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Biallelic SORD mutations cause one of the most frequent forms of recessive hereditary neuropathy, estimated to affect approximately 10,000 patients in North America and Europe alone. Pathogenic SORD loss-of-function changes in the encoded enzyme sorbitol dehydrogenase result in abnormally high sorbitol levels in cells and serum. How sorbitol accumulation leads to peripheral neuropathy remains to be elucidated. A reproducible animal model for SORD neuropathy is essential to illuminate the pathogenesis of SORD deficiency and for preclinical studies of potential therapies. Therefore, we have generated a Sord knockout (KO), Sord-/-, Sprague Dawley rat, to model the human disease and to investigate the pathophysiology underlying SORD deficiency. We have characterized the phenotype in these rats with a battery of behavioral tests as well as biochemical, physiological, and comprehensive histological examinations. Sord-/- rats had remarkably increased levels of sorbitol in serum, cerebrospinal fluid (CSF), and peripheral nerve. Moreover, serum from Sord-/- rats contained significantly increased levels of neurofilament light chain, NfL, an established biomarker for axonal degeneration. Motor performance significantly declined in Sord-/- animals starting at â¼7 months of age. Gait analysis evaluated with video motion tracking confirmed abnormal gait patterns in the hindlimbs. Motor nerve conduction velocities of the tibial nerves were slowed. Light and electron microscopy of the peripheral nervous system revealed degenerating myelinated axons, de- and remyelinated axons, and a likely pathognomonic finding - enlarged "ballooned" myelin sheaths. These findings mainly affected myelinated motor axons; myelinated sensory axons were largely spared. In summary, Sord-/- rats develop a motor-predominant neuropathy that closely resembles the human phenotype. Our studies revealed novel significant aspects of SORD deficiency, and this model will lead to an improved understanding of the pathophysiology and the therapeutic options for SORD neuropathy.
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DNA-based therapeutics have emerged as a revolutionary approach for addressing the treatment gap in rare inherited conditions by targeting the fundamental genetic causes of disease. Charcot-Marie-Tooth (CMT) disease, a group of inherited neuropathies, represents one of the most prevalent Mendelian disease groups in neurology and is characterized by diverse genetic etiology. Axonal forms of CMT, known as CMT2, are caused by dominant mutations in over 30 different genes which lead to degeneration of lower motor neuron axons. Recent advances in antisense oligonucleotide (ASO) therapeutics have shown promise in targeting neurodegenerative disorders. Here we elucidate pathomechanistic changes contributing to variant specific molecular phenotypes in CMT2E, caused by a single nucleotide substitution (p.N98S) in the neurofilament light chain gene (NEFL). We used a patient-derived pluripotent stem cell (iPSC)-induced motor neuron model, which recapitulates several cellular and biomarker phenotypes associated with CMT2E. Using an ASO treatment strategy targeting a heterozygous gain-of-function variant, we aimed to resolve molecular phenotypic changes observed in the CMT2E p.N98S subtype. To determine ASO therapeutic potential, we employed our treatment strategy in iPSC-derived motor neurons and used established as well as novel biomarkers of peripheral nervous system axonal degeneration. Our findings have demonstrated a significant decrease in clinically relevant biomarkers of axonal degeneration, presenting the first clinically viable genetic therapeutic for CMT2E. Similar strategies could be used to develop precision medicine approaches for otherwise untreatable gain of function inherited disorders.
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COQ7 encodes a hydroxylase responsible for the penultimate step of coenzyme Q10 (CoQ10) biosynthesis in mitochondria. CoQ10 is essential for multiple cellular functions, including mitochondrial oxidative phosphorylation, lipid metabolism, and reactive oxygen species homeostasis. Mutations in COQ7 have been previously associated with primary CoQ10 deficiency, a clinically heterogeneous multisystemic mitochondrial disorder. We identified COQ7 biallelic variants in nine families diagnosed with distal hereditary motor neuropathy with upper neuron involvement, expending the clinical phenotype associated with defects in this gene. A recurrent p.Met1? change was identified in five families from Brazil with evidence of a founder effect. Fibroblasts isolated from patients revealed a substantial depletion of COQ7 protein levels, indicating protein instability leading to loss of enzyme function. High-performance liquid chromatography assay showed that fibroblasts from patients had reduced levels of CoQ10, and abnormal accumulation of the biosynthetic precursor DMQ10. Accordingly, fibroblasts from patients displayed significantly decreased oxygen consumption rates in patients, suggesting mitochondrial respiration deficiency. Induced pluripotent stem cell-derived motor neurons from patient fibroblasts showed significantly increased levels of extracellular neurofilament light protein, indicating axonal degeneration. Our findings indicate a molecular pathway involving CoQ10 biosynthesis deficiency and mitochondrial dysfunction in patients with distal hereditary motor neuropathy. Further studies will be important to evaluate the potential benefits of CoQ10 supplementation in the clinical outcome of the disease.
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Enfermedades Mitocondriales , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Neuronas Motoras/metabolismo , Mutación/genética , Ubiquinona/genéticaRESUMEN
Lipid analysis at the molecular species level represents a valuable opportunity for clinical applications due to the essential roles that lipids play in metabolic health. However, a comprehensive and high-throughput lipid profiling remains challenging given the lipid structural complexity and exceptional diversity. Herein, we present an 'omic-scale targeted LC-MS/MS approach for the straightforward and high-throughput quantification of a broad panel of complex lipid species across 26 lipid (sub)classes. The workflow involves an automated single-step extraction with 2-propanol, followed by lipid analysis using hydrophilic interaction liquid chromatography in a dual-column setup coupled to tandem mass spectrometry with data acquisition in the timed-selective reaction monitoring mode (12 min total run time). The analysis pipeline consists of an initial screen of 1903 lipid species, followed by high-throughput quantification of robustly detected species. Lipid quantification is achieved by a single-point calibration with 75 isotopically labeled standards representative of different lipid classes, covering lipid species with diverse acyl/alkyl chain lengths and unsaturation degrees. When applied to human plasma, 795 lipid species were measured with median intra- and inter-day precisions of 8.5 and 10.9%, respectively, evaluated within a single and across multiple batches. The concentration ranges measured in NIST plasma were in accordance with the consensus intervals determined in previous ring-trials. Finally, to benchmark our workflow, we characterized NIST plasma materials with different clinical and ethnic backgrounds and analyzed a sub-set of sera (n = 81) from a clinically healthy elderly population. Our quantitative lipidomic platform allowed for a clear distinction between different NIST materials and revealed the sex-specificity of the serum lipidome, highlighting numerous statistically significant sex differences.
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Lípidos , Espectrometría de Masas en Tándem , Anciano , Femenino , Humanos , Masculino , Cromatografía Liquida , Espectrometría de Masas en Tándem/métodos , Lípidos/análisis , Plasma/química , Suero/químicaRESUMEN
[Figure: see text].
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Autofagia , Beclina-1/farmacología , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Beclina-1/uso terapéutico , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Contracción Miocárdica , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéuticoRESUMEN
The liver-derived, circulating transport protein transthyretin (TTR) is the cause of systemic hereditary (ATTRv) and wild-type (ATTRwt) amyloidosis. TTR stabilization and knockdown are approved therapies to mitigate the otherwise lethal disease course. To date, the variety in phenotypic penetrance is not fully understood. This systematic review summarizes the current literature on TTR pathophysiology with its therapeutic implications. Tetramer dissociation is the rate-limiting step of amyloidogenesis. Besides destabilizing TTR mutations, other genetic (RBP4, APCS, AR, ATX2, C1q, C3) and external (extracellular matrix, Schwann cell interaction) factors influence the type of onset and organ tropism. The approved small molecule tafamidis stabilizes the tetramer and significantly decelerates the clinical course. By sequence-specific mRNA knockdown, the approved small interfering RNA (siRNA) patisiran and antisense oligonucleotide (ASO) inotersen both significantly reduce plasma TTR levels and improve neuropathy and quality of life compared to placebo. With enhanced hepatic targeting capabilities, GalNac-conjugated siRNA and ASOs have recently entered phase III clinical trials. Bivalent TTR stabilizers occupy both binding groves in vitro, but have not been tested in trials so far. Tolcapone is another stabilizer with the potential to cross the blood-brain barrier, but its half-life is short and liver failure a potential side effect. Amyloid-directed antibodies and substances like doxycycline aim at reducing the amyloid load, however, none of the yet developed antibodies has successfully passed clinical trials. ATTR-amyloidosis has become a model disease for pathophysiology-based treatment. Further understanding of disease mechanisms will help to overcome the remaining limitations, including application burden, side effects, and blood-brain barrier permeability.
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Amiloidosis Familiar/tratamiento farmacológico , Amiloidosis Familiar/genética , Prealbúmina/efectos de los fármacos , Amiloide/antagonistas & inhibidores , Amiloide/biosíntesis , Amiloide/genética , Amiloidosis Familiar/fisiopatología , Animales , Técnicas de Silenciamiento del Gen , Humanos , Prealbúmina/genéticaRESUMEN
The MS blastomere produces one-third of the body wall muscles (BWMs) in the C. elegans embryo. MS-derived BWMs require two distinct cell-cell interactions, the first inhibitory and the second, two cell cycles later, required to overcome this inhibition. The inductive interaction is not required if the inhibitory signal is absent. Although the Notch receptor GLP-1 was implicated in both interactions, the molecular nature of the two signals was unknown. We now show that zygotically expressed MOM-2 (Wnt) is responsible for both interactions. Both the inhibitory and the activating interactions require precise spatiotemporal expression of zygotic MOM-2, which is dependent upon two distinct Notch signals. In a Notch mutant defective only in the inductive interaction, MS-derived BWMs can be restored by preventing zygotic MOM-2 expression, which removes the inhibitory signal. Our results suggest that the inhibitory interaction ensures the differential lineage specification of MS and its sister blastomere, whereas the inductive interaction promotes the expression of muscle-specifying genes by modulating TCF and ß-catenin levels. These results highlight the complexity of cell fate specification by cell-cell interactions in a rapidly dividing embryo.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Receptores Notch/metabolismo , Animales , Animales Modificados Genéticamente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Blastómeros/citología , Blastómeros/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Linaje de la Célula/genética , Linaje de la Célula/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inducción Embrionaria/genética , Inducción Embrionaria/fisiología , Factores de Transcripción GATA/genética , Factores de Transcripción GATA/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes de Helminto , Péptidos y Proteínas de Señalización Intracelular/genética , Modelos Biológicos , Músculos/embriología , Mutación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Notch/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Canales de Sodio/genética , Canales de Sodio/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción TCF/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/genética , Vía de Señalización Wnt/fisiología , Cigoto/citología , Cigoto/metabolismo , beta Catenina/metabolismoRESUMEN
Vertebrate ß-catenin has two functions, as a structural component of the adherens junction in cell adhesion and as the T-cell factor (TCF) transcriptional coactivator in canonical Wnt (wingless-related integration site) signaling. These two functions are split between three of the four ß-catenin-related proteins present in the round worm Caenorhabditis elegans. The fourth ß-catenin-related protein, WRM-1, exhibits neither of these functions. Instead, WRM-1 binds the MAPK loss of intestine 1 (LIT-1), and these two proteins have been shown to be essential for the transcription of Wnt target genes by phosphorylating and regulating the nuclear level of the sole worm TCF protein. We showed previously that WRM-1 binds to worm TCF and functions as the substrate-binding subunit for LIT-1. In this study, we show that phosphorylation of T220 in the activation loop is essential for LIT-1 kinase activity in vivo and in vitro. T220 can be phosphorylated either through LIT-1 autophosphorylation or directly by the upstream MAP3K MOM-4. Our data support a model in which WRM-1, which can undergo homotypic interaction, binds LIT-1 and thereby generates a kinase complex in which LIT-1 molecules are situated in a conformation enabling autophosphorylation as well as promoting phosphorylation of the T220 residue by MOM-4. In addition, we show that WRM-1 is essential for the translocation of the LIT-1 kinase complex to the nucleus, the site of its TCF substrate. To our knowledge, this is the first report of a MAP3K directly activating a MAPK by phosphorylation within the activation loop. This study should help uncover novel and as yet underappreciated functions of vertebrate ß-catenin.
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Proteínas de Caenorhabditis elegans/química , Proteínas del Citoesqueleto/química , Proteínas de la Membrana/química , Proteínas Serina-Treonina Quinasas/química , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , beta Catenina/química , beta Catenina/metabolismoRESUMEN
Maladaptive behavioral responses characteristic of post-traumatic stress disorders are notably resistant to treatment. We hypothesized that the pharmacological disruption of memories activated during reconsolidation might reverse established stress-induced increases in depression-like behaviors and cocaine reward. C57BL/6J mice were subjected to repeated social defeat stress (SDS), and examined for time spent immobile in a subsequent forced swim test (FST). An additional set of SDS-exposed mice were place-conditioned with cocaine, and tested for cocaine-conditioned place preference (CPP). All stress-exposed mice were then subjected to a single additional trial of SDS while under the influence of propranolol or cycloheximide to disrupt memory reconsolidation, then given one additional FST or CPP test the next day. Mice subjected to repeated SDS subsequently demonstrated increases in time spent immobile in the FST or in the cocaine-paired chamber. Vehicle-treatment followed by additional SDS exposure did not alter these behaviors, but propranolol or cycloheximide treatment reversed each of the potentiated responses in a dose-dependent manner. Overall, these results demonstrate that while repeated exposure to a social defeat stressor subsequently increased depression-like behavior and cocaine-CPP, disruption of traumatic memories made labile by re-exposure to SDS during reconsolidation may have therapeutic value in the treatment of established post-traumatic stress disorder-related behaviors.
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Cocaína/farmacología , Condicionamiento Psicológico/efectos de los fármacos , Trastorno Depresivo/fisiopatología , Inhibidores de Captación de Dopamina/farmacología , Memoria/fisiología , Estrés Psicológico/fisiopatología , Antagonistas Adrenérgicos beta/farmacología , Animales , Condicionamiento Psicológico/fisiología , Cicloheximida/farmacología , Dominación-Subordinación , Relación Dosis-Respuesta a Droga , Masculino , Memoria/efectos de los fármacos , Ratones Endogámicos C57BL , Pruebas Neuropsicológicas , Propranolol/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Percepción Espacial/efectos de los fármacos , Percepción Espacial/fisiología , Estrés Psicológico/psicología , NataciónRESUMEN
Ethanol and 3, 4-Methylenedioxymethamphetamine (MDMA) are popular recreational drugs widely abused by adolescents that may induce neurotoxic processes associated with behavioural alterations. Adolescent CD1 mice were subjected to ethanol intake using the drinking in the dark (DID) procedure, acute MDMA or a combination. Considering that both drugs of abuse cause oxidative stress in the brain, protein oxidative damage in different brain areas was analysed 72 h after treatment using a proteomic approach. Damage to specific proteins in treated animals was significant in the hippocampus but not in the prefrontal cortex. The damaged hippocampus proteins were then identified by mass spectrometry, revealing their involvement in energy metabolism, structural function, axonal outgrowth and stability, and neurotransmitter release. Mice treated with MDMA displayed higher oxidative damage than ethanol-treated mice. To determine whether this oxidative damage was affecting hippocampus activity, declarative memory was evaluated at 72 h after treatment using the object recognition assay and the radial arm maze. Although acquisition in the radial arm maze was not impaired by ethanol intake, MDMA treatment impaired long-term memory in both tests. Therefore, oxidative damage to specific proteins observed under MDMA treatment affects important cellular function on the hippocampus that may contribute to declarative memory deficits.
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Etanol/toxicidad , Hipocampo/efectos de los fármacos , Trastornos de la Memoria/inducido químicamente , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Estrés Oxidativo/efectos de los fármacos , Animales , Hipocampo/metabolismo , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Proteínas de la Membrana/metabolismo , Trastornos de la Memoria/metabolismo , Ratones , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/fisiologíaRESUMEN
Mitochondria are essential for the health and viability of both motor and sensory neurons and their axons. Processes that disrupt their normal distribution and transport along axons will likely cause peripheral neuropathies. Similarly, mutations in mtDNA or nuclear encoded genes result in neuropathies that either stand alone or are part of multisystem disorders. This chapter focuses on the more common genetic forms and characteristic clinical phenotypes of "mitochondrial" peripheral neuropathies. We also explain how these various mitochondrial abnormalities cause peripheral neuropathy. In a patient with a neuropathy either due to a mutation in a nuclear or an mtDNA gene, clinical investigations aim to characterize the neuropathy and make an accurate diagnosis. In some patients, this may be relatively straightforward, where a clinical assessment and nerve conduction studies followed by genetic testing is all that is needed. In others, multiple investigations including a muscle biopsy, CNS imaging, CSF analysis, and a wide range of metabolic and genetic tests in blood and muscle may be needed to establish diagnosis.
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Enfermedad de Charcot-Marie-Tooth , Enfermedades Mitocondriales , Humanos , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedades Mitocondriales/genética , Mitocondrias/genética , Axones/patología , ADN Mitocondrial , MutaciónRESUMEN
The macrocyclic tetrapeptide CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) and its D-Trp isomer exhibit kappa opioid receptor (KOR) antagonism which prevents stress-induced reinstatement of extinguished cocaine-conditioned place preference. Here, we evaluated the effects of substitution of Trp and D-Trp on the peptides' opioid activity, antinociceptive tolerance, and the ability to prevent relapse to extinguished drug-CPP. Six analogs were synthesized using a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, efficacy in the [35S]GTPγS assay, metabolic stability in mouse liver microsomes, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance, and conditioned place preference (CPP) were also assessed in vivo, and the ameliorating effect of analogs on the reinstatement of extinguished cocaine-place preference was assessed. Substitutions of other D-amino acids for D-Trp did not affect (or in one case increased) KOR affinity, while two of the three substitutions of an L-amino acid for Trp decreased KOR affinity. In contrast, all but one substitution increased mu opioid receptor (MOR) affinity in vitro. The metabolic stabilities of the analogs were similar to those of their respective parent peptides, with analogs containing a D-amino acid being much more rapidly metabolized than those containing an L-amino acid in this position. In vivo, CJ-15,208 analogs demonstrated antinociception, although potencies varied over an 80-fold range and the mediating opioid receptors differed by substitution. KOR antagonism was lost for all but the D-benzothienylalanine analog, and the 2'-naphthylalanine analog instead demonstrated significant delta opioid receptor (DOR) antagonism. Introduction of DOR antagonism coincided with reduced acute opioid antinociceptive tolerance and prevented stress-induced reinstatement of extinguished cocaine-CPP.
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Biallelic SORD mutations cause one of the most frequent forms of recessive hereditary neuropathy, estimated to affect approximately 10,000 patients in North America and Europe alone. Pathogenic SORD loss-of-function changes in the encoded enzyme sorbitol dehydrogenase result in abnormally high sorbitol levels in cells and serum. How sorbitol accumulation leads to peripheral neuropathy remains to be elucidated. A reproducible animal model for SORD neuropathy is essential to illuminate the pathogenesis of SORD deficiency and for preclinical studies of potential therapies. Therefore, we have generated a Sord knockout (KO), Sord -/- , Sprague Dawley rat, to model the human disease and to investigate the pathophysiology underlying SORD deficiency. We have characterized the phenotype in these rats with a battery of behavioral tests as well as biochemical, physiological, and comprehensive histological examinations. Sord -/- rats had remarkably increased levels of sorbitol in serum, cerebral spinal fluid (CSF), and peripheral nerve. Moreover, serum from Sord -/- rats contained significantly increased levels of neurofilament light chain, NfL, an established biomarker for axonal degeneration. Motor performance significantly declined in Sord -/- animals starting at â¼7 months of age. Gait analysis evaluated with video motion tracking confirmed abnormal gait patterns in the hindlimbs. Motor nerve conduction velocities of the tibial nerves were slowed. Light and electron microscopy of the peripheral nervous system revealed degenerating myelinated axons, de- and remyelinated axons, and a likely pathognomonic finding - enlarged "ballooned" myelin sheaths. These findings mainly affected myelinated motor axons; myelinated sensory axons were largely spared. In summary, Sord -/- rats develop a motor-predominant neuropathy that closely resembles the human phenotype. Our studies revealed novel significant aspects of SORD deficiency, and this model will lead to an improved understanding of the pathophysiology and the therapeutic options for SORD neuropathy.
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In the canonical Wnt pathway, signaling results in the stabilization and increased levels of ß-catenin in responding cells. ß-catenin then enters the nucleus, functioning as a coactivator for the Wnt effector, TCF/LEF protein. In the absence of Wnt signaling, TCF is complexed with corepressors, together repressing Wnt target genes. In C. elegans, Wnt signaling specifies the E blastomere to become the endoderm precursor. Activation of endoderm genes in E requires not only an increase in ß-catenin level, but a concomitant decrease in the nuclear level of POP-1, the sole C. elegans TCF. A decrease in nuclear POP-1 levels requires Wnt-induced phosphorylation of POP-1 and 14-3-3 protein-mediated nuclear export. Nuclear POP-1 levels remain high in the sister cell of E, MS, where POP-1 represses the expression of endoderm genes. Here we express three vertebrate TCF proteins (human TCF4, mouse LEF1 and Xenopus TCF3) in C. elegans embryos and compare their localization, repression and activation functions to POP-1. All three TCFs are localized to the nucleus in C. elegans embryos, but none undergoes Wnt-induced nuclear export. Although unable to undergo Wnt-induced nuclear export, human TCF4, but not mouse LEF1 or Xenopus TCF3, can repress endoderm genes in MS, in a manner very similar to POP-1. This repressive activity requires that human TCF4 recognizes specific promoter sequences upstream of endoderm genes and interacts with C. elegans corepressors. Domain swapping identified two regions of POP-1 that are sufficient to confer nuclear asymmetry to human TCF4 when swapped with its corresponding domains. Despite undergoing Wnt-induced nuclear export, the human TCF4/POP-1 chimeric protein continues to function as a repressor for endoderm genes in E, a result we attribute to the inability of hTCF4 to bind to C. elegans ß-catenin. Our results reveal a higher degree of species specificity among TCF proteins for coactivator interactions than for corepressor interactions, and uncover a basic difference between how POP-1 and human TCF4 steady state nuclear levels are regulated.
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Caenorhabditis elegans/embriología , Factores de Transcripción TCF/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Caenorhabditis elegans/metabolismo , Proteínas Co-Represoras/fisiología , Regulación del Desarrollo de la Expresión Génica , Ratones , Unión Proteica/fisiología , Proteínas Wnt/fisiología , Proteínas de Xenopus/metabolismo , beta Catenina/fisiologíaRESUMEN
Between 2012 and 2014, 715 green coffee samples were gathered by Almacafé S.A. (Bogotá, Colombia) from 27 countries. These were analysed at the nuclear magnetic resonance (NMR) laboratory at Universidad del Valle (Cali, Colombia). Over 1000 methanolic coffee extracts were prepared and 4563 spectra were acquired in a fully automatic manner using a 400 MHz NMR spectrometer (Bruker Biospin, Germany). The dataset spans the variance that could be expected for an industrial application of origin monitoring, including samples from different harvest times, collected over several years, and processed by at least two distinct operators. The resulting 1D and 2D spectra can be used to develop and evaluate feature extraction methods, multivariate algorithms, and automation monitoring techniques. They can also be used as datasets for teaching, or as a reference for new studies of similar samples and approaches.
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Despite the success of combined antiretroviral therapy (cART) in reducing viral load, a substantial portion of Human Immunodeficiency Virus (HIV)+ patients report chronic pain. The exact mechanism underlying this co-morbidity even with undetectable viral load remains unknown, but the transactivator of transcription (HIV-Tat) protein is of particular interest. Functional HIV-Tat protein is observed even in cerebrospinal fluid of patients who have an undetectable viral load. It is hypothesized that Tat protein exposure is sufficient to induce neuropathic pain-like manifestations via both activation of microglia and generation of oxidative stress. iTat mice conditionally expressed Tat(1-86) protein in the central nervous system upon daily administration of doxycycline (100 mg/kg/d, i.p., up to 14 days). The effect of HIV-Tat protein exposure on the well-being of the animal was assessed using sucrose-evoked grooming and acute nesting behavior for pain-depressed behaviors, and the development of hyperalgesia assessed with warm-water tail-withdrawal and von Frey assays for thermal hyperalgesia and mechanical allodynia, respectively. Tissue harvested at select time points was used to assess ex vivo alterations in oxidative stress, astrocytosis and microgliosis, and blood-brain barrier integrity with assays utilizing fluorescence-based indicators. Tat protein induced mild thermal hyperalgesia but robust mechanical allodynia starting after 4 days of exposure, reaching a nadir after 7 days. Changes in nociceptive processing were associated with reduced sucrose-evoked grooming behavior without altering acute nesting behavior, and in spinal cord dysregulated free radical generation as measured by DCF fluorescence intensity, altered immunohistochemical expression of the gliotic markers, Iba-1 and GFAP, and increased permeability of the blood-brain barrier to the small molecule fluorescent tracer, sodium fluorescein, in a time-dependent manner. Pretreatment with the anti-inflammatory, indomethacin (1 mg/kg/d, i.p.), the antioxidant, methylsulfonylmethane (100 mg/kg/d i.p.), or the immunomodulatory agent, dimethylfumarate (100 mg/kg/d p.o.) thirty minutes prior to daily injections of doxycycline (100 mg/kg/d i.p.) over 7 days significantly attenuated the development of Tat-induced mechanical allodynia. Collectively, the data suggests that even acute exposure to HIV-1 Tat protein at pathologically relevant levels is sufficient to produce select neurophysiological and behavioral manifestations of chronic pain consistent with that reported by HIV-positive patients.
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Dolor Crónico , Infecciones por VIH , Humanos , Ratones , Animales , Antioxidantes/farmacología , VIH , Transactivadores , Dolor Crónico/tratamiento farmacológico , Antiinflamatorios , Productos del Gen tat , Infecciones por VIH/tratamiento farmacológico , SacarosaRESUMEN
BACKGROUND/AIM: The main objective of this study was to describe the profile of patients who were benefitted in a collective effort to perform liver biopsies in Bahia, Brazil. METHOD: A cross-sectional study was conducted with a sample composed of all the patients who were submitted to liver biopsy during a collective effort carried out in Bahia between July 2007 and November 2009. At the time of the procedure, date on the age and gender of patients and the reason for performing the biopsy were recorded. Data on the degree of fibrosis and the presence of co-morbidities. Following statistical analysis, the frequency of the liver diseases that led to the biopsy procedure was described, and the profile of the patients was stratified into groups according to the most prevalent etiologies. RESULTS: Of the 550 patients evaluated, 55.3% were men and 44.7% women. Mean age was 46.63 ± 11.59 years and there was no statistically significant difference in age between males and females. Of the 550 patients, 72% had hepatitis C and the mean age of these patients was 48.49 ± 10.1 years, significantly higher than the mean age of the patients with hepatitis B (40.41 ± 12.43 years). Furthermore, 70.7% of the patients with hepatitis C were between 41 and 60 years of age. The most frequent fibrosis grade was F2 (44%) and the prevalence of advanced fibrosis was 27.7%. Overall, 85 patients, most of them men, had some degree of iron overload. With respect to the safety of the biopsy procedure, severe complications occurred in only two patients. CONCLUSION: Hepatitis C is the predominant liver disease that demanded liver biopsy. The profile of the patients who benefitted from this collective effort is similar to that of patients in the rest of the country. Moreover, non-Ultrasonography guided liver biopsy is safe and the collective effort to carry out liver biopsies in Bahia was found to be a viable venture.
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Biopsia/normas , Hepatopatías/epidemiología , Hígado/patología , Adulto , Biopsia/efectos adversos , Brasil/epidemiología , Distribución de Chi-Cuadrado , Comorbilidad , Estudios Transversales , Femenino , Hepatitis B/epidemiología , Hepatitis B/patología , Hepatitis C/epidemiología , Hepatitis C/patología , Hepatitis Autoinmune/epidemiología , Hepatitis Autoinmune/patología , Humanos , Hipertensión Portal/epidemiología , Hipertensión Portal/patología , Cirrosis Hepática/epidemiología , Cirrosis Hepática/patología , Hepatopatías/diagnóstico , Hepatopatías/patología , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Derivación y Consulta/normas , Medición de Riesgo , Factores de Riesgo , Índice de Severidad de la Enfermedad , Factores de TiempoRESUMEN
Hereditary transthyretin (TTR) amyloidosis (ATTRv) is an autosomal dominant, systemic disease transmitted by amyloidogenic mutations in the TTR gene. To prevent the otherwise fatal disease course, TTR stabilizers and mRNA silencing antisense drugs are currently approved treatment options. With 90% of the amyloidogenic protein produced by the liver, disease progression including polyneuropathy and cardiomyopathy, the two most prominent manifestations, can successfully be halted by hepatic drug targeting or-formerly-liver transplantation. Certain TTR variants, however, favor disease manifestations in the central nervous system (CNS) or eyes, which is mostly associated with TTR production in the choroid plexus and retina. These compartments cannot be sufficiently reached by any of the approved medications. From liver-transplanted patients, we have learned that with longer lifespans, such CNS manifestations become more relevant over time, even if the underlying TTR mutation is not primarily associated with such. Are we therefore creating a new phenotype? Prolonging life will most likely lead to a shift in the phenotypic spectrum, enabling manifestations like blindness, dementia, and cerebral hemorrhage to come out of the disease background. To overcome the first therapeutic limitation, the blood-brain barrier, we might be able to learn from other antisense drugs currently being used in research or even being approved for primary neurodegenerative CNS diseases like spinal muscular atrophy or Alzheimer's disease. But what effects will unselective CNS TTR knock-down have considering its role in neuroprotection? A potential approach to overcome this second limitiation might be allele-specific targeting, which is, however, still far from clinical trials. Ethical standpoints underline the need for seamless data collection to enable more evidence-based decisions and for thoughtful consenting in research and clinical practice. We conclude that the current advances in treating ATTRv amyloidosis have become a meaningful example for mechanism-based treatment. With its great success in improving patient life spans, we will still have to face new challenges including shifts in the phenotype spectrum and the ongoing need for improved treatment precision. Further investigation is needed to address these closed barriers and open questions.
RESUMEN
Expanding metabolome coverage to include complex lipids and polar metabolites is essential in the generation of well-founded hypotheses in biological assays. Traditionally, lipid extraction is performed by liquid-liquid extraction using either methyl-tert-butyl ether (MTBE) or chloroform, and polar metabolite extraction using methanol. Here, we evaluated the performance of single-step sample preparation methods for simultaneous extraction of the complex lipidome and polar metabolome from human plasma. The method performance was evaluated using high-coverage Hydrophilic Interaction Liquid Chromatography-ESI coupled to tandem mass spectrometry (HILIC-ESI-MS/MS) methodology targeting a panel of 1159 lipids and 374 polar metabolites. The criteria used for method evaluation comprised protein precipitation efficiency, and relative MS signal abundance and repeatability of detectable lipid and polar metabolites in human plasma. Among the tested methods, the isopropanol (IPA) and 1-butanol:methanol (BUME) mixtures were selected as the best compromises for the simultaneous extraction of complex lipids and polar metabolites, allowing for the detection of 584 lipid species and 116 polar metabolites. The extraction with IPA showed the greatest reproducibility with the highest number of lipid species detected with the coefficient of variation (CV) < 30%. Besides this difference, both IPA and BUME allowed for the high-throughput extraction and reproducible measurement of a large panel of complex lipids and polar metabolites, thus warranting their application in large-scale human population studies.