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Background: Short tandem repeats (STRs) are highly variable elements that play a pivotal role in multiple genetic diseases and the regulation of gene expression. Long-read sequencing (LRS) offers a potential solution to genome-wide STR analysis. However, characterizing STRs in human genomes using LRS on a large population scale has not been reported. Methods: We conducted the large LRS-based STR analysis in 193 unrelated samples of the Chinese population and performed genome-wide profiling of STR variation in the human genome. The repeat dynamic index (RDI) was introduced to evaluate the variability of STR. We sourced the expression data from the Genotype-Tissue Expression to explore the tissue specificity of highly variable STRs related genes across tissues. Enrichment analyses were also conducted to identify potential functional roles of the high variable STRs. Results: This study reports the large-scale analysis of human STR variation by LRS and offers a reference STR database based on the LRS dataset. We found that the disease-associated STRs (dSTRs) and STRs associated with the expression of nearby genes (eSTRs) were highly variable in the general population. Moreover, tissue-specific expression analysis showed that those highly variable STRs related genes presented the highest expression level in brain tissues, and enrichment pathways analysis found those STRs are involved in synaptic function-related pathways. Conclusion: Our study profiled the genome-wide landscape of STR using LRS and highlighted the highly variable STRs in the human genome, which provide a valuable resource for studying the role of STRs in human disease and complex traits.
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Background: Structural variations (SVs) are common genetic alterations in the human genome that could cause different phenotypes and diseases, including cancer. However, the detection of structural variations using the second-generation sequencing was limited by its short read length, which restrained our understanding of structural variations. Methods: In this study, we developed a 28-gene panel for long-read sequencing and employed it to Oxford Nanopore Technologies and Pacific Biosciences platforms. We analyzed structural variations in the 28 breast cancer-related genes through long-read genomic and transcriptomic sequencing of tumor, para-tumor, and blood samples in 19 breast cancer patients. Results: Our results showed that some somatic SVs were recurring among the selected genes, though the majority of them occurred in the non-exonic region. We found evidence supporting the existence of hotspot regions for SVs, which extended our previous understanding that they exist only for single nucleotide variations. Conclusion: In conclusion, we employed long-read genomic and transcriptomic sequencing to identify SVs from breast cancer patients and proved that this approach holds great potential in clinical application.
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Lately, Drosophila has been favored as a model in sleep and circadian rhythm research due to its conserved mechanism and easily manageable operation. These studies have revealed the sophisticated parameters in whole-day sleep profiles of Drosophila, drawing connections between Drosophila sleep and human sleep. In this study, we tested several sleep deprivation protocols (mechanical shakes and light interruptions) on Drosophila and delineated their influences on Drosophila sleep. We applied a daytime light-deprivation protocol (DD) mimicking jet-lag to screen drugs that alleviate sleep deprivation. Characteristically, classical sleep-aid compounds exhibited different forms of influence: phenobarbital and pentobarbital modified total sleep time, while melatonin only shortened the latency to sleep. Such results construct the basis for further research on sleep benefits in other treatments in Drosophila. We screened seven herb extracts, and found very diverse results regarding their effect on sleep regulation. For instance, Panax notoginseng and Withania somnifera extracts displayed potent influence on total sleep time, while Melissa officinalis increased the number of sleep episodes. By comparing these treatments, we were able to rank drug potency in different aspects of sleep regulation. Notably, we also confirmed the presence of sleep difficulties in a Drosophila Alzheimer's disease (AD) model with an overexpression of human Abeta, and recognized clear differences between the portfolios of drug screening effects in AD flies and in the control group. Overall, potential drug candidates and receipts for sleep problems can be identified separately for normal and AD Drosophila populations, outlining Drosophila's potential in drug screening tests in other populations if combined with the use of other genetic disease tools.
Assuntos
Extratos Vegetais/farmacologia , Privação do Sono/tratamento farmacológico , Transtornos do Sono-Vigília/tratamento farmacológico , Sono/fisiologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/genética , Animais , Ritmo Circadiano/efeitos dos fármacos , Modelos Animais de Doenças , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica/genética , Humanos , Melatonina/farmacologia , Mutação , Panax notoginseng/química , Fenobarbital/farmacologia , Extratos Vegetais/química , Sono/efeitos dos fármacos , Sono/genética , Privação do Sono/genética , Privação do Sono/fisiopatologia , Transtornos do Sono-Vigília/genética , Transtornos do Sono-Vigília/fisiopatologia , Withania/químicaRESUMO
Alzheimer's disease (AD) is an age-related neurodegenerative disease with unknown mechanism that is characterized by the aggregation of abnormal proteins and dysfunction of immune responses. In this study, an integrative approach employing in silico analysis and wet-lab experiment was conducted to estimate the degrees of innate immune system relevant gene expression, neurotoxic Aß42 generation and neuronal apoptosis in normal Drosophila melanogaster and a transgenic model of AD. Results demonstrated mRNA levels of antimicrobial peptide (AMP) genes gradually increased with age in wild-type flies, while which exhibited a trend for an initial decrease followed by subsequent increase during aging in the AD group. Time series and correlation analysis illustrated indicated a potential relationship between variation in AMP expression and Aß42 concentration. In conclusion, our study provides evidence for abnormal gene expression of AMPs in AD flies with age, which is distinct from the expression profiles in the normal aging process. Aberrant AMP expression may participate in the onset and development of AD by inducing or accelerating Aß deposition. These findings suggest that AMPs may serve as potential diagnostic biomarkers and therapeutic targets. However, further studies are required to elucidate the pathological effects and underlying mechanisms of AMP dysregulation in AD progression.
Assuntos
Envelhecimento/genética , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Regulação da Expressão Gênica , Proteínas Citotóxicas Formadoras de Poros/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/genética , Encéfalo/metabolismo , Bases de Dados Genéticas , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunidade Inata/genética , Neurônios/metabolismoRESUMO
Herbal extracts have been extensively used worldwide for their application on memory improvement, especially among aged and memory-deficit populations. In the present study, the memory loss induced by human Abeta protein over-expression in fruitfly Alzheimer's disease (AD) model was rescued by multiple extracts from Gardenia jasminoides. Three extracts that rich with gardenia yellow, geniposide, and gardenoside components showed distinct rescue effect on memory loss. Further investigation on adding gardenoside into a formula of Ganoderma lucidum, Panax notoginseng and Panax ginseng (GPP) also support its therapeutic effects on memory improvement. Interestingly, the application of GPP and gardenoside did not alter the accumulation of Abeta proteins but suppressed the expression of immune-related genes in the brain. These results revealed the importance and relevancy of anti-inflammation process and the underlying mechanisms on rescuing memory deficits, suggesting the potential therapeutic use of the improved GPP formulation in improving cognition in defined population in the future.
Assuntos
Doença de Alzheimer , Cognição/efeitos dos fármacos , Gardenia/química , Imunidade Inata/efeitos dos fármacos , Extratos Vegetais/farmacologia , Doença de Alzheimer/tratamento farmacológico , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Iridoides/química , Iridoides/isolamento & purificação , Iridoides/farmacologia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Reação em Cadeia da PolimeraseRESUMO
Intrinsic electric activities of neurons play important roles in establishing and refining neural circuits during development. However, how the underlying ionic currents undergo postembryonic reorganizations remains largely unknown. Using acutely dissociated neurons from larval, pupal, and adult Drosophila brains, we show drastic re-assemblies and compensatory regulations of voltage-gated (IKv) and Ca2+-activated (IK(Ca)) K+ currents during postembryonic development. Larval and adult neurons displayed prominent fast-inactivating IKv, mediated by the Shaker (Sh) channel to a large extent, while in the same neurons IK(Ca) was far smaller in amplitude. In contrast, pupal neurons were characterized by large sustained IKv and prominent IK(Ca), encoded predominantly by the slowpoke (slo) gene. Surprisingly, deletion of Sh in the ShM null mutant removed inactivating, transient IKv from large portions of neurons at all stages. Interestingly, elimination of Sh currents was accompanied by upregulation of non-Sh transient IKv. In comparison, the slo1 mutation abolished the vast majority of IK(Ca), particularly at the pupal stage. Strikingly, the deficiency of IK(Ca) in slo pupae was compensated by the transient component of IKv mediated by Sh channels. Thus, IK(Ca) appears to play critical roles in pupal development and its absence induces functional compensations from a specific transient IKv current. While mutants lacking either Sh or slo currents survived normally, Sh;;slo double mutants deficient in both failed to survive through pupal metamorphosis. Together, our data highlight significant reorganizations and homeostatic compensations of K+ currents during postembryonic development and uncover previously unrecognized roles for Sh and slo in this plastic process.
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Drosophila/fisiologia , Neurogênese/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Canais de Potássio/metabolismo , Animais , Homeostase/fisiologiaRESUMO
Dact1 (Dapper/Frodo), an intracellular phosphoprotein that binds Dishevelled, catenins, and other signaling proteins, is expressed in the developing and mature mammalian CNS, but its function there is unknown. Dact1 colocalized with synaptic markers and partitioned to postsynaptic fractions from cultured mouse forebrain neurons. Hippocampal neurons from Dact1 knock-out mice had simpler dendritic arbors and fewer spines than hippocampal neurons from wild-type littermates. This correlated with reductions in excitatory synapses and miniature EPSCs, whereas inhibitory synapses were not affected. Loss of Dact1 resulted in a decrease in activated Rac, and recombinant expression of either Dact1 or constitutively active Rac, but not Rho or Cdc42, rescued dendrite and spine phenotypes in Dact1 mutant neurons. Our findings suggest that, during neuronal differentiation, Dact1 plays a critical role in a molecular pathway promoting Rac activity underlying the elaboration of dendrites and the establishment of spines and excitatory synapses.
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Espinhas Dendríticas/fisiologia , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neurônios/citologia , Sinapses/fisiologia , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Células Cultivadas , Espinhas Dendríticas/ultraestrutura , Proteína 4 Homóloga a Disks-Large , Potenciais Pós-Sinápticos Excitadores/genética , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Proteínas de Fluorescência Verde/genética , Guanilato Quinases , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas de Ligação a RNA , Coloração pela Prata/métodos , Frações Subcelulares/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
The maintenance of spine and synapse number during development is critical for neuronal circuit formation and function. Here we show that delta-catenin, a component of the cadherin-catenin cell adhesion complex, regulates spine and synapse morphogenesis during development. Genetic ablation or acute knockdown of delta-catenin leads to increases in spine and synapse density, accompanied by a decrease in tetrodotoxin induced spine plasticity. Our results indicate that delta-catenin may mediate conversion of activity-dependent signals to morphological spine plasticity. The functional role of delta-catenin in regulating spine density does not require binding to cadherins, but does require interactions with PDZ domain-containing proteins. We propose that the perturbations in spine and synaptic structure and function observed after depletion of delta-catenin during development may contribute to functional alterations in neural circuitry, the cognitive deficits observed in mutant mice, and the mental retardation pathology of Cri-du-chat syndrome.
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Moléculas de Adesão Celular/fisiologia , Espinhas Dendríticas/fisiologia , Hipocampo/crescimento & desenvolvimento , Morfogênese/fisiologia , Neurônios/fisiologia , Fosfoproteínas/fisiologia , Sinapses/fisiologia , Animais , Animais Recém-Nascidos , Cateninas , Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/genética , Células Cultivadas , Transtornos Cognitivos/genética , Transtornos Cognitivos/patologia , Espinhas Dendríticas/ultraestrutura , Hipocampo/ultraestrutura , Masculino , Camundongos , Camundongos Knockout , Plasticidade Neuronal/fisiologia , Neurônios/ultraestrutura , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Ratos , Sinapses/ultraestrutura , delta CateninaRESUMO
Localization of presynaptic components to synaptic sites is critical for hippocampal synapse formation. Cell adhesion-regulated signaling is important for synaptic development and function, but little is known about differentiation of the presynaptic compartment. In this study, we describe a pathway that promotes presynaptic development involving p120catenin (p120ctn), the cytoplasmic tyrosine kinase Fer, the protein phosphatase SHP-2, and beta-catenin. Presynaptic Fer depletion prevents localization of active zone constituents and synaptic vesicles and inhibits excitatory synapse formation and synaptic transmission. Depletion of p120ctn or SHP-2 similarly disrupts synaptic vesicle localization with active SHP-2, restoring synapse formation in the absence of Fer. Fer or SHP-2 depletion results in elevated tyrosine phosphorylation of beta-catenin. beta-Catenin overexpression restores normal synaptic vesicle localization in the absence of Fer or SHP-2. Our results indicate that a presynaptic signaling pathway through p120ctn, Fer, SHP-2, and beta-catenin promotes excitatory synapse development and function.
Assuntos
Moléculas de Adesão Celular/metabolismo , Hipocampo/enzimologia , Neurônios/enzimologia , Fosfoproteínas/metabolismo , Terminações Pré-Sinápticas/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transmissão Sináptica , beta Catenina/metabolismo , Animais , Axônios/enzimologia , Cateninas , Moléculas de Adesão Celular/genética , Células Cultivadas , Citoplasma/enzimologia , Potenciais Pós-Sinápticos Excitadores , Hipocampo/embriologia , Fosfoproteínas/genética , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Tirosina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Transfecção , beta Catenina/genética , Proteína rhoA de Ligação ao GTP/metabolismo , delta CateninaRESUMO
Myosin Va, an actin-based motor protein that transports intracellular cargos, can bundle actin in vitro. Whether myosin Va regulates cellular actin dynamics or cell migration remains unclear. To address this, we compared Chinese Hamster Ovary (CHO) cells that stably express GFP fused to either full length mouse myosin Va (GFP-M5) or heavy meromyosin Va (GFP-M5Delta). GFP-M5 and GFP-M5Delta co-immunoprecipitate with CHO myosin Va and serve as overexpression of wild-type and dominant negative mutants of myosin Va. Compared to non-expressing control cells, GFP-M5-overexpressing cells have peripheral endocytic vesicles, spread slowly after plating, as well as produce robust interior actin stress fibers, myosin II bundles, and focal adhesions. However, these cells display normal cell migration and lamellipodial dynamics. In contrast, GFP-M5Delta-expressing cells have perinuclear endocytic vesicles, produce thin interior actin and myosin bundles and contain no interior focal adhesions. In addition, these cells spread rapidly, migrate slowly and display reduced lamellipodial dynamics. Similarly, neurite outgrowth is compromised in neurons cultured from transgenic Drosophila that express M5Delta-dsRed and in neurons cultured from Drosophila that produce a tailless version of endogenous myosin V. Together, these data suggest that myosin Va overexpression induces actin bundles in vivo whereas the tailless version fails to bundle actin and disrupts cell motility.
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Movimento Celular , Citoesqueleto/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Subfragmentos de Miosina/metabolismo , Miosina Tipo V/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Vesículas Transportadoras/metabolismo , Actinas/metabolismo , Animais , Células CHO , Divisão Celular , Proliferação de Células , Forma Celular , Cricetinae , Cricetulus , Drosophila melanogaster , Endocitose , Proteínas de Fluorescência Verde/metabolismo , Imunoprecipitação , Camundongos , Proteínas Mutantes/metabolismo , Miosina Tipo II/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Environmental temperature is an important factor exerting pervasive influence on neuronal morphology and synaptic physiology. In the Drosophila brain, axonal arborization of mushroom body Kenyon cells was enhanced when flies were raised at high temperature (30 degrees C rather than 22 degrees C) for several days. Isolated embryonic neurons in culture that lacked cell-cell contacts also displayed a robust temperature-induced neurite outgrowth. This cell-autonomous effect was reflected by significantly increased high-order branching and enlarged growth cones. The temperature-induced morphological alterations were blocked by the Na+ channel blocker tetrodotoxin and a Ca2+ channel mutation but could be mimicked by raising cultures at room temperature with suppressed K+ channel activity. Physiological analyses revealed increased inward Ca2+ currents and decreased outward K+ currents, in conjunction with a distal shift in the site of action potential initiation and increased prevalence of TTX-sensitive spontaneous Ca2+ transients. Importantly, the overgrowth caused by both temperature and hyperexcitability K+ channel mutations were sensitive to genetic perturbations of cAMP metabolism. Thus, temperature acts in a cell-autonomous manner to regulate neuronal excitability and spontaneous activity. Presumably, activity-dependent Ca2+ accumulation triggers the cAMP cascade to confer the activity-dependent plasticity of neuronal excitability and growth.
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Encéfalo/crescimento & desenvolvimento , Sinalização do Cálcio/fisiologia , AMP Cíclico/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Animais , Encéfalo/citologia , Encéfalo/embriologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular/fisiologia , Membrana Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Cones de Crescimento/metabolismo , Cones de Crescimento/ultraestrutura , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Corpos Pedunculados/citologia , Corpos Pedunculados/embriologia , Corpos Pedunculados/crescimento & desenvolvimento , Mutação/genética , Neuritos/metabolismo , Neuritos/ultraestrutura , Neurônios/citologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/genética , Canais de Potássio/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , TemperaturaRESUMO
The cacophony (cac) locus in Drosophila encodes a Ca2+ channel alpha subunit, but little is known about properties of cac-mediated currents and functional consequences of cac mutations in central neurons. We found that, in Drosophila cultured neurons, Ca2+ currents were mediated predominantly by the cac channels. The cac channels contribute to low- and high-threshold, fast- and slow-inactivating types of Ca2+ currents, take part in membrane depolarization, and strongly activate Ca2+-activated K+ current [I(K(Ca))]. In cac neurons, unexpectedly, voltage-activated transient K+ current I(A) is upregulated to a level that matches I(K(Ca)) reduction, implicating a homeostatic regulation that was mimicked by chronic pharmacological blockade of Ca2+ currents in wild-type neurons. Among K+ channel transcripts, Shaker mRNA levels were preferentially increased in cac flies. However, Ca2+ current expression levels remained unaltered in several K+ channel mutants, illustrating a key role of cac in developmental regulation of Drosophila neuronal excitability.
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Canais de Cálcio/metabolismo , Proteínas de Drosophila/fisiologia , Homeostase/fisiologia , Neurônios/fisiologia , Canais de Potássio/metabolismo , Animais , Canais de Cálcio/fisiologia , Células Cultivadas , Drosophila , Canais de Potássio/fisiologiaRESUMO
Different K(+) currents participate in generating neuronal firing patterns. The Drosophila embryonic "giant" neuron culture system has facilitated current- and voltage-clamp recordings to correlate distinct excitability patterns with the underlying K(+) currents and to delineate the mutational effects of identified K(+) channels. Mutations of Sh and Shab K(+) channels removed part of inactivating I(A) and sustained I(K), respectively, and the remaining I(A) and I(K) revealed the properties of their counterparts, e.g., Shal and Shaw channels. Neuronal subsets displaying the delayed, tonic, adaptive, and damping spike patterns were characterized by different profiles of K(+) current voltage dependence and kinetics and by differential mutational effects. Shab channels regulated membrane repolarization and repetitive firing over hundreds of milliseconds, and Shab neurons showed a gradual decline in repolarization during current injection and their spike activities became limited to high-frequency, damping firing. In contrast, Sh channels acted on events within tens of milliseconds, and Sh mutations broadened spikes and reduced firing rates without eliminating any categories of firing patterns. However, removing both Sh and Shal I(A) by 4-aminopyridine converted the delayed to damping firing pattern, demonstrating their actions in regulating spike initiation. Specific blockade of Shab I(K) by quinidine mimicked the Shab phenotypes and converted tonic firing to a damping pattern. These conversions suggest a hierarchy of complexity in K(+) current interactions underlying different firing patterns. Different lineage-defined neuronal subsets, identifiable by employing the GAL4-UAS system, displayed different profiles of spike properties and K(+) current compositions, providing opportunities for mutational analysis in functionally specialized neurons.