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BACKGROUND: Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina's protocols. RESULTS: Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. CONCLUSIONS: A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results.
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Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Bordetella bronchiseptica/genética , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Genoma Bacteriano , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Análise de Sequência de DNA , Análise de Sequência de RNARESUMO
Skeletal muscle insulin resistance is considered to be the primary defect involved in type 2 diabetes mellitus (T2DM). Despite transcriptome studies in limited T2DM human subjects suggesting an association of T2DM with impaired oxidative phosphorylation in muscle, its molecular pathogenesis remains largely unknown. To identify dysregulated genes and gene networks that are associated with T2DM in human skeletal muscle, we examined expression patterns of 56,318 transcribed genes on 92 T2DM cases and 184 gender-, age- and race-matched non-diabetic controls from the Genotype-Tissue Expression (GTEx) database. RNA-Sequencing data suggest that diabetic skeletal muscle is characterized by decreased expression of genes that are related to insulin resistance (IRS2, MTOR, SLC2A4, and PPARA), carbohydrate, energy, and amino acid metabolism pathways (NDUFS1, NDUFA10, NDUFB4, NDUFB5, NDUFA5, NDUFB10, SDHB, SDHC, ATP5H, ATP5A, and ATP5J). Up-regulated genes in T2DM are mainly enriched in apoptosis pathways (TP53, GADD45A, TNFRSF10B, TP53AIP1, and PMAIP1), and notably include immune-related pathways suggestive of a response to various infectious diseases (C2, CFB, C4A, C4B, C1S, C1R, C3, HLA-DRA, HLA-DMA, HLA-DOA, and HLA-DPB1). These results confirm the essential regulation of impaired insulin signaling and oxidative phosphorylation in the muscle of T2DM patients, and provide novel molecular insights into the pathophysiological mechanisms of T2DM.
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Apoptose/imunologia , Diabetes Mellitus Tipo 2/imunologia , Resistência à Insulina/imunologia , Proteínas Musculares/imunologia , Músculo Esquelético/imunologia , Transcriptoma/imunologia , Humanos , Infecções/imunologia , Transdução de Sinais/imunologiaRESUMO
Three-dimensional (3D) brain organoids derived from human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), appear to recapitulate the brain's 3D cytoarchitectural arrangement and provide new opportunities to explore disease pathogenesis in the human brain. Human iPSC (hiPSC) reprogramming methods, combined with 3D brain organoid tools, may allow patient-derived organoids to serve as a preclinical platform to bridge the translational gap between animal models and human clinical trials. Studies using patient-derived brain organoids have already revealed novel insights into molecular and genetic mechanisms of certain complex human neurological disorders such as microcephaly, autism, and Alzheimer's disease. Furthermore, the combination of hiPSC technology and small-molecule high-throughput screening (HTS) facilitates the development of novel pharmacotherapeutic strategies, while transcriptome sequencing enables the transcriptional profiling of patient-derived brain organoids. Finally, the addition of CRISPR/Cas9 genome editing provides incredible potential for personalized cell replacement therapy with genetically corrected hiPSCs. This review describes the history and current state of 3D brain organoid differentiation strategies, a survey of applications of organoids towards studies of neurodevelopmental and neurodegenerative disorders, and the challenges associated with their use as in vitro models of neurological disorders.
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Encéfalo/patologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Doenças Neurodegenerativas/patologia , Transtornos do Neurodesenvolvimento/patologia , Organoides/fisiologia , Animais , Encéfalo/crescimento & desenvolvimento , Diferenciação Celular , Modelos Animais de Doenças , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/citologiaRESUMO
RNA sequencing (RNA-Seq), a revolutionary tool for transcriptome profiling, is becoming increasingly important for neuroscientists in studying the transcriptional landscape of the human brain. Studies using this next-generation sequencing technique have already revealed novel insights into the complexity of neurons in the human brain and pathogenesis of complex neurological diseases. In clinical neuroscience, RNA-Seq provides exciting opportunities for improving diagnosis and treatment of neurological diseases by facilitating the development of pharmacotherapies able to modulate gene expression. Furthermore, integrative whole genome sequencing and transcriptome sequencing can provide additional information for the functional role of mutated genes, prioritization of variants, and intron/exon splicing. This review describes the current state of RNA-Seq studies in neuropsychiatric disorders using post-mortem human brains, a brief survey of best practices for experimental design and sequencing data analysis, and the challenges associated with its application in the human brain.
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Encéfalo/metabolismo , Transtornos Mentais/genética , Análise de Sequência de RNA/métodos , Perfilação da Expressão Gênica/métodos , Humanos , Projetos de Pesquisa , Estatística como Assunto/métodos , Sequenciamento Completo do Genoma/métodosRESUMO
Tumor-associated macrophages (TAMs) are the major component of tumor-infiltrating leukocytes. TAMs are heterogeneous, with distinct phenotypes influenced by the microenvironment surrounding tumor tissues. Decoy receptor 3 (DcR3), a member of the TNFR superfamily, is overexpressed in tumor cells and is capable of modulating host immunity as either a neutralizing decoy receptor or an effector molecule. Upregulation of DcR3 has been observed to correlate with a poor prognosis in various cancers. However, the mechanisms underlying the DcR3-mediated tumor-promoting effect remain unclear. We previously demonstrated that DcR3 modulates macrophage activation toward an M2-like phenotype in vitro and that DcR3 downregulates MHC class II expression in TAMs via epigenetic control. To investigate whether DcR3 promotes tumor growth, CT26-DcR3 stable transfectants were established. Compared with the vector control clone, DcR3-transfectants grew faster and resulted in TAM infiltration. We further generated CD68 promoter-driven DcR3 transgenic (Tg) mice to investigate tumor growth in vivo. Compared with wild-type mice, macrophages isolated from DcR3-Tg mice displayed higher levels of IL-10, IL-1ra, Ym1, and arginase activity, whereas the expression of IL-12, TNF-α, IL-6, NO, and MHC class II was downregulated. Significantly enhanced tumor growth and spreading were observed in DcR3-Tg mice, and the enhanced tumor growth was abolished by arginase inhibitor N-ω-hydroxy-l-norarginine and histone deacetylase inhibitor sodium valproate. These results indicated that induction of TAMs is an important mechanism for DcR3-mediated tumor progression. Our findings also suggest that targeting DcR3 might help in the development of novel treatment strategies for tumors with high DcR3 expression.
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Diferenciação Celular/imunologia , Progressão da Doença , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/patologia , Membro 6b de Receptores do Fator de Necrose Tumoral/fisiologia , Regulação para Cima/imunologia , Adenocarcinoma/imunologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Feminino , Humanos , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Camundongos TransgênicosRESUMO
Diatoms, efficient carbon capture organisms, contribute to 20% of global carbon fixation and 40% of ocean primary productivity, garnering significant attention to their growth. Despite their significance, the synthesis mechanism of polyamines (PAs), especially spermidine (Spd), which are crucial for growth in various organisms, remains unexplored in diatoms. This study reveals the vital role of Spd, synthesized through the spermidine synthase (SDS)-based pathway, in the growth of the diatom Phaeodactylum tricornutum. PtSDS1 and PtSDS2 in the P. tricornutum genome were confirmed as SDS enzymes through enzyme-substrate selectivity assays. Their distinct activities are governed primarily by the Y79 active site. Overexpression of a singular gene revealed that PtSDS1, PtSDS2, and PtSAMDC from the SDS-based synthesis pathway are all situated in the cytoplasm, with no significant impact on PA content or diatom growth. Co-overexpression of PtSDS1 and PtSAMDC proved essential for elevating Spd levels, indicating multifactorial regulation. Elevated Spd content promotes diatom growth, providing a foundation for exploring PA functions and regulation in diatoms.
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Diatomáceas , Diatomáceas/genética , Diatomáceas/metabolismo , Espermidina Sintase/genética , Espermidina Sintase/metabolismo , Poliaminas/metabolismo , Vias Biossintéticas , GenomaRESUMO
Background: Chronic kidney disease (CKD) is a critical public health issue with a huge financial burden for both patients and society worldwide. Unfortunately, there are currently no efficacious therapies to prevent or delay the progression of end-stage renal disease (ESRD). Traditional Chinese medicine practices have shown that Cordyceps militaris (C. militaris) mycelia have a variety of pharmacologically useful properties, including antitumor, immunomodulation, and hepatoprotection. However, the effect of mycelial C. militaris on CKD remains unclear. Methods: Here, we investigated the effects of C. militaris mycelia on mice with CKD using four types of media: HKS, HKS with vitamin A (HKS + A), CM, and CM with vitamin A (CM + A). Results: The results at day 10 revealed that the levels of blood urea nitrogen (BUN) were significantly lower in the HKS (41%), HKS + A (41%), and CM + A (34%) groups compared with those in the corresponding control groups (nephrectomic mice). The level of serum creatinine in the HKS + A group decreased by 35% at day 10, whereas the levels in the HKS and CM + A groups decreased only by 14% and 13%, respectively, on day 30. Taken together, this is the first report using four new media (HKS, HKS + A, CM, and CM + A medium) for C. militaris mycelia. Each medium of mycelial C. militaris on CKD exhibits specific effect on BUN, serum creatinine, body weight, total protein, and uric acid. Conclusions: Taken together, this is the first report using four new media (HKS, HKS + A, CM, and CM + A medium) for C. militaris mycelia. Each medium of mycelial C. militaris on CKD exhibits specific effects on BUN, serum creatinine, body weight, total protein, and uric acid. We concluded that treatment with C. militaris mycelia cultured in HKS or CM + A medium could potentially prevent the deterioration of kidney function in mice with CKD.
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Prenatal cocaine exposure induces cytoarchitectural changes in the embryonic neocortex; however, the biological mechanisms and type of cortical neurons involved in these changes are not known. Previously, we found that neural progenitor proliferation in the neocortical ventricular zone (VZ) is inhibited by cocaine; here, we examine the changes in cortical neurogenesis and migration of glutamate and GABA neurons induced by prenatal cocaine exposure. Pregnant rats received 20 mg/kg of cocaine intraperitoneally twice at an interval of 12 h during three periods of neocortical neurogenesis. Neocortical area and distribution of developing neurons were examined by counting Tuj1+, glutamate+, or GABA+ cells in different areas of the cerebral cortex. Cocaine decreased neocortical area by reducing the size of the Tuj1+ layer, but only when administered during early periods of neocortical neurogenesis. The number of glutamatergic neurons was increased in the VZ but was decreased in the outer cortical laminae. Although the number of GABA+ neurons in the VZ of both the neocortex and ganglionic eminences was unchanged, GABA+ cells decreased in all other neocortical laminae. Tangential migration of GABA+ cells was also disrupted by cocaine. These findings suggest that in utero cocaine exposure disturbs radial migration of neocortical neurons, possibly because of decreased radial glia guiding support through enhanced differentiation of neocortical VZ progenitors. Cocaine interrupts radial migration of both glutamatergic and GABAergic neurons within the neocortex, in addition to the tangential migration of GABAergic neurons from the subcortical telecephalon. This may result in abnormal neocortical cytoarchitecture and concomitant adverse functional effects.
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Movimento Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Cocaína/farmacologia , Ácido Glutâmico/metabolismo , Neurônios/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Feminino , Imuno-Histoquímica , Neurônios/metabolismo , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Motor and cognitive decline as part of the normal aging process is linked to alterations in synaptic plasticity and reduction of adult neurogenesis in the dorsal striatum. Neuroinflammation, particularly in the form of microglial activation, is suggested to contribute to these age-associated changes. OBJECTIVE AND METHODS: To explore the molecular basis of alterations in striatal function during aging we analyzed RNA-Seq data for 117 postmortem human dorsal caudate samples and 97 putamen samples acquired through GTEx. RESULTS: Increased expression of neuroinflammatory transcripts including TREM2, MHC II molecules HLA-DMB, HLA-DQA2, HLA-DPA1, HLA-DPB1, HLA-DMA and HLA-DRA, complement genes C1QA, C1QB, CIQC and C3AR1, and MHCI molecules HLA-B and HLA-F was identified. We also identified down-regulation of transcripts involved in neurogenesis, synaptogenesis, and synaptic pruning, including DCX, CX3CL1, and CD200, and the canonical WNTs WNT7A, WNT7B, and WNT8A. The canonical WNT signaling pathway has previously been shown to mediate adult neurogenesis and synapse formation and growth. Recent findings also highlight the link between WNT/ß-catenin signaling and inflammation pathways. CONCLUSIONS: These findings suggest that age-dependent attenuation of canonical WNT signaling plays a pivotal role in regulating striatal plasticity during aging. Dysregulation of WNT/ß-catenin signaling via astrocyte-microglial interactions is suggested to be a novel mechanism that drives the decline of striatal neurogenesis and altered synaptic connectivity and plasticity, leading to a subsequent decrease in motor and cognitive performance with age. These findings may aid in the development of therapies targeting WNT/ß-catenin signaling to combat cognitive and motor impairments associated with aging.
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Doenças Neuroinflamatórias , Via de Sinalização Wnt , Perfilação da Expressão Gênica , Humanos , Glicoproteínas de Membrana , Neurogênese/fisiologia , Plasticidade Neuronal/genética , Receptores Imunológicos , Via de Sinalização Wnt/genéticaRESUMO
Decoy receptor 3 (DcR3) is a member of the TNF receptor superfamily and is up-regulated in tumors originating from a diversity of lineages. DcR3 is capable of promoting angiogenesis, inducing dendritic cell apoptosis, and modulating macrophage differentiation. Since tumor-associated macrophages (TAMs) are the major infiltrating leukocytes in most malignant tumors, we used microarray technology to investigate whether DcR3 contributes to the development of TAMs. Among the DcR3-modulated genes expressed by TAMs, those that encode proteins involved in MHC class II (MHC-II)-dependent antigen presentation were down-regulated substantially, together with the master regulator of MHC-II expression (the class II transactivator, CIITA). The ERK- and JNK-induced deacetylation of histones associated with the CIITA promoters was responsible for DcR3-mediated down-regulation of MHC-II expression. Furthermore, the expression level of DcR3 in cancer cells correlated inversely with HLA-DR levels on TAMs and with the overall survival time of pancreatic cancer patients. The role of DcR3 in the development of TAMs was further confirmed using transgenic mice overexpressing DcR3. This elucidates the molecular mechanism of impaired MHC-II-mediated antigen presentation by TAMs, and raises the possibility that subversion of TAM-induced immunosuppression via inhibition of DcR3 expression might represent a target for the design of new therapeutics.
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Epigênese Genética , Genes MHC da Classe II/genética , Macrófagos/metabolismo , Neoplasias/imunologia , Proteínas Nucleares/genética , Membro 6b de Receptores do Fator de Necrose Tumoral/fisiologia , Transativadores/genética , Animais , Apresentação de Antígeno/genética , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras GenéticasRESUMO
Cordyceps militaris (C. militaris) is a fungus with a long history of widespread use in folk medicine, and its biological and medicinal functions are well studied. A crucial pharmacological effect of C. militaris is immunomodulation. In this review, we catalog the immunomodulatory effects of different extracts of C. militaris, namely total extracts, polysaccharides and cordycepin. Total extracts obtained using water or 50% ethyl alcohol and polysaccharides from C. militaris were discovered to tend to promote type 1 immunity, whereas total extracts obtained using 70-80% ethyl alcohol and cordycepin from C. militaris were more likely to promote type 2 immunity. This article is the first to classify the immunomodulatory effects of different extracts of C. militaris. In addition, we discovered a relationship between different segments or extracts and differing types of immunity. This review can provide the readers a comprehensive understanding on the immunomodulatory effects of the precious folk medicine and guidance on its use for both health people and those with an immunodeficiency.
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To evaluate the expression of immune checkpoint genes, their concordance with expression of IFNγ, and to identify potential novel ICP related genes (ICPRG) in colorectal cancer (CRC), the biological connectivity of six well documented ("classical") ICPs (CTLA4, PD1, PDL1, Tim3, IDO1, and LAG3) with IFNγ and its co-expressed genes was examined by NGS in 79 CRC/healthy colon tissue pairs. Identification of novel IFNγ- induced molecules with potential ICP activity was also sought. In our study, the six classical ICPs were statistically upregulated and correlated with IFNγ, CD8A, CD8B, CD4, and 180 additional immunologically related genes in IFNγ positive (FPKM > 1) tumors. By ICP co-expression analysis, we also identified three IFNγ-induced genes [(IFNγ-inducible lysosomal thiol reductase (IFI30), guanylate binding protein1 (GBP1), and guanylate binding protein 4 (GBP4)] as potential novel ICPRGs. These three genes were upregulated in tumor compared to normal tissues in IFNγ positive tumors, co-expressed with CD8A and had relatively high abundance (average FPKM = 362, 51, and 25, respectively), compared to the abundance of the 5 well-defined ICPs (Tim3, LAG3, PDL1, CTLA4, PD1; average FPKM = 10, 9, 6, 6, and 2, respectively), although IDO1 is expressed at comparably high levels (FPKM = 39). We extended our evaluation by querying the TCGA database which revealed the commonality of IFNγ dependent expression of the three potential ICPRGs in 638 CRCs, 103 skin cutaneous melanomas (SKCM), 1105 breast cancers (BC), 184 esophageal cancers (ESC), 416 stomach cancers (STC), and 501 lung squamous carcinomas (LUSC). In terms of prognosis, based on Pathology Atlas data, correlation of GBP1 and GBP4, but not IFI30, with 5-year survival rate was favorable in CRC, BC, SKCM, and STC. Thus, further studies defining the role of IFI30, GBP1, and GBP4 in CRC are warranted.
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Neoplasias da Mama/genética , Colo/fisiologia , Neoplasias Colorretais/genética , Interferon gama/metabolismo , Melanoma/genética , Neoplasias Cutâneas/genética , Neoplasias Gástricas/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/mortalidade , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/mortalidade , Feminino , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas de Checkpoint Imunológico/genética , Masculino , Melanoma/imunologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Prognóstico , Neoplasias Cutâneas/imunologia , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/mortalidade , Análise de Sobrevida , Melanoma Maligno CutâneoRESUMO
Producing dopaminergic (DA) neurons is a major goal of human embryonic stem cell (hESC) research. DA neurons can be differentiated from hESC by coculture with the mouse PA6 stromal cell line; this differentiation-inducing effect is termed stromal-derived inducing activity (SDIA). The molecular and biochemical nature of SDIA is, however, unknown. Various studies have suggested that SDIA involves either a fixation-resistant component located on the PA6 cell surface or factors secreted into the medium by PA6 cells. To address this question, hESC were cocultured with PA6 cells for 12 days and then further differentiated with sonic hedgehog homolog, fibroblast growth factor-8, and glial cell line-derived neurotrophic factor. After 18 days, 34% of cells were tyrosine hydroxylase (TH)+. When PA6 cells were fixed or irradiated, the number of TH+ cells was decreased by threefold, whereas mitomycin-c treatment of feeder cells decreased the number of TH+ cells by 32%. The neural-inducing effect of PA6 cells, as monitored by beta-III-tubulin expression, was minimally affected by mitomycin-c treatment or fixation but was decreased 50% by irradiation. Medium conditioned by PA6 cells was ineffective in differentiating TH+ cells when used alone. Conditioned medium combined with heparin and/or fixed PA6 cells produced TH+ cell differentiation, although less effectively than PA6 cell coculture. Thus, PA6 cell surface activity is required for neural differentiation of hESC, but secreted factors are required for the specific DA neuron-inducing effect. Disclosure of potential conflicts of interest is found at the end of this article.
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Diferenciação Celular/fisiologia , Dopamina/fisiologia , Células-Tronco Embrionárias/citologia , Neurônios/citologia , Neurônios/fisiologia , Células Estromais/citologia , Animais , Técnicas de Cultura de Células/métodos , Técnicas de Cocultura , Meios de Cultura , Células-Tronco Embrionárias/fisiologia , Humanos , Imuno-Histoquímica , Camundongos , Células Estromais/fisiologiaRESUMO
Prenatal substance exposure is a growing public health concern worldwide. Although the opioid crisis remains one of the most prevalent addiction problems in our society, abuse of cocaine, methamphetamines, and other illicit drugs, particularly amongst pregnant women, are nonetheless significant and widespread. Evidence demonstrates prenatal drug exposure can affect fetal brain development and thus can have long-lasting impact on neurobehavioral and cognitive performance later in life. In this review, we highlight research examining the most prevalent drugs of abuse and their effects on brain development with a focus on endoplasmic reticulum stress and oxidative stress signaling pathways. A thorough exploration of drug-induced cellular stress mechanisms during prenatal brain development may provide insight into therapeutic interventions to combat effects of prenatal drug exposure.
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BACKGROUND: Prenatal exposure of the developing brain to cocaine causes morphological and behavioral abnormalities. Recent studies indicate that cocaine-induced proliferation inhibition and/or apoptosis in neural progenitor cells may play a pivotal role in causing these abnormalities. To understand the molecular mechanism through which cocaine inhibits cell proliferation in neural progenitors, we sought to identify the molecules that are responsible for mediating the effect of cocaine on cell cycle regulation. METHODS AND FINDINGS: Microarray analysis followed by quantitative real-time reverse transcription PCR was used to screen cocaine-responsive and cell cycle-related genes in a neural progenitor cell line where cocaine exposure caused a robust anti-proliferative effect by interfering with the G1-to-S transition. Cyclin A2, among genes related to the G1-to-S cell cycle transition, was most strongly down-regulated by cocaine. Down-regulation of cyclin A was also found in cocaine-treated human primary neural and A2B5+ progenitor cells, as well as in rat fetal brains exposed to cocaine in utero. Reversing cyclin A down-regulation by gene transfer counteracted the proliferation inhibition caused by cocaine. Further, we found that cocaine-induced accumulation of reactive oxygen species, which involves N-oxidation of cocaine via cytochrome P450, promotes cyclin A down-regulation by causing an endoplasmic reticulum (ER) stress response, as indicated by increased phosphorylation of eIF2alpha and expression of ATF4. In the developing rat brain, the P450 inhibitor cimetidine counteracted cocaine-induced inhibition of neural progenitor cell proliferation as well as down-regulation of cyclin A. CONCLUSIONS: Our results demonstrate that down-regulation of cyclin A underlies cocaine-induced proliferation inhibition in neural progenitors. The down-regulation of cyclin A is initiated by N-oxidative metabolism of cocaine and consequent ER stress. Inhibition of cocaine N-oxidative metabolism by P450 inhibitors may provide a preventive strategy for counteracting the adverse effects of cocaine on fetal brain development.
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Cocaína/farmacologia , Neurônios/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Cimetidina/farmacologia , Ciclina A/genética , Ciclina A/metabolismo , Ciclina A2 , Regulação para Baixo , Feminino , Humanos , Fosforilação , Gravidez , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
BACKGROUND AND PURPOSE: Human embryonic stem cells (hESC) are considered a renewable source of dopamine producing neurons, and are of particular interest for their potential clinical use in Parkinson's disease. In this study, we characterized human dopaminergic neurons generated by stromal-derived inducing activity (SDIA) from BG01V2, a strain of human embryonic stem cell line, BG01, characterized by a chromosome 17 trisomy. Similar chromosomal changes have been repeatedly observed in hESC cultures in different laboratories, indicating the importance of chromosome 17 for growth and adaptation of hESC to culture. METHODS: We investigated in vitro proliferation of differentiating cells using a BrDU incorporation assay, and monitored the cell population in long term cultures. Despite the cytogenetic abnormality, TH+ neurons were postmitotic at all stages of differentiation. After 30 days of differentiation, cell division ceased in 91% of the overall population of cells in the culture, indicating intact cell cycle regulation. RESULTS: Expression of midbrain specific marker genes (Otx2, Pax5, Msx-1) showed differentiation of hESC-derived neural progenitor cells into midbrain specific dopamine neurons. These neurons expressed the dopamine transporter (DAT), and displayed functional DAT activity and electrical excitability. CONCLUSIONS: TH+ cells derived from the BG01V2 hESC line using SDIA are postmitotic and have functional characteristics of normal dopaminergic neurons.
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Diferenciação Celular/fisiologia , Dopamina/metabolismo , Células-Tronco Embrionárias/fisiologia , Neurônios/fisiologia , Actinas/metabolismo , Bromodesoxiuridina/metabolismo , Linhagem Celular , Proliferação de Células , Cromossomos Humanos Par 17 , Cocaína/análogos & derivados , Cocaína/farmacocinética , Técnicas de Cocultura , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Humanos , Potenciais da Membrana/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Otx/metabolismo , Técnicas de Patch-Clamp/métodos , Ligação Proteica/efeitos dos fármacos , Fatores de Tempo , Trítio/farmacocinética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The polysaccharides in many plants are attracting worldwide attention because of their biological activities and medical properties, such as anti-viral, anti-oxidative, antichronic inflammation, anti-hypertensive, immunomodulation, and neuron-protective effects, as well as anti-tumor activity. Denodrobium species, a genus of the family orchidaceae, have been used as herbal medicines for hundreds of years in China due to their pharmacological effects. These effects include nourishing the Yin, supplementing the stomach, increasing body fluids, and clearing heat. Recently, numerous researchers have investigated possible active compounds in Denodrobium species, such as lectins, phenanthrenes, alkaloids, trigonopol A, and polysaccharides. Unlike those of other plants, the biological effects of polysaccharides in Dendrobium are a novel research field. In this review, we focus on these novel findings to give readers an overall picture of the intriguing therapeutic potential of polysaccharides in Dendrobium, especially those of the four commonly-used Denodrobium species: D. huoshanense, D. offininale, D. nobile, and D. chrysotoxum.
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Polissacarídeos/farmacologia , Animais , Linhagem Celular Tumoral , Dendrobium/classificação , HumanosRESUMO
Dengue fever has become an imminent threat to international public health because of global warming and climate change. The World Health Organization proclaimed that more than 50% of the world's population is at risk of dengue virus (DENV) infection. Therefore, developing a clinically approved vaccine and effective therapeutic remedy for treating dengue fever is imperative. Peptide drug development has become a novel pharmaceutical research field. This article reviews various peptidesbased antimicrobial agents targeting three pathways involved in the DENV lifecycle. Specifically, they are peptide vaccines from immunomodulation, peptide drugs that inhibit virus entry, and peptide drugs that interfere with viral replication. Many antiviral peptide studies against DENV have been conducted in animal model trials, and progression to clinical trials for these promising peptide drugs is anticipated.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antivirais/farmacologia , Vacinas contra Dengue/imunologia , Vírus da Dengue/efeitos dos fármacos , Dengue/tratamento farmacológico , Peptídeos/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Antivirais/uso terapêutico , Dengue/imunologia , Dengue/prevenção & controle , Descoberta de Drogas , Humanos , Terapia de Alvo Molecular , Peptídeos/uso terapêutico , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/imunologia , Replicação Viral/efeitos dos fármacosRESUMO
Skin disorders are among most common complications associated with type 2 diabetes mellitus (T2DM). Although T2DM patients are known to have increased risk of infections and other T2DM-related skin disorders, their molecular mechanisms are largely unknown. This study aims to identify dysregulated genes and gene networks that are associated with T2DM in human skin. We compared the expression profiles of 56,318 transcribed genes on 74 T2DM cases and 148 gender- age-, and race-matched non-diabetes controls from the Genotype-Tissue Expression (GTEx) database. RNA-Sequencing data indicates that diabetic skin is characterized by increased expression of genes that are related to immune responses (CCL20, CXCL9, CXCL10, CXCL11, CXCL13, and CCL18), JAK/STAT signaling pathway (JAK3, STAT1, and STAT2), tumor necrosis factor superfamily (TNFSF10 and TNFSF15), and infectious disease pathways (OAS1, OAS2, OAS3, and IFIH1). Genes in cell adhesion molecules pathway (NCAM1 and L1CAM) and collagen family (PCOLCE2 and COL9A3) are downregulated, suggesting structural changes in the skin of T2DM. For the first time, to the best of our knowledge, this pioneer analytic study reports comprehensive unbiased gene expression changes and dysregulated pathways in the non-diseased skin of T2DM patients. This comprehensive understanding derived from whole-genome expression profiles could advance our knowledge in determining molecular targets for the prevention and treatment of T2DM-associated skin disorders.
Assuntos
Diabetes Mellitus Tipo 2/genética , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Pele/imunologia , Sequenciamento Completo do Genoma/métodos , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/imunologia , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Análise de Sequência de RNA , Pele/químicaRESUMO
Because of unavoidable confounding variables in the direct study of human subjects, it has been difficult to unravel the effects of prenatal cocaine exposure on the human fetal brain, as well as the cellular and biochemical mechanisms involved. Here, we propose a novel approach using a human pluripotent stem cell (hPSC)-based 3D neocortical organoid model. This model retains essential features of human neocortical development by encompassing a single self-organized neocortical structure, without including an animal-derived gelatinous matrix. We reported previously that prenatal cocaine exposure to rats during the most active period of neural progenitor proliferation induces cytoarchitectural changes in the embryonic neocortex. We also identified a role of CYP450 and consequent oxidative ER stress signaling in these effects. However, because of differences between humans and rodents in neocorticogenesis and brain CYP metabolism, translation of the research findings from the rodent model to human brain development is uncertain. Using hPSC 3D neocortical organoids, we demonstrate that the effects of cocaine are mediated through CYP3A5-induced generation of reactive oxygen species, inhibition of neocortical progenitor cell proliferation, induction of premature neuronal differentiation, and interruption of neural tissue development. Furthermore, knockdown of CYP3A5 reversed these cocaine-induced pathological phenotypes, suggesting CYP3A5 as a therapeutic target to mitigate the deleterious neurodevelopmental effects of prenatal cocaine exposure in humans. Moreover, 3D organoid methodology provides an innovative platform for identifying adverse effects of abused psychostimulants and pharmaceutical agents, and can be adapted for use in neurodevelopmental disorders with genetic etiologies.