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Sonic hedgehog signaling regulates processes of embryonic development across multiple tissues, yet factors regulating context-specific Shh signaling remain poorly understood. Exome sequencing of families with polymicrogyria (disordered cortical folding) revealed multiple individuals with biallelic deleterious variants in TMEM161B, which encodes a multi-pass transmembrane protein of unknown function. Tmem161b null mice demonstrated holoprosencephaly, craniofacial midline defects, eye defects, and spinal cord patterning changes consistent with impaired Shh signaling, but were without limb defects, suggesting a CNS-specific role of Tmem161b. Tmem161b depletion impaired the response to Smoothened activation in vitro and disrupted cortical histogenesis in vivo in both mouse and ferret models, including leading to abnormal gyration in the ferret model. Tmem161b localizes non-exclusively to the primary cilium, and scanning electron microscopy revealed shortened, dysmorphic, and ballooned ventricular zone cilia in the Tmem161b null mouse, suggesting that the Shh-related phenotypes may reflect ciliary dysfunction. Our data identify TMEM161B as a regulator of cerebral cortical gyration, as involved in primary ciliary structure, as a regulator of Shh signaling, and further implicate Shh signaling in human gyral development.
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Furões , Proteínas Hedgehog , Animais , Feminino , Humanos , Camundongos , Gravidez , Sistema Nervoso Central/metabolismo , Cílios/genética , Cílios/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Camundongos Knockout , Transdução de SinaisRESUMO
Corticospinal neurons (CSN) are centrally required for skilled voluntary movement, which necessitates that they establish precise subcerebral connectivity with the brainstem and spinal cord. However, molecular controls regulating specificity of this projection targeting remain largely unknown. We previously identified that developing CSN subpopulations exhibit striking axon targeting specificity in the spinal white matter. These CSN subpopulations with segmentally distinct spinal projections are also molecularly distinct; a subset of differentially expressed genes between these distinct CSN subpopulations regulate differential axon projection targeting. Rostrolateral CSN extend axons exclusively to bulbar-cervical segments (CSNBC-lat), while caudomedial CSN (CSNmedial) are more heterogeneous, with distinct, intermingled subpopulations extending axons to either bulbar-cervical or thoraco-lumbar segments. Here, we report, in male and female mice, that Cerebellin 1 (Cbln1) is expressed specifically by CSN in medial, but not lateral, sensorimotor cortex. Cbln1 shows highly dynamic temporal expression, with Cbln1 levels in CSN highest during the period of peak axon extension toward thoraco-lumbar segments. Using gain-of-function experiments, we identify that Cbln1 is sufficient to direct thoraco-lumbar axon extension by CSN. Misexpression of Cbln1 in CSNBC-lat either by in utero electroporation, or by postmitotic AAV-mediated gene delivery, redirects these axons past their normal bulbar-cervical targets toward thoracic segments. Further, Cbln1 overexpression in postmitotic CSNBC-lat increases the number of CSNmedial axons that extend past cervical segments into the thoracic cord. Collectively, these results identify that Cbln1 functions as a potent molecular control over thoraco-lumbar CSN axon extension, part of an integrated network of controls over segmentally-specific CSN axon projection targeting.SIGNIFICANCE STATEMENT Corticospinal neurons (CSN) exhibit remarkable diversity and precision of axonal projections to targets in the brainstem and distinct spinal segments; the molecular basis for this targeting diversity is largely unknown. CSN subpopulations projecting to distinct targets are also molecularly distinguishable. Distinct subpopulations degenerate in specific motor neuron diseases, further suggesting that intrinsic molecular differences might underlie differential vulnerability to disease. Here, we identify a novel molecular control, Cbln1, expressed by CSN extending axons to thoraco-lumbar spinal segments. Cbln1 is sufficient, but not required, for CSN axon extension toward distal spinal segments, and Cbln1 expression is controlled by recently identified, CSN-intrinsic regulators of axon extension. Our results identify that Cbln1, together with other regulators, coordinates segmentally precise CSN axon targeting.
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Axônios , Medula Espinal , Feminino , Masculino , Animais , Camundongos , Axônios/fisiologia , Medula Espinal/fisiologia , Neurônios/fisiologia , Neuritos , Proteínas do Tecido Nervoso/metabolismo , Precursores de Proteínas/metabolismoRESUMO
Complete genome sequencing has identified millions of DNA changes that differ between humans and chimpanzees. Although a subset of these changes likely underlies important phenotypic differences between humans and chimpanzees, it is currently difficult to distinguish causal from incidental changes and to map specific phenotypes to particular genome locations. To facilitate further genetic study of human-chimpanzee divergence, we have generated human and chimpanzee autotetraploids and allotetraploids by fusing induced pluripotent stem cells (iPSCs) of each species. The resulting tetraploid iPSCs can be stably maintained and retain the ability to differentiate along ectoderm, mesoderm, and endoderm lineages. RNA sequencing identifies thousands of genes whose expression differs between humans and chimpanzees when assessed in single-species diploid or autotetraploid iPSCs. Analysis of gene expression patterns in interspecific allotetraploid iPSCs shows that human-chimpanzee expression differences arise from substantial contributions of both cis-acting changes linked to the genes themselves and trans-acting changes elsewhere in the genome. To enable further genetic mapping of species differences, we tested chemical treatments for stimulating genome-wide mitotic recombination between human and chimpanzee chromosomes, and CRISPR methods for inducing species-specific changes on particular chromosomes in allotetraploid cells. We successfully generated derivative cells with nested deletions or interspecific recombination on the X chromosome. These studies confirm an important role for the X chromosome in trans regulation of expression differences between species and illustrate the potential of this system for more detailed cis and trans mapping of the molecular basis of human and chimpanzee evolution.
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Fusão Celular/métodos , Mapeamento Cromossômico/métodos , Variação Genética , Genômica , Células-Tronco Pluripotentes Induzidas/fisiologia , Pan troglodytes/genética , Animais , Evolução Molecular , Genoma , Humanos , Ploidias , Especificidade da Espécie , TranscriptomaRESUMO
Bipolar disorder (BD) and schizophrenia (SCZ) are highly heritable diseases that affect more than 3% of individuals worldwide. Genome-wide association studies have strongly and repeatedly linked risk for both of these neuropsychiatric diseases to a 100 kb interval in the third intron of the human calcium channel gene CACNA1C. However, the causative mutation is not yet known. We have identified a human-specific tandem repeat in this region that is composed of 30 bp units, often repeated hundreds of times. This large tandem repeat is unstable using standard polymerase chain reaction and bacterial cloning techniques, which may have resulted in its incorrect size in the human reference genome. The large 30-mer repeat region is polymorphic in both size and sequence in human populations. Particular sequence variants of the 30-mer are associated with risk status at several flanking single-nucleotide polymorphisms in the third intron of CACNA1C that have previously been linked to BD and SCZ. The tandem repeat arrays function as enhancers that increase reporter gene expression in a human neural progenitor cell line. Different human arrays vary in the magnitude of enhancer activity, and the 30-mer arrays associated with increased psychiatric disease risk status have decreased enhancer activity. Changes in the structure and sequence of these arrays likely contribute to changes in CACNA1C function during human evolution and may modulate neuropsychiatric disease risk in modern human populations.
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Transtorno Bipolar/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único/genética , Esquizofrenia/genética , Sequências de Repetição em Tandem/genética , Canais de Cálcio Tipo L/genética , Genoma Humano/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Íntrons/genéticaRESUMO
The obstetrical conditions placenta accreta spectrum (PAS) and placenta previa are a significant source of pregnancy-associated morbidity and mortality, yet the specific molecular and cellular underpinnings of these conditions are not known. In this study, we identified misregulated gene expression patterns in tissues from placenta previa and percreta (the most extreme form of PAS) compared with control cases. By comparing this gene set with existing placental single-cell and bulk RNA-Seq datasets, we show that the upregulated genes predominantly mark extravillous trophoblasts. We performed immunofluorescence on several candidate molecules and found that PRG2 and AQPEP protein levels are upregulated in both the fetal membranes and the placental disk in both conditions. While this increased AQPEP expression remains restricted to trophoblasts, PRG2 is mislocalized and is found throughout the fetal membranes. Using a larger patient cohort with a diverse set of gestationally aged-matched controls, we validated PRG2 as a marker for both previa and PAS and AQPEP as a marker for only previa in the fetal membranes. Our findings suggest that the extraembryonic tissues surrounding the conceptus, including both the fetal membranes and the placental disk, harbor a signature of previa and PAS that is characteristic of EVTs and that may reflect increased trophoblast invasiveness.
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Proteína Básica Maior de Eosinófilos/genética , Membranas Extraembrionárias/metabolismo , Regulação da Expressão Gênica , Metaloproteases/genética , Placenta Acreta/metabolismo , Placenta Prévia/metabolismo , Proteoglicanas/genética , Proteína Básica Maior de Eosinófilos/metabolismo , Feminino , Humanos , Metaloproteases/metabolismo , Gravidez , Proteoglicanas/metabolismoRESUMO
Little is known about the role of non-coding regions in the etiology of autism spectrum disorder (ASD). We examined three classes of non-coding regions: human accelerated regions (HARs), which show signatures of positive selection in humans; experimentally validated neural VISTA enhancers (VEs); and conserved regions predicted to act as neural enhancers (CNEs). Targeted and whole-genome analysis of >16,600 samples and >4,900 ASD probands revealed that likely recessive, rare, inherited variants in HARs, VEs, and CNEs substantially contribute to ASD risk in probands whose parents share ancestry, which enriches for recessive contributions, but modestly contribute, if at all, in simplex family structures. We identified multiple patient variants in HARs near IL1RAPL1 and in VEs near OTX1 and SIM1 and showed that they change enhancer activity. Our results implicate both human-evolved and evolutionarily conserved non-coding regions in ASD risk and suggest potential mechanisms of how regulatory changes can modulate social behavior.
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Transtorno do Espectro Autista , Humanos , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/epidemiologia , Predisposição Genética para Doença , Elementos Facilitadores Genéticos/genética , Masculino , Evolução Molecular , FemininoRESUMO
BACKGROUND: Geographical variations in mood and psychotic disorders have been found in upper-income countries. We looked for geographic variation in these disorders in Colombia, a middle-income country. We analyzed electronic health records from the Clínica San Juan de Dios Manizales (CSJDM), which provides comprehensive mental healthcare for the one million inhabitants of Caldas. METHODS: We constructed a friction surface map of Caldas and used it to calculate the travel-time to the CSJDM for 16,295 patients who had received an initial diagnosis of mood or psychotic disorder. Using a zero-inflated negative binomial regression model, we determined the relationship between travel-time and incidence, stratified by disease severity. We employed spatial scan statistics to look for patient clusters. RESULTS: We show that travel-times (for driving) to the CSJDM are less than 1 h for ~50% of the population and more than 4 h for ~10%. We find a distance-decay relationship for outpatients, but not for inpatients: for every hour increase in travel-time, the number of expected outpatient cases decreases by 20% (RR = 0.80, 95% confidence interval [0.71, 0.89], p = 5.67E-05). We find nine clusters/hotspots of inpatients. CONCLUSIONS: Our results reveal inequities in access to healthcare: many individuals requiring only outpatient treatment may live too far from the CSJDM to access healthcare. Targeting of resources to comprehensively identify severely ill individuals living in the observed hotspots could further address treatment inequities and enable investigations to determine factors generating these hotspots.
The frequencies of mental disorders vary by geographic region. Investigating such variations may lead to more equitable access to mental healthcare and to scientific discoveries that reveal specific localized factors that contribute to the causes of mental illness. This study examined the frequency of three disorders with a major impact on public health schizophrenia, bipolar disorder, and major depressive disorder by analyzing electronic health records from a hospital providing comprehensive mental health care for a large region in Colombia. We show that individuals receiving outpatient care mainly live relatively near the facility. Those receiving inpatient care live throughout the region, but cluster in a few scattered locations. Future research could lead to strategies for more equitable provision of mental healthcare in Colombia and identify environmental or genetic factors that affect the likelihood that someone will develop one of these disorders.
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The majority of people with HIV live in sub-Saharan Africa, where HIV epidemics are generalized. For these epidemics to develop, populations need to be mobile. However, population-level mobility has not yet been studied in the context of the development of generalized HIV epidemics. Here we do so by studying historical migration data from Botswana which has one of the most severe generalized HIV epidemics worldwide; in 2021, HIV prevalence was 21%. The country reported its first AIDS case in 1985 when it began to rapidly urbanize. We hypothesize that, during the development of Botswana's epidemic, the population was highly mobile and there were substantial urban-to-rural and rural-to-urban migratory flows. We test this hypothesis by conducting a network analysis using a historical time series (1981 to 2011) of micro-census data from Botswana. We found 10% of the population moved their residency annually, complex migration networks connected urban with rural areas, and there were very high rates of rural-to-urban migration. Notably, we also found mining towns were both important in-flow and out-flow migration hubs; consequently, there was a very high turnover of residents in towns. Our results support our hypothesis, and together, provide one explanation for the development of Botswana's generalized epidemic.
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The majority of people with HIV live in sub-Saharan Africa, where epidemics are generalized. For these epidemics to develop, populations need to be mobile. However, the role of population-level mobility in the development of generalized HIV epidemics has not been studied. Here we do so by studying historical migration data from Botswana, which has one of the most severe generalized HIV epidemics worldwide; HIV prevalence was 21% in 2021. The country reported its first AIDS case in 1985 when it began to rapidly urbanize. We hypothesize that, during the development of Botswana's epidemic, the population was extremely mobile and the country was highly connected by substantial migratory flows. We test this mobility hypothesis by conducting a network analysis using a historical time series (1981-2011) of micro-census data from Botswana. Our results support our hypothesis. We found complex migration networks with very high rates of rural-to-urban, and urban-to-rural, migration: 10% of the population moved annually. Mining towns (where AIDS cases were first reported, and risk behavior was high) were important in-flow and out-flow migration hubs, suggesting that they functioned as 'core groups' for HIV transmission and dissemination. Migration networks could have dispersed HIV throughout Botswana and generated the current hyperendemic epidemic.
Over 25 million people in sub-Saharan Africa live with HIV. After reporting its first AIDS case in 1985, Botswana is one of the most severely affected countries in the region, with one in five adults now living with HIV. Movement of the population is likely to have contributed to a geographically dispersed, and high-prevalence, HIV epidemic in Botswana. Since 1985, urbanization, rapid economic and population growth, and migration have transformed Botswana. Yet, few studies have analyzed the role of population-level movement patterns in the spread of HIV during this time. By studying micro-census data from Botswana between 1981 and 2011, Song et al. found that the country's population was highly mobile during this period. Reconstructions of internal migration patterns show very high rates of rural-to-urban and urban-to-rural migration, with 10% of Botswana's population moving each year. The first reported AIDS cases in Botswana occurred in mining towns and cities where high-risk behavior was prevalent. These areas were also migration hubs during this period and could have contributed to the rapid spread of HIV throughout the country as infected individuals moved back to rural districts. Understanding human migration patterns and how they affect the spread of infectious diseases using current data could help public health authorities in Botswana and additional sub-Saharan African countries design control strategies for HIV and other important infections that occur in the region.
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Epidemias , Infecções por HIV , Humanos , Botsuana/epidemiologia , Assunção de Riscos , Fatores de Tempo , Infecções por HIV/epidemiologiaRESUMO
Little is known about the role of noncoding regions in the etiology of autism spectrum disorder (ASD). We examined three classes of noncoding regions: Human Accelerated Regions (HARs), which show signatures of positive selection in humans; experimentally validated neural Vista Enhancers (VEs); and conserved regions predicted to act as neural enhancers (CNEs). Targeted and whole genome analysis of >16,600 samples and >4900 ASD probands revealed that likely recessive, rare, inherited variants in HARs, VEs, and CNEs substantially contribute to ASD risk in probands whose parents share ancestry, which enriches for recessive contributions, but modestly, if at all, in simplex family structures. We identified multiple patient variants in HARs near IL1RAPL1 and in a VE near SIM1 and showed that they change enhancer activity. Our results implicate both human-evolved and evolutionarily conserved noncoding regions in ASD risk and suggest potential mechanisms of how changes in regulatory regions can modulate social behavior.
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Importance: Polymicrogyria is the most commonly diagnosed cortical malformation and is associated with neurodevelopmental sequelae including epilepsy, motor abnormalities, and cognitive deficits. Polymicrogyria frequently co-occurs with other brain malformations or as part of syndromic diseases. Past studies of polymicrogyria have defined heterogeneous genetic and nongenetic causes but have explained only a small fraction of cases. Objective: To survey germline genetic causes of polymicrogyria in a large cohort and to consider novel polymicrogyria gene associations. Design, Setting, and Participants: This genetic association study analyzed panel sequencing and exome sequencing of accrued DNA samples from a retrospective cohort of families with members with polymicrogyria. Samples were accrued over more than 20 years (1994 to 2020), and sequencing occurred in 2 stages: panel sequencing (June 2015 to January 2016) and whole-exome sequencing (September 2019 to March 2020). Individuals seen at multiple clinical sites for neurological complaints found to have polymicrogyria on neuroimaging, then referred to the research team by evaluating clinicians, were included in the study. Targeted next-generation sequencing and/or exome sequencing were performed on probands (and available parents and siblings) from 284 families with individuals who had isolated polymicrogyria or polymicrogyria as part of a clinical syndrome and no genetic diagnosis at time of referral from clinic, with sequencing from 275 families passing quality control. Main Outcomes and Measures: The number of families in whom genetic sequencing yielded a molecular diagnosis that explained the polymicrogyria in the family. Secondarily, the relative frequency of different genetic causes of polymicrogyria and whether specific genetic causes were associated with co-occurring head size changes were also analyzed. Results: In 32.7% (90 of 275) of polymicrogyria-affected families, genetic variants were identified that provided satisfactory molecular explanations. Known genes most frequently implicated by polymicrogyria-associated variants in this cohort were PIK3R2, TUBB2B, COL4A1, and SCN3A. Six candidate novel polymicrogyria genes were identified or confirmed: de novo missense variants in PANX1, QRICH1, and SCN2A and compound heterozygous variants in TMEM161B, KIF26A, and MAN2C1, each with consistent genotype-phenotype relationships in multiple families. Conclusions and Relevance: This study's findings reveal a higher than previously recognized rate of identifiable genetic causes, specifically of channelopathies, in individuals with polymicrogyria and support the utility of exome sequencing for families affected with polymicrogyria.
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Polimicrogiria , Humanos , Polimicrogiria/diagnóstico por imagem , Polimicrogiria/genética , Sequenciamento do Exoma , Estudos Retrospectivos , Mutação de Sentido Incorreto , Irmãos , Proteínas do Tecido Nervoso/genética , Conexinas/genéticaRESUMO
The cognitive abilities of humans are distinctive among primates, but their molecular and cellular substrates are poorly understood. We used comparative single-nucleus transcriptomics to analyze samples of the middle temporal gyrus (MTG) from adult humans, chimpanzees, gorillas, rhesus macaques, and common marmosets to understand human-specific features of the neocortex. Human, chimpanzee, and gorilla MTG showed highly similar cell-type composition and laminar organization as well as a large shift in proportions of deep-layer intratelencephalic-projecting neurons compared with macaque and marmoset MTG. Microglia, astrocytes, and oligodendrocytes had more-divergent expression across species compared with neurons or oligodendrocyte precursor cells, and neuronal expression diverged more rapidly on the human lineage. Only a few hundred genes showed human-specific patterning, suggesting that relatively few cellular and molecular changes distinctively define adult human cortical structure.
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Cognição , Hominidae , Neocórtex , Lobo Temporal , Animais , Humanos , Perfilação da Expressão Gênica , Gorilla gorilla/genética , Hominidae/genética , Hominidae/fisiologia , Macaca mulatta/genética , Pan troglodytes/genética , Filogenia , Transcriptoma , Neocórtex/fisiologia , Especificidade da Espécie , Lobo Temporal/fisiologiaRESUMO
For precise motor control, distinct subpopulations of corticospinal neurons (CSN) must extend axons to distinct spinal segments, from proximal targets in the brainstem and cervical cord to distal targets in thoracic and lumbar spinal segments. We find that developing CSN subpopulations exhibit striking axon targeting specificity in spinal white matter, which establishes the foundation for durable specificity of adult corticospinal circuitry. Employing developmental retrograde and anterograde labeling, and their distinct neocortical locations, we purified developing CSN subpopulations using fluorescence-activated cell sorting to identify genes differentially expressed between bulbar-cervical and thoracolumbar-projecting CSN subpopulations at critical developmental times. These segmentally distinct CSN subpopulations are molecularly distinct from the earliest stages of axon extension, enabling prospective identification even before eventual axon targeting decisions are evident in the spinal cord. This molecular delineation extends beyond simple spatial separation of these subpopulations in the cortex. Together, these results identify candidate molecular controls over segmentally specific corticospinal axon projection targeting.
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Axônios/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Crescimento Neuronal , Tratos Piramidais/metabolismo , Córtex Sensório-Motor/metabolismo , Substância Branca/metabolismo , Fatores Etários , Animais , Receptores de Proteínas Morfogenéticas Ósseas/genética , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Separação Celular , Feminino , Citometria de Fluxo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Rastreamento Neuroanatômico , Tratos Piramidais/crescimento & desenvolvimento , Córtex Sensório-Motor/crescimento & desenvolvimento , Transcrição Gênica , Substância Branca/crescimento & desenvolvimentoRESUMO
Human accelerated regions (HARs) are the fastest-evolving regions of the human genome, and many are hypothesized to function as regulatory elements that drive human-specific gene regulatory programs. We interrogate the in vitro enhancer activity and in vivo epigenetic landscape of more than 3,100 HARs during human neurodevelopment, demonstrating that many HARs appear to act as neurodevelopmental enhancers and that sequence divergence at HARs has largely augmented their neuronal enhancer activity. Furthermore, we demonstrate PPP1R17 to be a putative HAR-regulated gene that has undergone remarkable rewiring of its cell type and developmental expression patterns between non-primates and primates and between non-human primates and humans. Finally, we show that PPP1R17 slows neural progenitor cell cycle progression, paralleling the cell cycle length increase seen predominantly in primate and especially human neurodevelopment. Our findings establish HARs as key components in rewiring human-specific neurodevelopmental gene regulatory programs and provide an integrated resource to study enhancer activity of specific HARs.
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Encéfalo/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Animais , Evolução Biológica , Epigenômica , Evolução Molecular , Furões , Humanos , Macaca , Camundongos , Pan troglodytesRESUMO
Biosynthesis of the pyrimidine nucleotide uridine monophosphate (UMP) is essential for cell proliferation and is achieved by the activity of convergent de novo and salvage metabolic pathways. Here we report the development and application of a cell-based metabolic modifier screening platform that leverages the redundancy in pyrimidine metabolism for the discovery of selective UMP biosynthesis modulators. In evaluating a library of protein kinase inhibitors, we identified multiple compounds that possess nucleotide metabolism modifying activity. The JNK inhibitor JNK-IN-8 was found to potently inhibit nucleoside transport and engage ENT1. The PDK1 inhibitor OSU-03012 (also known as AR-12) and the RAF inhibitor TAK-632 were shown to inhibit the therapeutically relevant de novo pathway enzyme DHODH and their affinities were unambiguously confirmed through in vitro assays and co-crystallization with human DHODH.
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Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Nucleosídeos de Pirimidina/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Di-Hidro-Orotato Desidrogenase , Desenho de Fármacos , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Humanos , Simulação de Dinâmica Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/químicaRESUMO
A potent class of isoquinoline-based α-N-heterocyclic carboxaldehyde thiosemicarbazone (HCT) compounds has been rediscovered; based upon this scaffold, three series of antiproliferative agents were synthesized through iterative rounds of methylation and fluorination modifications, with anticancer activities being potentiated by physiologically relevant levels of copper. The lead compound, HCT-13, was highly potent against a panel of pancreatic, small cell lung carcinoma, prostate cancer, and leukemia models, with IC50 values in the low-to-mid nanomolar range. Density functional theory (DFT) calculations showed that fluorination at the 6-position of HCT-13 was beneficial for ligand-copper complex formation, stability, and ease of metal-center reduction. Through a chemical genomics screen, we identify DNA damage response/replication stress response (DDR/RSR) pathways, specifically those mediated by ataxia-telangiectasia and Rad3-related protein kinase (ATR), as potential compensatory mechanism(s) of action following HCT-13 treatment. We further show that the cytotoxicity of HCT-13 is copper-dependent, that it promotes mitochondrial electron transport chain (mtETC) dysfunction, induces production of reactive oxygen species (ROS), and selectively depletes guanosine nucleotide pools. Lastly, we identify metabolic hallmarks for therapeutic target stratification and demonstrate the in vivo efficacy of HCT-13 against aggressive models of acute leukemias in mice.
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PURPOSE: Studies suggest that melatonin may prevent delirium, a condition of acute brain dysfunction occurring in 20%-30% of hospitalized older adults that is associated with increased morbidity and mortality. We examined the effect of melatonin on delirium prevention in hospitalized older adults while measuring sleep parameters as a possible underlying mechanism. METHODS: This was a randomized clinical trial measuring the impact of 3 mg of melatonin nightly on incident delirium and both objective and subjective sleep in inpatients age ≥65 years, admitted to internal medicine wards (non-intensive care units). Delirium incidence was measured by bedside nurses using the confusion assessment method. Objective sleep measurements (nighttime sleep duration, total sleep time per 24 hours, and sleep fragmentation as determined by average sleep bout length) were obtained via actigraphy. Subjective sleep quality was measured using the Richards Campbell Sleep Questionnaire. RESULTS: Delirium occurred in 22.2% (8/36) of subjects who received melatonin vs in 9.1% (3/33) who received placebo (Pâ¯=â¯.19). Melatonin did not significantly change objective or subjective sleep measurements. Nighttime sleep duration and total sleep time did not differ between subjects who became delirious vs those who did not, but delirious subjects had more sleep fragmentation (sleep bout length 7.0 ± 3.0 vs 9.5 ± 5.3 min; Pâ¯=â¯.03). CONCLUSIONS: Melatonin given as a nightly dose of 3 mg did not prevent delirium in non-intensive care unit hospitalized patients or improve subjective or objective sleep.
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Antioxidantes/administração & dosagem , Delírio/prevenção & controle , Hospitalização , Melatonina/administração & dosagem , Sono , Idoso , Idoso de 80 Anos ou mais , California/epidemiologia , Delírio/epidemiologia , Método Duplo-Cego , Feminino , Humanos , Masculino , Privação do Sono/epidemiologiaRESUMO
Measures of regional brain volumes, which can be derived from magnetic resonance imaging (MRI) images by dividing a brain into its constituent parts, can be used as structural indicators of many different neuroanatomical diseases and disorders, including alcoholism. Reducing the time and cost required for brain segmentation would greatly facilitate both clinical and research endeavors. In the present study, we compared two segmentation methods to measure brain volumes in alcoholic and nonalcoholic control subjects: 1) an automated system (FreeSurfer) and 2) a semi-automated, supervised system (Cardviews, developed by the Center for Morphometric Analysis [CMA] at Massachusetts General Hospital), which requires extensive staff and oversight. The participants included 32 abstinent alcoholics (19 women) and 37 demographically matched, nonalcoholic controls (17 women). Brain scans were acquired in a 3 Tesla MRI scanner. The FreeSurfer and CMA methods showed good agreement for the lateral ventricles, cerebral white matter, caudate, and thalamus. In general, the larger the brain structure, the closer the agreement between the methods, except for the cerebral cortex, which showed large between-method differences. However, several other discrepancies existed between the FreeSurfer and CMA volume measures of alcoholics' brains. The CMA volumes, but not FreeSurfer, demonstrated that the thalamus, caudate, and putamen were significantly smaller in male alcoholics as compared with male controls. Additionally, the hippocampus was significantly smaller in alcoholic women compared with women controls. In general, correlation between methods was lowest in male alcoholic subjects, who also showed the greatest abnormalities. These results suggest that although many brain structures can be segmented reliably by CMA and FreeSurfer, low correlations between methods in some regions may be due to morphological changes in the brains of alcoholics.
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We have previously demonstrated that mutated DNA derived from the circulation can be detected in urine and predominantly exists as DNA fragments <1 kb. To preferentially isolate the trans-renal DNA from urine, we developed a method using carboxylated beads to separate high-MW (1 kb or larger) from low-MW DNA in urine. A primer set for 18s rRNA (generating a PCR product of 872 bp) was designed and optimized for real-time PCR quantification of high-MW DNA templates. To evaluate the method, urine samples from 5 volunteers with no known diseases and 36 patients with various colorectal diseases were collected and tested. It was found that the average removal efficiency of high-MW DNA from total urine DNA using carboxylated beads is 92.72%+/- 1.42%. Furthermore, compared with using total urine DNA, our method provides a greater ability to detect mutated K-ras in the urine of colorectal cancer patients. The concurrence of K-ras mutations detected in disease tissue and the corresponding urine specimen is significantly higher (P= 0.0015) when the samples were enriched in low-MW DNA.