmium, but little is known about their long-distance transport between plant organs. A modified bacterial gamma-glutamylcysteine synthetase (ECS) gene, S1ptECS, was expressed in the shoots of the ECS-deficient, heavy-metal-sensitive cad2-1 mutant of Arabidopsis (Arabidopsis thaliana). S1ptECS directed strong ECS protein expression in the shoots, but no ECS was detected in the roots of transgenic plant lines. The S1ptECS gene restored full mercury tolerance and partial cadmium tolerance to the mutant and enhanced arsenate tolerance significantly beyond wild-type levels. After arsenic treatment, the root concentrations of gamma-glutamylcysteine (EC), PC2, and PC3 peptides in a S1ptECS-complemented cad2-1 line increased 6- to 100-fold over the mutant levels and were equivalent to wild-type concentrations. The shoot and root levels of glutathione were 2- to 5-fold above those in wild-type plants, with or without treatment with toxicants. Thus, EC and perhaps glutathione are efficiently transported from shoots to roots. The possibility that EC or other PC pathway intermediates may act as carriers for the long-distance phloem transport and subsequent redistribution of thiol-reactive toxins and nutrients in plants is discussed.

Arsenic and mercury tolerance and cadmium sensitivity in Arabidopsis plants expressing bacterial gamma-glutamylcysteine synthetase.

Cysteine sulfhydryl-rich peptide thiols are believed to play important roles in the detoxification of many heavy metals and metalloids such as arsenic, mercury, and cadmium in plants. The gamma-glutamylcysteine synthetase (gamma-ECS) catalyzes the synthesis of the dipeptidethiol gamma-glu-cys (gamma-EC), the first step in the biosynthesis of phytochelatins (PCs). Arabidopsis thaliana, engineered to express the bacterial gamma-ECS gene under control of a strong constitutive actin regulatory sequence (A2), expressed gamma-ECS at levels approaching 0.1% of total protein. In response to arsenic, mercury, and cadmium stresses, the levels of gamma-EC and its derivatives, glutathione (GSH) and PCs, were increased in the A2::ECS transgenic plants to three- to 20-fold higher concentrations than the increases that occurred in wild-type (WT). Compared to cadmium and mercury treatments, arsenic treatment most significantly increased levels of gamma-EC and PCs in both the A2::ECS transgenic and WT plants. The A2::ECS transgenic plants were highly resistant to arsenic and weakly resistant to mercury. Although exposure to cadmium produced three- to fivefold increases in levels of gamma-EC-related peptides in the A2::ECS lines, these plants were significantly more sensitive to Cd(II) than WT and trace levels of Cd(II) blocked resistance to arsenic and mercury. A few possible mechanisms for gamma-ECS-enhanced arsenic and mercury resistance and cadmium hypersensitivity are discussed.