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Argininosuccinate lyase (ASL) is a key enzyme integral to the hepatic urea cycle which is required for ammonia detoxification, and the citrulline-nitric oxide (NO) cycle for NO production. ASL deficient patients present with argininosuccinic aciduria (ASA), an inherited metabolic disease with hyperammonaemia and a chronic systemic phenotype with neurocognitive impairment and chronic liver disease. ASL deficiency as an inherited model of systemic NO deficiency, shows enhanced nitrosative and oxidative stress. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics andin vivopositron emission tomography (PET) imaging using (S)-4-(3-18F-fluoropropyl)-L-glutamate ([18F]FSPG). Upregulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways.hASLmRNA encapsulated in lipid nanoparticles corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis and glutathione metabolism and ameliorating chronic liver disease. We further present [18F]FSPG PET as a novel non-invasive diagnostic tool to assess liver disease and therapeutic efficacy in ASA. These findings support clinical translation of mRNA therapy for ASA.

PDXK encodes for a pyridoxal kinase, which converts inactive B6 vitamers to the active cofactor pyridoxal 5'-phosphate (PLP). Recently, biallelic pathogenic variants in PDXK were shown to cause axonal Charcot-Marie-Tooth disease with optic atrophy that responds to PLP supplementation. We present two affected siblings carrying a novel biallelic missense PDXK variant with a similar phenotype with earlier onset. After detection of a novel PDXK variant using Whole Exome Sequencing, we confirmed pathogenicity through in silico protein structure analysis, determination of pyridoxal kinase activity using liquid chromatography-tandem mass spectrometry, and measurement of plasma PLP concentrations using high performance liquid chromatography. Our in silico analysis shows a potential effect on PDXK dimer stability, as well as a putative effect on posttranslational ubiquitination that is predicted to lead to increased protein degradation. We demonstrate that the variant leads to almost complete loss of PDXK enzymatic activity and low PLP levels. Our patients' early diagnosis and prompt PLP replacement restored the PLP plasma levels, enabling long-term monitoring of clinical outcomes. We recommend that patients presenting with similar phenotype should be screened for PDXK mutations, as this is a rare opportunity for treatment.

Peroxisomal fatty acid α-oxidation is an essential pathway for the degradation of β-carbon methylated fatty acids such as phytanic acid. One enzyme in this pathway is 2-hydroxyacyl CoA lyase (HACL1), which is responsible for the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA. Hacl1 deficient mice do not present with a severe phenotype, unlike mice deficient in other α-oxidation enzymes such as phytanoyl-CoA hydroxylase deficiency (Refsum disease) in which neuropathy and ataxia are present. Tissues from wild-type and Hacl1−/− mice fed a high phytol diet were obtained for proteomic and lipidomic analysis. There was no phenotype observed in these mice. Liver, brain, and kidney tissues underwent trypsin digestion for untargeted proteomic liquid chromatography-mass spectrometry analysis, while liver tissues also underwent fatty acid hydrolysis, extraction, and derivatisation for fatty acid gas chromatography-mass spectrometry analysis. The liver fatty acid profile demonstrated an accumulation of phytanic and 2-hydroxyphytanic acid in the Hacl1−/− liver and significant decrease in heptadecanoic acid. The liver proteome showed a significant decrease in the abundance of Hacl1 and a significant increase in the abundance of proteins involved in PPAR signalling, peroxisome proliferation, and omega oxidation, particularly Cyp4a10 and Cyp4a14. In addition, the pathway associated with arachidonic acid metabolism was affected; Cyp2c55 was upregulated and Cyp4f14 and Cyp2b9 were downregulated. The kidney proteome revealed fewer significantly upregulated peroxisomal proteins and the brain proteome was not significantly different in Hacl1−/− mice. This study demonstrates the powerful insight brought by proteomic and metabolomic profiling of Hacl1−/− mice in better understanding disease mechanism in fatty acid α-oxidation disorders.

Both plasma and cerebrospinal fluid (CSF) are rich in cholesterol and its metabolites. Here we describe in detail a methodology for the identification and quantification of multiple sterols including oxysterols and sterol-acids found in these fluids. The method is translatable to any laboratory with access to liquid chromatography – tandem mass spectrometry. The method exploits isotope-dilution mass spectrometry for absolute quantification of target metabolites. The method is applicable for semi-quantification of other sterols for which isotope labelled surrogates are not available and approximate quantification of partially identified sterols. Values are reported for non-esterified sterols in the absence of saponification and total sterols following saponification. In this way absolute quantification data is reported for 17 sterols in the NIST SRM 1950 plasma along with semi-quantitative data for 8 additional sterols and approximate quantification for one further sterol. In a pooled (CSF) sample used for internal quality control, absolute quantification was performed on 10 sterols, semi-quantification on 9 sterols and approximate quantification on a further three partially identified sterols. The value of the method is illustrated by confirming the sterol phenotype of a patient suffering from ACOX2 deficiency, a rare disorder of bile acid biosynthesis, and in a plasma sample from a patient suffering from cerebrotendinous xanthomatosis, where cholesterol 27-hydroxylase is deficient., Graphical abstract Image 1, Highlights • Absolute quantification of oxysterols and cholestenoic acids. • Methodology applicable to plasma and cerebrospinal fluid. • Data generated for non-esterified and total sterols. • Diastereoisomers at C-24 and C-25 separated and quantified.

Resumen del póster presentado al 18th International Congress of the Society for Melanoma Research, celebrado online del 28 al 31 de octubre de 2021., Melanoma is a very aggressive skin cancer, known for its high degree of plasticity and heterogeneity that confers high metastatic capabilities and resistance to therapies. Increasing evidences indicate that melanoma progression is not only regulated by mutation-driven mechanisms but also by non-genetic mechanisms that play an important role in melanoma phenotypic plasticity. The reactivation of the expression of neural crest genes in melanoma is associated to progression and resistance to therapies. Among those, epithelial-to-mesenchymal transcription factors (EMT-TFs) have been proposed to regulate reversible switches between phenotypic states driving melanoma progression. However the role of Prrx 1, an important EMT inducer also expressed during neural crest development, has not been investigated in detail. We have found that Prrx1 is expressed in melanoma and that Prrx1 downregulation induces cell cycle retention and reduces cell proliferation and cancer stem-like properties in melanoma cells. We have also generated a clinically relevant melanoma mouse model that cannot reactivate Prrx 1 expression in melanocytes to address the contribution ofthis EMT-TF to melanoma initiation, progression and responses to therapies.

Trabajo presentado al 17th Meeting of the Spanish Society for Developmental Biology (SEBD), celebrado de forma virtual del 18 al 20 de noviembre de 2020.

Trabajo presentado al Seminario de Neurobiología del Desarrollo, celebrado online el 12 de mayo de 2020.

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18-interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor (GEF) complex. 12 of these 28 interactions are supported by prior reports and we have directly validated novel interactions with SEC22A, TMCO4 and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites (MCSs), interactors included groups of microtubule/membrane-remodelling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP (emopamil binding protein) is a Δ8-Δ7 sterol isomerase and OSBPL2/ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We find that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Further,de novocholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated. Our data demonstrate that GEF-dependent Rab-interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Alicante del 23 al 26 de octubre de 2019., Melanoma is a very aggressive skin cancer due to its high metastatic abilities. Melanoma arises from melanocytes that derive from highly motile neural crest progenitors that colonize the body during development. The high degree of cell plasticity underlying melanoma metastatic abilities and resistance to therapies is a hallmark of these cells. Although, EMT-TFs (epithelial-to-mesenchymal transition transcription factors) are key regulators of epithelial plasticity in development and in aberrant processes such as fibrosis and cancer, their role in melanoma is far from being understood. E-cadherin loss is a hallmark of EMT, and although melanocytes are not epithelial cells, they express E-cadherin that is lost as melanoma develops, indicating that EMT-TFs may contribute to melanoma initiation. In addition, the reactivation of the expression of neural crest genes in melanoma cells is associated to tumour progression and EMT-TFs reprogramming has been proposed to regulate reversible switches between two phenotypic states (differentiated vs invasive) proposed to control melanoma progression. However, the functional contribution of EMT-TFs, to melanoma initiation and metastasis remains to be elucidated. Our preliminary results indicate that Snail1 and Prrx1, two EMT-TFs expressed in the neural crest among others are expressed in melanomas. We are currently establishing mouse models and functional studies to address their contribution to melanoma biology.

Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Alicante del 23 al 26 de octubre de 2019., Transcription factors that trigger Epithelial-to-Mesenchymal Transition (EMT-TFs) are fundamental for the development of multiple tissues and organs. They can be reactivated in pathological conditions resulting in a profound remodeling of epithelial phenotypes, leading to cancer cell dissemination or functional loss in organ fibrosis. Different EMT-TFs can promote distinct EMT programs and, in addition, both embryonic and cancer cells express different combinations of EMT-TFs. Due to this intrinsic complexity, it is unclear how EMT-TFs are coordinated to orchestrate heterogeneous EMT programs, especially the highly plastic and reversible intermediate or partial EMT (pEMT) states. Here, we used in silico, in vitro and in vivo approaches to decipher the relationship between the expression of different EMT-TFs and the resulting phenotypes. (i) Using in silico analysis, we identify a stereotypic EMT-TFs expression-code accompanying the epithelial, hybrid epithelial/mesenchymal (E-M) and mesenchymal (M) phenotypes. (ii) In cultured epithelial kidney cells, TGF-b-induced EMT can associate with partial or full EMT programmes with a stereotypic EMT-TFs expression code. Manipulation of EMT-TFs expression confirms the sequential recruitment of EMT-TFs to fine-tune EMT states. (iii) In renal fibrosis, an in vivo model of pEMT, damaged tubular epithelial cells acquire a stable pEMT phenotype upon the reactivation of specific EMT-TFs, and reminiscent of E-M hybrid phenotype profile identified in our in silico and in vitro analyses. In summary, this work provides evidence for specific combinatorial EMT-TFs codes that regulate the heterogeneity and plasticity of EMT programs leading to different pathological outcomes.

Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Alicante del 23 al 26 de octubre de 2019., También presentado al 16th Christmas Meeting del Instituto de Neurociencias, celebrado en Alicante del 19 al 20 de diciembre de 2019., The epithelial-mesenchymal transition (EMT) endows cells with migratory and invasive properties and it is crucial for the formation of many tissues and organs during embryonic development. This cellular program is triggered after the activation of transcription factors, referred to as EMT-TFs. In the adult, the reactivation of the EMT program contributes to the progression of diseases like fibrosis and cancer (Nieto et al. Cell, 2016). Prrx1, identified as a novel EMT-TF in our lab, produces three different splice isoforms, whose functions remain to be characterized. We have tested the ability of each of these isoforms to induce EMT in vitro, and only the longest isoform (Prrx1L) is able to do so. To test the role of the different isoforms in vivo, we have generated isoform-specific mutant mouse lines that we are characterizing using the phenotype of the Prrx1 null mice in the skeleton and the vasculature (J.F. Martin et al. Genes & Development, 1995, K. Ihida-Stansbury et al. Circulation research, 2004). Using the postnatal retina as a model to study vasculogenesis, we have observed that Prrx1 is specifically expressed in mural cells. Consistent with this expression pattern, we have seen that the Prrx1 isoforms are essential for the integrity of the vasculature in the P6 retina and adult lungs. We are using the in vivo matrigel plug assay to dissect the function of Prrx1 isoforms in mural cells and their interaction with endothelial cells.

تتناول هذه الدراسة موضوع حماية المكلفين بضريبة الدخل في فلسطين، سواء في قانون ضريبة الدخل رقم (17) لسنة 2004م الساري فعليا في قطاع غزة أو القرار بقانون رقم(8) لسنة 2011م الساري في الضفة الغربية. وقد اعتمدت الدراسة على المنهج المقارن القائم على التحليل للمواقف التشريعية التي تنظم الحماية القضائية للمكلفين، وتظهر التباين التشريعي في مستوى الحماية التي يوفرها لهم المشرع الفلسطيني وغيره من المشرعين، كما تبين الدراسة مدى تحقق التوازن المطلوب بين طرفي العلاقة الضريبية، سواء أكان التوازن في مرحلة الاعتراض الإداري أم في مرحلة الطعن القضائي، لما لذلك من أثر مباشر في رفع مستوى الامتثال الضريبي، وخفض مستويات التهرب الضريبي. وقد خلصت الدراسة إلى عدد من النتائج، أهمها: أن المشرع الفلسطيني لم يوفر الحماية الكافية للمكلف بالضريبة على الدخل، ولم يحقق التوازن بين طرفي العلاقة الضريبية. كما انتهت إلى عدد من التوصيات، أهمها: ضرورة إجراء التعديل التشريعي اللازم لإعادة بناء منظومة القضاء الضريبي بحيث يتم: تشكيل محكمة بداية ضريبية ذات اختصاص عام، وتشكيل محكمة استئناف ضريبي، وتشكيل نيابة ضريبية، وتأهيل المحاكم والنيابة الضريبية للنظر في الجوانب الضريبية كافة، جزائية كانت أو حقوقية أو إدارية، مع تأهيل الكادر تأهيلا علميا كافيا للاضطلاع بهذا الدور.