Function and regulation of phenylalanine and tyrosine hydroxylases from human and Caenorhabditis elegans. With focus in evolutionary aspects and development of new therapies

The family of the aromatic amino acid hydroxylases (AAAH) is well studied in mammals. It includes four members, i.e. phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH) and the tryptophan hydroxylases (TPH1 and 2). These enzymes share important features, such as domain organization, three dimensional structure and mechanism of the reaction. The AAAH have important functions and are related to genetic human diseases. PAH, expressed in liver, is in charge of L-Phe catabolism from the diet and mutations in the PAH gene lead to phenylketonuria (PKU), a paradigm for genetic metabolic diseases. TH and the TPHs are enzymes of the neuroendocrine system, that carry out the rate limiting steps in the synthesis of neurotransmitters and hormones, i.e. catecholamines (TH) and serotonin and melatonin (TPHs). Mutations in TH and the TPHs genes are also involved in important neurological diseases and disorders, such as some forms of dystonia and parkinsonism in the case of TH and mood disorders in TPH. The nematode Caenorhabditis elegans is a model organism widely used in biology. We here present the expression and characterization of two AAAH from the nematode, PAH and TH, in order to get insights into evolution of structure, function and regulation in this enzymatic family. In the case of PAH we found functional and molecular similarities between C. elegans PAH (cePAH) and human PAH (hPAH), although they display important differences in enzymatic activity regulation, especially regarding the regulation exerted by the substrate, L-Phe. Both the preactivation and the positive cooperativity induced by the substrate on mammalian PAHs were absent in cePAH. In vivo experiments with knock-out worms bearing a deletion in the pah gene (pah-1) demonstrated that cePAH is involved in the synthesis of a melanin-like compound that localizes in the cuticle. The study of the recombinant TH from C. elegans (ceTH) in comparison with human TH (hTH) also revealed important differences at the level of short-term activity regulation. Basic regulatory mechanisms for hTH, such as substrate inhibition and feed-back inhibition by the end catecholamine products, appear to be absent in ceTH, suggesting a less tight regulation of enzymatic activity in the worm. But interestingly, ceTH was effectively phosphorylated by cAMPdependent protein kinase (PKA) at Ser35, though this modification did not translate into activation of the enzyme in synergy with the feed-back inhibition by catecholamines, as it is the case for phosphorylation of hTH at the equivalent Ser40. We hypothesised that phosphorylation of ceTH could regulate the interaction with other proteins and/or control subcellular localization. Supplementation with BH4 has been recently established as a therapeutic intervention for PKU. A main effect of the cofactor is the stabilisation of misfolded PKU mutants, and BH4 appears to function as a natural chaperone molecule. Since BH4 is a shared cofactor by all the AAAH we set to investigate the effect of BH4 supplementation on rat brain TH. Higher doses of BH4 than those currently used for the treatment of PKU were needed to increase the cofactor concentration in brain, most probably due to the selectivity of the blood-brain-barrier (BBB). This indicates that the current treatments using lower doses of BH4 (up to 20 mg/kg/day) are not expected to affect neuronal TH and TPH2. An increment of total TH protein and activity was measured in the brains of wild-type (wt) mice upon treatment with 100 mg BH4/kg/day, suggesting that BH4 also functions as a natural chaperone in the case of TH. In agreement with these effects, in vitro experiments also showed the capability of BH4 to stabilise TH. Finally, the screening of chemical libraries of small organic compounds is arising as a promising tool to find specific stabilisers of proteins (i.e. pharmacological chaperones). In the case of PAH, four stabilising compounds (compounds I-IV) were found in a previous study, revealing their potential as therapeutic pharmacological chaperones for PKU. As in the case of BH4, it was interesting to study the effect of these four molecules upon neuronal TH and TPH2. We found that compound III stabilized the three AAAH investigated, whereas the other compounds exerted different enzyme specific effects. In vivo studies with supplemented mice revealed the potential of compound III to treat TH-associated diseases. These results are important not only for the development of new specific therapies, but also to unravel enzyme specific/non specific ligand binding in the AAAH family

Inflammatory and non-inflammatory monocytes as novel prognostic biomarkers of survival in SOD1G93A mouse model of Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) has lately become a suitable scenario to study the interplay between the hematopoietic system and disease progression. Recent studies in C9orf72 null mice have demonstrated that C9orf72 is necessary for the normal function of myeloid cells. In this study, we aimed to analyze in depth the connection between the hematopoietic system and secondary lymphoid (spleen) and non-lymphoid (liver and skeletal muscle) organs and tissues along the disease progression in the transgenic SOD1G93A mice. Our findings suggested that the inflammatory response due to the neurodegeneration in this animal model affected all three organs and tissues, especially the liver and the skeletal muscle. However, the liver was able to compensate this inflammatory response by means of the action of non-inflammatory monocytes, while in the skeletal muscle inflammatory monocytes prompted a further inflammation process until the terminal state of the animals. Interestingly, in blood, a positive correlation was found between non-inflammatory monocytes and survival of the transgenic SOD1G93A mice, while the contrary (a negative correlation) was found in the case of inflammatory monocytes, supporting their potential role as biomarkers of disease progression and survival in this animal model. These findings could prompt future translational studies in ALS patients, promoting the identification of new reliable biomarkers of disease progression

A first approach for an evidence-based in vitro digestion method to adjust pancreatic enzyme replacement therapy in cystic fibrosis

[EN] Background Patients with cystic fibrosis have to take enzymatic supplements to allow for food digestion. However, an evidence-based method to adjust Pancreatic Enzyme Replacement Therapy (PERT) is inexistent, and lipid content of meals is used as a rough criterion. Objective In this study, an in vitro digestion model was set up to determine the theoretical optimal dose (TOD) of enzymatic supplement for a selection of foods, which is the dose that allows for maximum lipolysis extent. Methods A static in vitro digestion model was applied to simulate digestion of eight foods covering a wide range of lipid contents. First, the dose of the enzymatic supplement was fixed at 2000 lipase units per gram of fat (LU/g fat) using intestinal pH and bile salt concentration as variables. Second, intestinal pH and bile salt concentrations were fixed and the variable was the dose of the enzymatic supplement. Lipolysis extent was determined by measuring the free fatty acids released from initial triglycerides content of foods after digestion. Results in terms of percentage of lipolysis extent were fitted into a linear-mixed segmented model and the deducted equations were used to predict the TOD to reach 90% of lipolysis in every food. In addition, the effect of intestinal pH and bile salt concentration were investigated. Results The predictive equations obtained for the assessed foods showed that lipolysis was not only dependent on the dose of the enzyme supplement or the lipid content. Moreover, intestinal pH and bile salt concentration had significant effects on lipolysis. Therefore an evidence-based model can be developed taking into account these variables. Conclusions Depending on food characteristics, a specific TOD should be assigned to achieve an optimal digestion extent. This work represents a first step towards an evidence-based method for PERT dosing, which will be applied in an in vivo setting to validate its efficacy.This work was fully funded by the European Union and the Horizon 2020 Research and Innovation Framework Programme (PHC-26-2014 call Self management of health and disease: citizen engagement and mHealth) under grant number 643806.Calvo-Lerma, J.; Fornes-Ferrer, V.; Peinado Pardo, I.; Heredia Gutiérrez, AB.; Ribes-Koninckx C.; Andrés Grau, AM. (2019). A first approach for an evidence-based in vitro digestion method to adjust pancreatic enzyme replacement therapy in cystic fibrosis. PLoS ONE. 14(2):1-14. https://doi.org/10.1371/journal.pone.0212459S114142Lesmes, U., & McClements, D. J. (2012). Controlling lipid digestibility: Response of lipid droplets coated by β-lactoglobulin-dextran Maillard conjugates to simulated gastrointestinal conditions. Food Hydrocolloids, 26(1), 221-230. doi:10.1016/j.foodhyd.2011.05.011Humbert, L., Rainteau, D., Tuvignon, N., Wolf, C., Seksik, P., Laugier, R., & Carrière, F. (2018). Postprandial bile acid levels in intestine and plasma reveal altered biliary circulation in chronic pancreatitis patients. Journal of Lipid Research, 59(11), 2202-2213. doi:10.1194/jlr.m084830Lamothe, S., Azimy, N., Bazinet, L., Couillard, C., & Britten, M. (2014). Interaction of green tea polyphenols with dairy matrices in a simulated gastrointestinal environment. Food Funct., 5(10), 2621-2631. doi:10.1039/c4fo00203bMuggeo, V. & Muggeo, V. M. R. Segmented mixed models with random changepoints in R Working paper (2016)

Does technology-based interventions in psychosis improved functioning and quality of life? A systematic review and meta-analysis

Introduction: Technology-based interventions (TBIs), including computer and Internet-based interventions, mobile interventions, health applications, social media interventions, and interventions using technological devices, could become a useful, effective, accessible, and cost-effective approach (Berry et al., 2016; Firth, 2016) to complement conventional interventions for psychosi

Glofitamab, a Novel, Bivalent CD20-Targeting T-Cell–Engaging Bispecific Antibody, Induces Durable Complete Remissions in Relapsed or Refractory B-Cell Lymphoma: A Phase I Trial

Glofitamab; B-Cell Lymphoma; RelapsedGlofitamab; Linfoma de células B; RecaídaGlofitamab; Limfoma de cèl·lules B; RecaigudaPURPOSE Glofitamab is a T-cell–engaging bispecific antibody possessing a novel 2:1 structure with bivalency for CD20 on B cells and monovalency for CD3 on T cells. This phase I study evaluated glofitamab in relapsed or refractory (R/R) B-cell non-Hodgkin lymphoma (B-NHL). Data for single-agent glofitamab, with obinutuzumab pretreatment (Gpt) to reduce toxicity, are presented. METHODS Seven days before the first dose of glofitamab (0.005-30 mg), all patients received 1,000 mg Gpt. Dose-escalation steps were determined using a Bayesian continuous reassessment method with overdose control. Primary end points were safety, pharmacokinetics, and the maximum tolerated dose of glofitamab. RESULTS Following initial single-patient cohorts, 171 patients were treated within conventional multipatient cohorts and received at least one dose of glofitamab. This trial included heavily pretreated patients with R/R B-NHL; most were refractory to prior therapy (155; 90.6%) and had received a median of three prior therapies. One hundred and twenty-seven patients (74.3%) had diffuse large B-cell lymphoma, transformed follicular lymphoma, or other aggressive histology, and the remainder had indolent lymphoma subtypes. Five (2.9%) patients withdrew from treatment because of adverse events. Cytokine release syndrome occurred in 86 of 171 (50.3%) patients (grade 3 or 4: 3.5%); two (1.2%) patients experienced grade 3, transient immune effector cell–associated neurotoxicity syndrome-like symptoms. The overall response rate was 53.8% (complete response [CR], 36.8%) among all doses and 65.7% (CR, 57.1%) in those dosed at the recommended phase II dose. Of 63 patients with CR, 53 (84.1%) have ongoing CR with a maximum of 27.4 months observation. CONCLUSION In patients with predominantly refractory, aggressive B-NHL, glofitamab showed favorable activity with frequent and durable CRs and a predictable and manageable safety profile

Pilot study of an online intervention for young people with a first psychotic episode: Thinkapp

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Caenorhabditis elegans AGXT-1 is a mitochondrial and temperature-adapted ortholog of peroxisomal human AGT1: New insights into between-species divergence in glyoxylate metabolism

In humans, glyoxylate is an intermediary product of metabolism, whose concentration is finely balanced. Mutations in peroxisomal alanine:glyoxylate aminotransferase (hAGT1) cause primary hyperoxaluria type 1 (PH1), which results in glyoxylate accumulation that is converted to toxic oxalate. In contrast, glyoxylate is used by the nematode Caenorhabditis elegans through a glyoxylate cycle to by-pass the decarboxylation steps of the tricarboxylic acid cycle and thus contributing to energy production and gluconeogenesis from stored lipids. To investigate the differences in glyoxylate metabolism between humans and C. elegans and to determine whether the nematode might be a suitable model for PH1, we have characterized here the predicted nematode ortholog of hAGT1 (AGXT-1) and compared its molecular properties with those of the human enzyme. Both enzymes form active PLP-dependent dimers with high specificity towards alanine and glyoxylate, and display similar three-dimensional structures. Interestingly, AGXT-1 shows 5-fold higher activity towards the alanine/glyoxylate pair than hAGT1. Thermal and chemical stability of AGXT-1 is lower than that of hAGT1, suggesting temperature-adaptation of the nematode enzyme linked to the lower optimal growth temperature of C. elegans. Remarkably, in vivo experiments demonstrate the mitochondrial localization of AGXT-1 in contrast to the peroxisomal compartmentalization of hAGT1. Our results support the view that the different glyoxylate metabolism in the nematode is associated with the divergent molecular properties and subcellular localization of the alanine:glyoxylate aminotransferase activity.This work was supported by the Spanish Ministry of Science and Innovation (CSD2009-00088, BIO2012-34937 and SAF2011-23933), the Junta de Andalucia (P11-CTS-7187), and the Oxalosis and Hyperoxaluria Foundation (OHF2012 to B.C.). A.L.P. acknowledges a Ramon y Cajal research contract (RyC2009-04147) from the Spanish Ministry of Science and Innovation and the University of Granada. N. M-T acknowledges a FPI predoctoral fellowship from the Spanish Ministry of Science and Innovation. A.C.C. and N.T. were supported by the grant IOS-1353845 from the National Science Foundation (NSF). N.T. acknowledges the Tetelman Fellowship for International Research on the Sciences awarded by Yale University.Peer Reviewe

Evaluation of dietary supplementation of a novel microbial muramidase on gastrointestinal functionality and growth performance in broiler chickens

This study was conducted to assess the effect of dietary supplementation of Muramidase 007 to broiler chickens on gastrointestinal functionality, evaluating growth performance, apparent ileal digestibility, intestinal histomorphology, vitamin A in plasma and cecal microbiota. A total of 480 one-day male chicks (Ross 308) were distributed in 16 pens allocated in 2 experimental diets: the control diet (CTR) without feed enzymes, coccidiostat or growth promoters, and the experimental diet (MUR): CTR supplemented with 35,000 units (LSU(F))/kg of the Muramidase 007. Digesta and tissue samples were obtained on days 9 and 36 of the study. A lower feed conversion ratio was observed in the MUR treatment. Apparent ileal digestibility of DM, organic matter and energy were improved by Muramidase 007. It was also observed that MUR improved digestibility of total fatty acids, mono-unsaturated fatty acids and poly-unsaturated fatty acids, and content of vitamin A in plasma at day 9 (P < 0.05). Histomorphological analysis of jejunum samples revealed no differences in the villus height or crypt depth; but a higher number of goblet cells and intraepithelial lymphocytes at day 36 with MUR. No differences were observed in plate counts of enterobacteria or Lactobacillus along the gastrointestinal tract, neither on the cecal short-chain fatty acids. An statistical trend was observed for reduction of cecal clostridia at day 9 for MUR. Analysis of cecal microbiota structure by 16S rRNA gene sequencing revealed relevant changes correlated to age. At day 9, broilers receiving MUR showed decreased alpha diversity compared to CTR that was not detected at day 36. Changes in specific taxonomic groups with an increase in Lactobacillus genus were identified. In conclusion, evaluation of the variables in this study indicates that dietary Muramidase 007 contributes to improve feed conversation ratio and gastrointestinal function in broiler chickens. Effects could have been mediated by slight shifts observed in the intestinal microbiota. More studies are guaranteed to fully understand the mechanisms involved.info:eu-repo/semantics/publishedVersio

Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule

Significant improvements in behavioral and electrophysiological results, motoneuron survival and anti-apoptotic/survival-activated pathways were observed with BDNF-TTC treatment. However, no synergistic effect was found for this fusion molecule. Although BDNF in the fusion molecule is capable of activating autocrine and neuroprotective pathways, TTC treatment alone yielded similar neuroprotection. Therefore, an accurate study of the neuroprotective effects of TTC fusion molecules should be performed to obtain a better understanding of its effect