RESUMEN
Dietary lipids play an essential role in regulating the function of the gut microbiota and gastrointestinal tract, and these luminal interactions contribute to mediating host metabolism. Palmitic Acid Hydroxy Stearic Acids (PAHSAs) are a family of lipids with antidiabetic and anti-inflammatory properties, but whether the gut microbiota contributes to their beneficial effects on host metabolism is unknown. Here, we report that treating chow-fed female and male germ-free (GF) mice with PAHSAs improves glucose tolerance, but these effects are lost upon high fat diet (HFD) feeding. However, transfer of feces from PAHSA-treated, but not vehicle-treated, chow-fed conventional mice increases insulin sensitivity in HFD-fed GF mice. Thus, the gut microbiota is necessary for, and can transmit, the insulin-sensitizing effects of PAHSAs in HFD-fed GF male mice. Analyses of the cecal metagenome and lipidome of PAHSA-treated mice identified multiple lipid species that associate with the gut commensal Bacteroides thetaiotaomicron (Bt) and with insulin sensitivity resulting from PAHSA treatment. Supplementing live, and to some degree, heat-killed Bt to HFD-fed female mice prevented weight gain, reduced adiposity, improved glucose tolerance, fortified the colonic mucus barrier and reduced systemic inflammation compared to HFD-fed controls. These effects were not observed in HFD-fed male mice. Furthermore, ovariectomy partially reversed the beneficial Bt effects on host metabolism, indicating a role for sex hormones in mediating the Bt probiotic effects. Altogether, these studies highlight the fact that PAHSAs can modulate the gut microbiota and that the microbiota is necessary for the beneficial metabolic effects of PAHSAs in HFD-fed mice.
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Dieta Alta en Grasa , Microbioma Gastrointestinal , Resistencia a la Insulina , Obesidad , Animales , Masculino , Femenino , Ratones , Microbioma Gastrointestinal/efectos de los fármacos , Obesidad/metabolismo , Obesidad/microbiología , Obesidad/etiología , Dieta Alta en Grasa/efectos adversos , Ratones Endogámicos C57BL , Ácidos Esteáricos/metabolismo , Ácido Palmítico/metabolismo , Heces/microbiología , Ratones ObesosRESUMEN
CONTEXT: Elevated 3-hydroxyisovaleryl-/2-methyl-3-hydroxybutyryl (C5-OH) acylcarnitine in blood can result from several genetic enzyme deficiencies: 3-methylcrotonyl CoA carboxylase deficiency, 3-hydroxy 3-methylglutaryl-CoA lyase deficiency, beta-ketothiolase deficiency, 2-methyl 3-hydroxybutyryl-CoA dehydrogenase deficiency, primary 3-methylglutaconic aciduria, multiple biotin-dependent carboxylase deficiencies and biotin metabolism disorders. Biochemical tests help differentiate these causes while molecular tests are usually required for definitive diagnosis. CASE DESCRIPTION: We reported an infant girl with newborn screen findings of elevated C5-OH acylcarnitine. She had further confirmational biochemical testing including plasma acylcarnitines, urine organic acids and urine acylglycines. Patient's urine organic acid profile showed markedly increased 3-hydroxyisovaleric acid and 3-methylcrotonylglycine. Urine acylglycine test reported a large increase of 3-methylcrotonylglycine and plasma acylcarnitine test repeated the finding of elevated C5-OH acylcarnitine together with propionyl acylcarnitine elevation. These results point to multiple biotin-dependent carboxylase deficiency. Molecular tests revealed a homozygous mutation in the holocarboxylase synthetase gene that is consistent with her biochemical test findings. This case demonstrated the critical role of newborn screen in identifying inborn errors of metabolism that may otherwise be missed and lead to severe morbidity later in life. It also showcased that both biochemical and molecular tests are essential tools in the diagnosis.
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Carnitina , Deficiencia de Holocarboxilasa Sintetasa , Humanos , Femenino , Carnitina/análogos & derivados , Carnitina/sangre , Carnitina/orina , Deficiencia de Holocarboxilasa Sintetasa/diagnóstico , Deficiencia de Holocarboxilasa Sintetasa/genética , Recién Nacido , LactanteRESUMEN
The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC)n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, but its role in disease pathogenesis is unknown. Here, we show alterations in glucose metabolic pathways and ATP levels in the brains of asymptomatic C9-BAC mice. We find that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also show that one of the arginine-rich DPRs (PR) could directly contribute to glucose metabolism and metabolic stress by inhibiting glucose uptake in neurons. Our findings provide a potential mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and suggest a feedforward loop model with potential opportunities for therapeutic intervention.
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Esclerosis Amiotrófica Lateral , Proteína C9orf72 , Demencia Frontotemporal , Glucosa , Fenotipo , Proteína de Unión al GTP ran , Animales , Ratones , Adenosina Trifosfato/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Encéfalo/metabolismo , Encéfalo/patología , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Modelos Animales de Enfermedad , Expansión de las Repeticiones de ADN/genética , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Glucosa/metabolismo , Ratones Transgénicos , Neuronas/metabolismo , Biosíntesis de Proteínas , Proteína de Unión al GTP ran/metabolismoRESUMEN
Conditional protein degradation tags (degrons) are usually >100 amino acids long or are triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous protein levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved a 36-amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex with PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame with SD40 using prime editing are degraded by otherwise inert PT-179. Cryo-electron microscopy structures of SD40 in complex with ligand-bound cereblon revealed mechanistic insights into the molecular basis of SD40's activity and specificity. Our efforts establish a system for continuous evolution of molecular glue complexes and provide ZF tags that overcome shortcomings associated with existing degrons.
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Degrones , Evolución Molecular Dirigida , Proteolisis , Ubiquitina-Proteína Ligasas , Dedos de Zinc , Microscopía por Crioelectrón , Talidomida/química , Ubiquitina-Proteína Ligasas/química , Ubiquitinación , Degrones/genética , Dedos de Zinc/genética , Quimera Dirigida a la Proteólisis , Evolución Molecular Dirigida/métodos , HumanosRESUMEN
Realizing the promise of prime editing for the study and treatment of genetic disorders requires efficient methods for delivering prime editors (PEs) in vivo. Here we describe the identification of bottlenecks limiting adeno-associated virus (AAV)-mediated prime editing in vivo and the development of AAV-PE vectors with increased PE expression, prime editing guide RNA stability and modulation of DNA repair. The resulting dual-AAV systems, v1em and v3em PE-AAV, enable therapeutically relevant prime editing in mouse brain (up to 42% efficiency in cortex), liver (up to 46%) and heart (up to 11%). We apply these systems to install putative protective mutations in vivo for Alzheimer's disease in astrocytes and for coronary artery disease in hepatocytes. In vivo prime editing with v3em PE-AAV caused no detectable off-target effects or significant changes in liver enzymes or histology. Optimized PE-AAV systems support the highest unenriched levels of in vivo prime editing reported to date, facilitating the study and potential treatment of diseases with a genetic component.
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Edición Génica , ARN Guía de Sistemas CRISPR-Cas , Ratones , Animales , Edición Génica/métodos , Hígado/metabolismo , Hepatocitos/metabolismo , Encéfalo , Sistemas CRISPR-CasRESUMEN
Dietary lipids play an essential role in regulating the function of the gut microbiota and gastrointestinal tract, and these luminal interactions contribute to mediating host metabolism. PAHSAs are a class of lipids with anti-diabetic and anti-inflammatory properties, but whether the gut microbiota contributes to their beneficial effects on host metabolism is unknown. Here, we report that treating high fat diet (HFD)-fed germ-free mice with PAHSAs does not improve insulin sensitivity. However, transfer of feces from PAHSA-treated, but not Vehicle-treated, chow-fed mice increases insulin-sensitivity in HFD-fed germ free mice. Thus, the gut microbiota is necessary for and can transmit the insulin-sensitizing effects of PAHSAs in HFD-fed germ-free mice. Functional analyses of the cecal metagenome and lipidome of PAHSA-treated mice identified multiple lipid species that associate with the gut commensal Bacteroides thetaiotaomicron ( Bt ) and with insulin sensitivity resulting from PAHSA treatment. Bt supplementation in HFD-fed female mice prevented weight gain, reduced adiposity, improved glucose tolerance, fortified the colonic mucus barrier and reduced systemic inflammation versus chow-fed controls, effects that were not observed in HFD-fed male mice. Furthermore, ovariectomy partially reversed the beneficial Bt effects on host metabolism, indicating a role for sex hormones in mediating probiotic effects. Altogether, these studies highlight the fact that lipids can modulate the gut microbiota resulting in improvement in host metabolism and that PAHSA-induced changes in the microbiota result in at least some of their insulin-sensitizing effects in female mice.
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Sickle-cell disease (SCD) is caused by an A·T-to-T·A transversion mutation in the ß-globin gene (HBB). Here we show that prime editing can correct the SCD allele (HBBS) to wild type (HBBA) at frequencies of 15%-41% in haematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Seventeen weeks after transplantation into immunodeficient mice, prime-edited SCD HSPCs maintained HBBA levels and displayed engraftment frequencies, haematopoietic differentiation and lineage maturation similar to those of unedited HSPCs from healthy donors. An average of 42% of human erythroblasts and reticulocytes isolated 17 weeks after transplantation of prime-edited HSPCs from four SCD patient donors expressed HBBA, exceeding the levels predicted for therapeutic benefit. HSPC-derived erythrocytes carried less sickle haemoglobin, contained HBBA-derived adult haemoglobin at 28%-43% of normal levels and resisted hypoxia-induced sickling. Minimal off-target editing was detected at over 100 sites nominated experimentally via unbiased genome-wide analysis. Our findings support the feasibility of a one-time prime editing SCD treatment that corrects HBBS to HBBA, does not require any viral or non-viral DNA template and minimizes undesired consequences of DNA double-strand breaks.
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Anemia de Células Falciformes , Edición Génica , Adulto , Humanos , Ratones , Animales , Sistemas CRISPR-Cas , Globinas beta/genética , Anemia de Células Falciformes/terapia , Anemia de Células Falciformes/genética , Células Madre Hematopoyéticas , Fenotipo , ADNRESUMEN
Acquired von Willebrand syndrome (AVWS) is a rare hematologic disorder characterized by quantitative or qualitative defects of von Willebrand factor (vWF), a protein crucial for normal hemostasis. AVWS has been described in association with several pathologic entities with varied mechanisms. Among these, lymphoproliferative disorders are the most common, with monoclonal gammopathy of undetermined significance (MGUS) being the most frequently reported. AVWS in this setting is commonly associated with the development of bleeding that is clinically challenging to manage due to accelerated clearance of vWF, limiting the utility of many conventional treatment modalities such as DDAVP or vWF/FVIII. We report a case of a 43-year-old male who was sent to our institution for new-onset easy bruising and laboratories concerning for von Willebrand disease (vWD). Further diagnostic workup revealed evidence of an IgG monoclonal gammopathy and findings suggestive of vWF inhibition. Ultimately, he was found to have monoclonal gammopathy of clinical significance (MGCS)-associated AVWS refractory to conventional treatment but responsive to lenalidomide and dexamethasone. This case suggests that lenalidomide may be suitable for patients with AVWS secondary to MGCS.
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Gammopatía Monoclonal de Relevancia Indeterminada , Paraproteinemias , Enfermedades de von Willebrand , Masculino , Humanos , Adulto , Enfermedades de von Willebrand/complicaciones , Enfermedades de von Willebrand/tratamiento farmacológico , Gammopatía Monoclonal de Relevancia Indeterminada/complicaciones , Gammopatía Monoclonal de Relevancia Indeterminada/tratamiento farmacológico , Factor de von Willebrand/metabolismo , Lenalidomida/uso terapéutico , Paraproteinemias/complicaciones , Paraproteinemias/tratamiento farmacológico , Paraproteinemias/diagnósticoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that primarily affects motor neurons and causes muscle atrophy, paralysis, and death. While a great deal of progress has been made in deciphering the underlying pathogenic mechanisms, no effective treatments for the disease are currently available. This is mainly due to the high degree of complexity and heterogeneity that characterizes the disease. Over the last few decades of research, alterations to bioenergetic and metabolic homeostasis have emerged as a common denominator across many different forms of ALS. These alterations are found at the cellular level (e.g., mitochondrial dysfunction and impaired expression of monocarboxylate transporters) and at the systemic level (e.g., low BMI and hypermetabolism) and tend to be associated with survival or disease outcomes in patients. Furthermore, an increasing amount of preclinical evidence and some promising clinical evidence suggests that targeting energy metabolism could be an effective therapeutic strategy. This review examines the evidence both for and against these ALS-associated metabolic alterations and highlights potential avenues for therapeutic intervention.
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Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Humanos , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Metabolismo Energético/fisiología , Homeostasis , Neuronas Motoras/patologíaRESUMEN
Branched fatty acid (FA) esters of hydroxy FAs (HFAs; FAHFAs) are recently discovered lipids that are conserved from yeast to mammals1,2. A subfamily, palmitic acid esters of hydroxy stearic acids (PAHSAs), are anti-inflammatory and anti-diabetic1,3. Humans and mice with insulin resistance have lower PAHSA levels in subcutaneous adipose tissue and serum1. PAHSA administration improves glucose tolerance and insulin sensitivity and reduces inflammation in obesity, diabetes and immune-mediated diseases1,4-7. The enzyme(s) responsible for FAHFA biosynthesis in vivo remains unknown. Here we identified adipose triglyceride lipase (ATGL, also known as patatin-like phospholipase domain containing 2 (PNPLA2)) as a candidate biosynthetic enzyme for FAHFAs using chemical biology and proteomics. We discovered that recombinant ATGL uses a transacylation reaction that esterifies an HFA with a FA from triglyceride (TG) or diglyceride to produce FAHFAs. Overexpression of wild-type, but not catalytically dead, ATGL increases FAHFA biosynthesis. Chemical inhibition of ATGL or genetic deletion of Atgl inhibits FAHFA biosynthesis and reduces the levels of FAHFA and FAHFA-TG. Levels of endogenous and nascent FAHFAs and FAHFA-TGs are 80-90 per cent lower in adipose tissue of mice in which Atgl is knocked out specifically in the adipose tissue. Increasing TG levels by upregulating diacylglycerol acyltransferase (DGAT) activity promotes FAHFA biosynthesis, and decreasing DGAT activity inhibits it, reinforcing TGs as FAHFA precursors. ATGL biosynthetic transacylase activity is present in human adipose tissue underscoring its potential clinical relevance. In summary, we discovered the first, to our knowledge, biosynthetic enzyme that catalyses the formation of the FAHFA ester bond in mammals. Whereas ATGL lipase activity is well known, our data establish a paradigm shift demonstrating that ATGL transacylase activity is biologically important.
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Aciltransferasas , Ésteres , Ácidos Grasos , Hidroxiácidos , Aciltransferasas/genética , Aciltransferasas/metabolismo , Tejido Adiposo/química , Tejido Adiposo/metabolismo , Animales , Diglicéridos , Esterificación , Ésteres/química , Ésteres/metabolismo , Ácidos Grasos/biosíntesis , Ácidos Grasos/química , Humanos , Hidroxiácidos/química , Hidroxiácidos/metabolismo , Resistencia a la Insulina , Ratones , TriglicéridosRESUMEN
Evaluating novel compounds for neuroprotective effects in animal models of traumatic brain injury (TBI) is a protracted, labor-intensive and costly effort. However, the present lack of effective treatment options for TBI, despite decades of research, shows the critical need for alternative methods for screening new drug candidates with neuroprotective properties. Because natural products have been a leading source of new therapeutic agents for human diseases, we used an in vitro model of stretch injury to rapidly assess pro-survival effects of three bioactive compounds, two isolated from natural products (clovanemagnolol [CM], vinaxanthone [VX]) and the third, a dietary compound (pterostilbene [PT]) found in blueberries. The stretch injury experiments were not used to validate drug efficacy in a comprehensive manner but used primarily, as proof-of-principle, to demonstrate that the neuroprotective potential of each bioactive agent can be quickly assessed in an immortalized hippocampal cell line in lieu of comprehensive testing in animal models of TBI. To gain mechanistic insights into potential molecular mechanisms of neuroprotective effects, we performed a pathway-specific PCR array analysis of the effects of CM on the rat hippocampus and microRNA sequencing analysis of the effects of VX and PT on cultured hippocampal progenitor neurons. We show that the neuroprotective properties of these natural compounds are associated with altered expression of several genes or microRNAs that have functional roles in neurodegeneration or cell survival. Our approach could help in quickly assessing multiple natural products for neuroprotective properties and expedite the process of new drug discovery for TBI therapeutics.
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Productos Biológicos , Lesiones Traumáticas del Encéfalo , Fármacos Neuroprotectores , Animales , Productos Biológicos/uso terapéutico , Línea Celular , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Fármacos Neuroprotectores/uso terapéutico , RatasRESUMEN
The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA breaks (DSBs) that lead to undesired consequences, including uncontrolled indel mixtures and chromosomal abnormalities. Here we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.
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Sistemas CRISPR-Cas , Edición Génica , Secuencia de Bases , Inversión Cromosómica , ADN/genética , Edición Génica/métodos , Humanos , ARN Guía de Kinetoplastida/genéticaRESUMEN
Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are endogenous lipids with antidiabetic and anti-inflammatory effects. Each FAHFA family consists of esters with different acyl chains and multiple isomers with branch points at different carbons. Some FAHFAs, including palmitic acid hydroxy stearic acids (PAHSAs), improve insulin sensitivity and glucose tolerance in mice by enhancing glucose-stimulated insulin secretion (GSIS), insulin-stimulated glucose transport, and insulin action to suppress hepatic glucose production and reducing adipose tissue inflammation. However, little is known about the biological effects of other FAHFAs. Here, we investigated whether PAHSAs, oleic acid hydroxy stearic acid, palmitoleic acid hydroxy stearic acid, and stearic acid hydroxy stearic acid potentiate GSIS in ß-cells and human islets, insulin-stimulated glucose uptake in adipocytes, and anti-inflammatory effects in immune cells. We also investigated whether they activate G protein-coupled receptor 40, which mediates the effects of PAHSAs on insulin secretion and sensitivity in vivo. We show that many FAHFAs potentiate GSIS, activate G protein-coupled receptor 40, and attenuate LPS-induced chemokine and cytokine expression and secretion and phagocytosis in immune cells. However, fewer FAHFAs augment insulin-stimulated glucose uptake in adipocytes. S-9-PAHSA, but not R-9-PAHSA, potentiated GSIS and glucose uptake, while both stereoisomers had anti-inflammatory effects. FAHFAs containing unsaturated acyl chains with higher branching from the carboxylate head group are more likely to potentiate GSIS, whereas FAHFAs with lower branching are more likely to be anti-inflammatory. This study provides insight into the specificity of the biological actions of different FAHFAs and could lead to the development of FAHFAs to treat metabolic and immune-mediated diseases.
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Ésteres/metabolismo , Ácidos Grasos/metabolismo , Adulto , Ésteres/química , Ácidos Grasos/química , Femenino , Glucosa/metabolismo , Humanos , Secreción de Insulina , Masculino , Persona de Mediana Edad , Estructura Molecular , EstereoisomerismoRESUMEN
Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.
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ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Desoxirribonucleasa I/metabolismo , Conformación de Ácido Nucleico , Conformación Proteica , Ingeniería de Proteínas/métodos , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Transactivadores/metabolismo , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Circoviridae/enzimología , Secuencia Conservada , Cristalografía por Rayos X , ADN Helicasas/química , ADN de Cadena Simple/química , Desoxirribonucleasa I/química , Biblioteca de Genes , Modelos Moleculares , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Virus de Plantas/enzimología , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Origen de Réplica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/química , Especificidad por Sustrato , Transactivadores/química , Proteínas Virales/químicaRESUMEN
Changes in chemical speciation of uranium oxides following storage under varied conditions of temperature and relative humidity are valuable for characterizing material provenance. In this study, subsamples of high purity α-UO3 were stored under four sets of controlled conditions of temperature and relative humidity over several years, and then measured periodically for chemical speciation. Powder X-ray diffraction (XRD) analysis and extended X-ray absorption fine structure spectroscopy confirm hydration of α-UO3 to a schoepite-like end product following storage under each of the varied storage conditions, but the species formed during exposure to the lower relative humidity and lower temperature condition follows different trends from those formed under the other three storage conditions (high relative humidity with high or low temperatures, and low relative humidity with a high temperature). Thermogravimetry coupled with XRD analysis was carried out to distinguish desorption pathways of water from the hydrated end products. Density functional theory calculations discern changes in the structure of α-UO3 following incorporation of 1, 2 or 3 H2O molecules or 1, 2 or 3 OH groups into the orthorhombic lattice, revealing differences in lattice constants, U-O bond lengths, and U-U distances. The collective results from this analysis are in contrast to analogous studies that report that U3O8 is oxidized and hydrated in air during storage under high relative humidity conditions.
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Thiopeptides are a class of natural products with untapped therapeutic potential. To expand the methods available for the scaled production of these antibiotics, we report the laboratory synthesis of micrococcin P1 showcasing thiazole forming reactions of cysteine derivatives and nitriles followed by oxidation. In most instances, this thiazole forming sequence does not require chromatography and proved scalable. Using this approach, 199 mg of micrococcin P1 was generated in a single synthetic sequence.
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Bacteriocinas/síntesis química , Cisteína/análogos & derivados , Nitrilos/química , Tiazoles/síntesis química , Cisteína/química , Tiazoles/químicaRESUMEN
A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease1-9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11-13 to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry14. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis.
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Ácidos y Sales Biliares/biosíntesis , Ácidos y Sales Biliares/química , Metabolómica , Microbiota/fisiología , Animales , Ácidos y Sales Biliares/metabolismo , Ácido Cólico/biosíntesis , Ácido Cólico/química , Ácido Cólico/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Vida Libre de Gérmenes , Humanos , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/microbiología , Ratones , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
Palmitic acid esters of hydroxy stearic acids (PAHSAs) are bioactive lipids with antiinflammatory and antidiabetic effects. PAHSAs reduce ambient glycemia and improve glucose tolerance and insulin sensitivity in insulin-resistant aged chow- and high-fat diet-fed (HFD-fed) mice. Here, we aimed to determine the mechanisms by which PAHSAs improve insulin sensitivity. Both acute and chronic PAHSA treatment enhanced the action of insulin to suppress endogenous glucose production (EGP) in chow- and HFD-fed mice. Moreover, chronic PAHSA treatment augmented insulin-stimulated glucose uptake in glycolytic muscle and heart in HFD-fed mice. The mechanisms by which PAHSAs enhanced hepatic insulin sensitivity included direct and indirect actions involving intertissue communication between adipose tissue and liver. PAHSAs inhibited lipolysis directly in WAT explants and enhanced the action of insulin to suppress lipolysis during the clamp in vivo. Preventing the reduction of free fatty acids during the clamp with Intralipid infusion reduced PAHSAs' effects on EGP in HFD-fed mice but not in chow-fed mice. Direct hepatic actions of PAHSAs may also be important, as PAHSAs inhibited basal and glucagon-stimulated EGP directly in isolated hepatocytes through a cAMP-dependent pathway involving Gαi protein-coupled receptors. Thus, this study advances our understanding of PAHSA biology and the physiologic mechanisms by which PAHSAs exert beneficial metabolic effects.
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Resistencia a la Insulina/fisiología , Hígado/efectos de los fármacos , Hígado/metabolismo , Estearatos/farmacología , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , AMP Cíclico/metabolismo , Dieta Alta en Grasa/efectos adversos , Glucagón/farmacología , Técnicas In Vitro , Lipólisis/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Estearatos/administración & dosificaciónRESUMEN
Palmitic acid esters of hydroxy stearic acids (PAHSAs) are endogenous antidiabetic and antiinflammatory lipids. Here, we show that PAHSAs protect against type 1 diabetes (T1D) and promote ß cell survival and function. Daily oral PAHSA administration to nonobese diabetic (NOD) mice delayed the onset of T1D and markedly reduced the incidence of T1D, whether PAHSAs were started before or after insulitis was established. PAHSAs reduced T and B cell infiltration and CD4+ and CD8+ T cell activation, while increasing Treg activation in pancreata of NOD mice. PAHSAs promoted ß cell proliferation in both NOD mice and MIN6 cells and increased the number of ß cells in NOD mice. PAHSAs attenuated cytokine-induced apoptotic and necrotic ß cell death and increased ß cell viability. The mechanism appears to involve a reduction of ER stress and MAPK signaling, since PAHSAs lowered ER stress in NOD mice, suppressed thapsigargin-induced PARP cleavage in human islets, and attenuated ERK1/2 and JNK1/2 activation in MIN6 cells. This appeared to be mediated in part by glucagon-like peptide 1 receptor (GLP-1R) and not the G protein-coupled receptor GPR40. PAHSAs also prevented impairment of glucose-stimulated insulin secretion and improved glucose tolerance in NOD mice. Thus, PAHSAs delayed the onset of T1D and reduced its incidence by attenuating immune responses and exerting direct protective effects on ß cell survival and function.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Diabetes Mellitus Tipo 1/inmunología , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Ácido Palmítico/farmacología , Ácidos Esteáricos/farmacología , Adulto , Anciano , Animales , Diabetes Mellitus Experimental/inmunología , Ésteres , Femenino , Prueba de Tolerancia a la Glucosa , Humanos , Sistema Inmunológico , Insulina/metabolismo , Islotes Pancreáticos/inmunología , Masculino , Ratones , Ratones Endogámicos NOD , Persona de Mediana Edad , Linfocitos T/inmunologíaRESUMEN
Reported is an experimental and computational investigation of the low temperature heat capacity, thermodynamic functions, and thermal conductivity of stoichiometric, polycrystalline CeO2. The experimentally measured heat capacity at T < 15 K provides an important correction to the historically accepted experimental values, and the low temperature thermal conductivity serves as the most comprehensive data set at T < 400 K available. Below 10 K, the heat capacity is observed to obey the Debye T3 law, with a Debye temperature of ΘD = 455 K. The entropy, enthalpy, and Gibbs free energy functions are obtained from the experimental heat capacity and compared with predictions from Hubbard-corrected density functional perturbation theory calculations using the Perdew, Burke, and Ernzerhof parameterization revised for solids. The thermal conductivity is determined using the Maldonado continuous measurement technique, along with laser flash analysis, and analyzed according to the Klemens-Callaway model.