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Brain insulin signaling controls peripheral energy metabolism and plays a key role in the regulation of mood and cognition. Epidemiological studies have indicated a strong connection between type 2 diabetes (T2D) and neurodegenerative disorders, especially Alzheimer's disease (AD), linked via dysregulation of insulin signaling, i.e., insulin resistance. While most studies have focused on neurons, here, we aim to understand the role of insulin signaling in astrocytes, a glial cell type highly implicated in AD pathology and AD progression. To this end, we created a mouse model by crossing 5xFAD transgenic mice, a well-recognized AD mouse model that expresses five familial AD mutations, with mice carrying a selective, inducible insulin receptor (IR) knockout in astrocytes (iGIRKO). We show that by age 6 mo, iGIRKO/5xFAD mice exhibited greater alterations in nesting, Y-maze performance, and fear response than those of mice with the 5xFAD transgenes alone. This was associated with increased Tau (T231) phosphorylation, increased Aß plaque size, and increased association of astrocytes with plaques in the cerebral cortex as assessed using tissue CLARITY of the brain in the iGIRKO/5xFAD mice. Mechanistically, in vitro knockout of IR in primary astrocytes resulted in loss of insulin signaling, reduced ATP production and glycolic capacity, and impaired Aß uptake both in the basal and insulin-stimulated states. Thus, insulin signaling in astrocytes plays an important role in the control of Aß uptake, thereby contributing to AD pathology, and highlighting the potential importance of targeting insulin signaling in astrocytes as a site for therapeutics for patients with T2D and AD.
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Doença de Alzheimer , Diabetes Mellitus Tipo 2 , Camundongos , Animais , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Astrócitos/metabolismo , Insulina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Camundongos Transgênicos , Fenótipo , Modelos Animais de DoençasRESUMO
GABAergic interneurons are poised with the capacity to shape circuit output via inhibitory gating. How early in the development of medial vestibular nucleus (MVN) are GABAergic neurons recruited for feedforward shaping of outputs to higher centers for spatial navigation? The role of early GABAergic transmission in assembling vestibular circuits for spatial navigation was explored by neonatal perturbation. Immunohistochemistry and confocal imaging were utilized to reveal the expression of parvalbumin (PV)-expressing MVN neurons and their perineuronal nets. Whole-cell patch-clamp recording, coupled with optogenetics, was conducted in vitro to examine the synaptic function of MVN circuitry. Chemogenetic targeting strategy was also employed in vivo to manipulate neuronal activity during navigational tests. We found in rats a neonatal critical period before postnatal day (P) 8 in which competitive antagonization of GABAergic transmission in the MVN retarded maturation of inhibitory neurotransmission, as evidenced by deranged developmental trajectory for excitation/inhibition ratio and an extended period of critical period-like plasticity in GABAergic transmission. Despite increased number of PV-expressing GABAergic interneurons in the MVN, optogenetic-coupled patch-clamp recording indicated null-recruitment of these neurons in tuning outputs along the ascending vestibular pathway. Such perturbation not only offset output dynamics of ascending MVN output neurons, but was further accompanied by impaired vestibular-dependent navigation in adulthood. The same perturbations were however non-consequential when applied after P8. Results highlight neonatal GABAergic transmission as key to establishing feedforward output dynamics to higher brain centers for spatial cognition and navigation.
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Navegação Espacial , Ratos , Animais , Interneurônios , Transmissão Sináptica , Núcleos Vestibulares/metabolismo , Neurônios GABAérgicosRESUMO
Hepatocellular carcinoma (HCC) is the fastest-growing cause of cancer-related deaths worldwide. Chronic inflammation and fibrosis are the greatest risk factors for the development of HCC. Although the cell of origin for HCC is uncertain, many theories believe this cancer may arise from liver progenitor cells or stem cells. Here, we describe the activation of hepatic stem cells that overexpress the cholecystokinin-B receptor (CCK-BR) after liver injury with either a DDC diet (0.1% 3, 5-diethoxy-carbonyl 1,4-dihydrocollidine) or a NASH-inducing CDE diet (choline-deficient ethionine) in murine models. Pharmacologic blockade of the CCK-BR with a receptor antagonist proglumide or knockout of the CCK-BR in genetically engineered mice during the injury diet reduces the expression of hepatic stem cells and prevents the formation of three-dimensional tumorspheres in culture. RNA sequencing of livers from DDC-fed mice treated with proglumide or DDC-fed CCK-BR knockout mice showed downregulation of differentially expressed genes involved in cell proliferation and oncogenesis and upregulation of tumor suppressor genes compared with controls. Inhibition of the CCK-BR decreases hepatic transaminases, fibrosis, cytokine expression, and alters the hepatic immune cell signature rendering the liver microenvironment less oncogenic. Furthermore, proglumide hastened recovery after liver injury by reversing fibrosis and improving markers of synthetic function. Proglumide is an older drug that is orally bioavailable and being repurposed for liver conditions. These findings support a promising therapeutic intervention applicable to patients to prevent the development of HCC and decrease hepatic fibrosis.NEW & NOTEWORTHY This investigation identified a novel pathway involving the activation of hepatic stem cells and liver oncogenesis. Receptor blockade or genetic disruption of the cholecystokinin-B receptor (CCK-BR) signaling pathway decreased the activation and proliferation of hepatic stem cells after liver injury without eliminating the regenerative capacity of healthy hepatocytes.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Camundongos , Animais , Receptor de Colecistocinina B/genética , Receptor de Colecistocinina B/metabolismo , Carcinoma Hepatocelular/patologia , Proglumida/farmacologia , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Fibrose , Células-Tronco/metabolismo , Carcinogênese/metabolismo , Transformação Celular Neoplásica/metabolismo , Colecistocinina/metabolismo , Microambiente TumoralRESUMO
Long-term continuous imaging of endogenous HClO burst is of great importance for the elucidation of various physiological or pathological processes. However, most of the currently reported HClO probes have failed to achieve this goal due to their insufficient photobleaching resistance under a laser source. Herein, a highly stable ratiometric probe, HFTC-HClO 1, which is capable of continuously monitoring endogenous HClO burst over a long period of time, has been judiciously developed. Briefly, the de novo development of HFTC-HClO 1 mainly involved three main steps: (1) novel coumarins (HFTC 1-5) were designed and synthesized; (2) the most stable scaffold, HFTC 3, was selected through dye screening and cell imaging validation; and (3) based on HFTC 3, three candidate HClO probes were constructed, and HFTC-HClO 1 was finally selected due to its superior sensing properties toward HClO. Furthermore, HFTC-HClO 1 can quantitatively measure HClO levels in various real samples with excellent recovery (>90.4%), and the use of HFTC-HClO 1-coated test strips for qualitative analysis of HClO in real samples was also achieved. In addition, the application of HFTC-HClO 1 for long-term continuous monitoring of intracellular HClO burst was successfully demonstrated. Significantly, HFTC-HClO 1 was able to visualize HClO generated in the rheumatoid arthritis mouse model.
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Corantes Fluorescentes , Ácido Hipocloroso , Camundongos , Animais , Ácido Hipocloroso/análise , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , CumarínicosRESUMO
BACKGROUND: Serum uric acid (SUA) is an important pathogenetic and prognostic factor for heart failure (HF). Gender differences are apparent in HF. Furthermore, gender differences also exist in the association between SUA and prognosis in various cardiovascular diseases. However, the gender difference for SUA in the prediction of long-term prognosis in HF is still ambiguous. METHODS: A total of 1593 HF patients (897 men, 696 women) from the National Health and Nutrition Examination Survey (NHANES) 1999-2018 cycle were enrolled in our final analysis. Participants were categorized according to gender-specific SUA tertile. We assessed the association between SUA and long-term prognosis of HF patients, defined as all-cause mortality and cardiovascular mortality, in different genders via Kaplan-Meier curve analysis, Cox proportional hazard model, and Fine-Gray competing risk model. The restricted cubic spline (RCS) was performed to investigate the dose-response relationship between SUA and outcomes. RESULTS: Gender differences exist in demographic characteristics, clinical parameters, laboratory tests, and medication of HF patients. After a median follow-up of 127 months (95% CI 120-134 months), there were 853 all-cause deaths (493 events in men, 360 events in women) and 361 cardiovascular deaths (206 events in men, 155 events in women). Kaplan-Meier analysis showed that SUA had gender difference in the prediction of cardiovascular mortality (Log-rank p < 0.001, for male, Log-rank p = 0.150, for female), but not in all-cause mortality. Multivariate Cox regression analysis revealed that elevated SUA levels were associated with higher all-cause mortality and cardiovascular mortality in men (HR 1.11, 95% CI 1.05-1.18, p < 0.001, for all-cause death; HR 1.18, 95% CI 1.09-1.28, p < 0.001, for cardiovascular death), but not in women (HR 1.05, 95% CI 0.98-1.12, p = 0.186, for all-cause death; HR 1.01, 95% CI 0.91-1.12, p = 0.902, for cardiovascular death). Even using non-cardiovascular death as a competitive risk, adjusted Fine-Gray model also illustrated that SUA was an independent predictor of cardiovascular death in men (SHR 1.17, 95% CI 1.08-1.27, p < 0.001), but not in women (SHR 0.98, 95% CI 0.87 - 1.10, p = 0.690). CONCLUSIONS: Gender differences in the association between SUA and long-term prognosis of HF existed. SUA was an independent prognostic predictor for long-term outcomes of HF in men, but not in women.
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Doenças Cardiovasculares , Insuficiência Cardíaca , Humanos , Masculino , Feminino , Ácido Úrico , Fatores Sexuais , Inquéritos Nutricionais , Fatores de Risco , Prognóstico , Insuficiência Cardíaca/tratamento farmacológicoRESUMO
After double fertilization, zygotic embryogenesis initiates a new life cycle, and stem cell homeostasis in the shoot apical meristem (SAM) and root apical meristem (RAM) allows plants to produce new tissues and organs continuously. Here, we report that mutations in DEAD-BOX RNA HELICASE 27 (RH27) affect zygote division and stem cell homeostasis in Arabidopsis (Arabidopsis thaliana). The strong mutant allele rh27-1 caused a zygote-lethal phenotype, while the weak mutant allele rh27-2 led to minor defects in embryogenesis and severely compromised stem cell homeostasis in the SAM and RAM. RH27 is expressed in embryos from the zygote stage, and in both the SAM and RAM, and RH27 is a nucleus-localized protein. The expression levels of genes related to stem cell homeostasis were elevated in rh27-2 plants, alongside down-regulation of their regulatory microRNAs (miRNAs). Further analyses of rh27-2 plants revealed reduced levels of a large subset of miRNAs and their pri-miRNAs in shoot apices and root tips. In addition, biochemical studies showed that RH27 associates with pri-miRNAs and interacts with miRNA-biogenesis components, including DAWDLE, HYPONASTIC LEAVES 1, and SERRATE. Therefore, we propose that RH27 is a component of the microprocessor complex and is critical for zygote division and stem cell homeostasis.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , MicroRNAs/metabolismo , Zigoto/metabolismoRESUMO
SET domain-containing 5 (SETD5), a member of protein lysine methyltransferase family, is expressed in multiple cancers, making it potential therapeutic targets. However, the role of SETD5 in colorectal cancer remains largely unknown. The expression of SETD5 in the 30 pairs colorectal cancer tissues samples and cell lines were determined by qRT-PCR. The functions of SETD5 was detected by knocked-down or overexpression in colorectal cancer cell lines SW480 and HCT116 cells. Cell proliferative activity, cell death, and stemness characteristics were assessed. BEZ235, a PI3K/AKT/mTOR pathway inhibitor, was used to perform rescue experiment to analyze whether SETD5 exerted its effects through activating PI3K/AKT/mTOR pathway. SETD5 was substantially upregulated in colorectal cancer, and correlated to metastasis and clinical stage of patients. Knockdown of SETD5 inhibited SW480 and HCT116 cell growth, as evidenced by the inhibition of cell viability and clone-forming. Moreover, Knockdown of SETD5 suppressed the capability of tumor sphere formation of SW480 and HCT116 cells, and reduced the expression of stemness-related proteins Nanog and Sox2. Further western blot analysis revealed that SETD5 knockdown inhibited the phosphorylation of proteins associated with the PI3K/AKT/mTOR pathway. In contrast, overexpression of SETD5 exerted the opposite effects. Mechanistically, by blocking PI3K/AKT/mTOR pathway with BEZ235, the effects of SETD5 overexpression on cell viability and Nanog and Sox2 protein expression were reversed. Our results substantiated that SETD5 functioned as an oncogene by promoting cell growth and stemness in colorectal cancer cells through activating the PI3K/AKT/mTOR signaling pathway.
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Rhodamines have emerged as a useful class of dye for bioimaging. However, intrinsic issues such as short emission wavelengths and small Stokes shifts limit their widespread applications in living systems. By taking advantage of the homoadamantane-fused tetrahydroquinoxaline (HFT) moiety as an electron donor, we developed a new class of asymmetric NIR rhodamine dyes, NNR1-7. These new dyes retained ideal photophysical properties from the classical rhodamine scaffold and showed large Stokes shifts (>80 nm) with improved chemo/photostability. We found that NNR1-7 specifically target cellular mitochondria with superior photobleaching resistance and improved tolerance for cell fixation compared to commercial mitochondria trackers. Based on NNR4, a novel NIR pH sensor (NNR4M) was also constructed and successfully applied for real-time monitoring of variations in lysosomal pH. We envision this design strategy would find broad applications in the development of highly stable NIR dyes with a large Stokes shift.
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Elétrons , Corantes Fluorescentes , Rodaminas/química , Corantes Fluorescentes/química , LisossomosRESUMO
BACKGROUND: The number of grains per panicle is an important factor in determining rice yield. The DST-OsCKX2 module has been demonstrated to regulate panicle development in rice by controlling cytokinin content. However, to date, how the function of DST-OsCKX2 module is regulated during panicle development remains obscure. RESULT: In this study, the ABNORMAL PANICLE 1 (ABP1), a severely allele of FRIZZY PANICLE (FZP), exhibits abnormal spikelets morphology. We show that FZP can repress the expression of DST via directly binding to its promotor. Consistently, the expression level of OsCKX2 increased and the cytokinin content decreased in the fzp mutant, suggesting that the FZP acts upstream of the DST-OsCKX2 to maintain cytokinin homeostasis in the inflorescence meristem. CONCLUSIONS: Our results indicate that FZP plays an important role in regulating spikelet development and grain number through mediating cytokinin metabolism.
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Oryza , Oryza/metabolismo , Inflorescência/genética , Citocininas/metabolismo , Grão Comestível/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Elaborate management on bubble shape and transportations depends on the balance between multiple physical behaviors for two-phase flow with Marangoni stress and the interface mass transfer. In this paper, a new model combining PFLBM (phase-field lattice Boltzmann method) and FDM ( finite-difference method) coupling with the ghost-cell method was built. The PFLBM-FDM was validated for the high accuracy, less computational cost, and low mass loss compared to other methods. Based on the PFLBM-FDM, a surfactant-laden bubble deformed and transported in a laminar Couette flow was investigated. The deformation ratio and transportation velocity were explored with different density ratios, surface tensions, shear velocities, and diffusion coefficients. The numerical results showed that the equilibrium state of the bubble deformation was decided only by the dimensionless numbers when the Sh number was higher than 100. Moreover, the transportation velocity of the bubble can be controlled by the balance between the Marangoni stress and shear velocity. When the Sh is lower than 100, the Marangoni stress from the surfactant is not a long-range force, which only works at the early flow. Otherwise, the Marangoni stress will be a long-range force that provides a persistent force to accelerate the bubble by â¼10%. Increasing ReH will further intensify the effect. Based on all the data, a correlation of the bubble deformation including with the densities of two fluids was obtained and the error range is less 5%.
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RATIONALE: The NLRP3 (NLR [NOD-like receptor] family, pyrin domain containing 3) inflammasome is an important driver of atherosclerosis. Our previous study shows that chaperone-mediated autophagy (CMA), one of the main lysosomal degradative process, has a regulatory role in lipid metabolism of macrophages. However, whether the NLRP3 inflammasome is regulated by CMA, and the role of CMA in atherosclerosis remains unclear. OBJECTIVE: To determine the role of CMA in the regulation of NLRP3 inflammasome and atherosclerosis. METHODS AND RESULTS: The expression of CMA marker, LAMP-2A (lysosome-associated membrane protein type 2A), was first analyzed in ApoE-/- mouse aortas and human coronary atherosclerotic plaques, and a significant downregulation of LAMP-2A in advanced atherosclerosis in both mice and humans was observed. To selectively block CMA, we generated macrophage-specific conditional LAMP-2A knockout mouse strains in C57BL/6 mice and ApoE-/- mice. Deletion of macrophage LAMP-2A accelerated atherosclerotic lesion formation in the aortic root and the whole aorta in ApoE-/- mice. Mechanistically, LAMP-2A deficiency promoted NLRP3 inflammasome activation and subsequent release of mature IL (interleukin)-1ß in macrophages and atherosclerotic plaques. Furthermore, gain-of-function studies verified that restoration of LAMP-2A levels in LAMP-2A-deficient macrophages greatly attenuated NLRP3 inflammasome activation. Importantly, we identified the NLRP3 protein as a CMA substrate and demonstrated that LAMP-2A deficiency did not affect the NLRP3 mRNA levels but hindered degradation of the NLRP3 protein through CMA pathway. CONCLUSIONS: CMA function becomes impaired during the progression of atherosclerosis, which increases NLRP3 inflammasome activation and secretion of IL-1ß, promoting vascular inflammation and atherosclerosis progression. Our study unveils a new mechanism by which NLRP3 inflammasome is regulated in macrophages and atherosclerosis, thus providing a new insight into the role of autophagy-lysosomal pathway in atherosclerosis. Pharmacological activation of CMA may provide a novel therapeutic strategy for atherosclerosis and other NLRP3 inflammasome/IL-1ß-driven diseases.
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Aterosclerose/metabolismo , Autofagia , Inflamassomos/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Interleucina-1beta/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genéticaRESUMO
KEY MESSAGE: OsVP1 and Sdr4 play an important role in regulating seed dormancy that involved in multiple metabolism and regulatory pathways. Seed dormancy and germination are critical agricultural traits influencing rice grain yield. Although there are some genes have identified previously, the comprehensive understanding based on transcriptome is still deficient. In this study, we generated mutants of two representative regulators of seed germination, Oryza sativa Viviparous1 (OsVP1) and Seed dormancy 4 (Sdr4), by CRISPR/Cas9 approach and named them cr-osvp1 and cr-sdr4. The weakened dormancy of mutants indicated that the functions of OsVP1 and Sdr4 are required for normal early seed dormancy. There were 4157 and 8285 differentially expressed genes (DEGs) were identified in cr-osvp1 vs. NIP and cr-sdr4 vs. NIP groups, respectively, with a large number of overlapped DEGs between two groups. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of common DEGs in two groups showed that genes related to carbohydrate metabolic, nucleoside metabolic, amylase activity and plant hormone signal transduction were involved in the dormancy regulation. These results suggest that OsVP1 and Sdr4 play an important role in regulating seed dormancy by multiple metabolism and regulatory pathways. The systematic analysis of the transcriptional level changes provides theoretical basis for the research of seed dormancy and germination in rice.
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Oryza , Dormência de Plantas , Dormência de Plantas/genética , Oryza/genética , Oryza/metabolismo , Germinação/genética , Perfilação da Expressão Gênica , Reguladores de Crescimento de Plantas/metabolismo , Transcriptoma/genética , Sementes/genética , Sementes/metabolismo , Regulação da Expressão Gênica de Plantas/genéticaRESUMO
The aim of this study is to investigate the role of CDGSH iron-sulfur domain 2 (CISD2) in colorectal cancer (CRC). The purpose of this study was to investigate the role of CDGSH iron-sulfur domain 2 (CISD2) in colorectal cancer (CRC) progression. The expression of CISD2 in CRC cell lines was measured by western blotting. Functional assays including MTT assays and colony formation assays were performed to explore the role of CISD2 in regulating tumor growth. Flow cytometry analysis was used to examine the percentage of apoptotic CRC cells. Expression of apoptosis-related gene, autophagy-related markers, and the protein included in Wnt/ß-Catenin signaling was also determined by western blotting. The in vivo role of CISD2 was also examined in a xenograft model. CISD2 expression was significantly increased in CRC cells. CISD2 promoted the CRC cell proliferation and inhibited the apoptosis and autophagy of CRC cells. Moreover, knockdown of CISD2 inhibited the activation of Wnt/ß-Catenin-signaling pathway. Knockdown of CISD2 inhibited the tumor growth in nude mice. CISD2 promoted colorectal cancer development by inhibiting CRC cell apoptosis and autophagy depending on activating Wnt/ß-Catenin-signaling pathway.
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Neoplasias Colorretais , beta Catenina , Animais , Camundongos , Humanos , Linhagem Celular Tumoral , beta Catenina/genética , beta Catenina/metabolismo , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias Colorretais/metabolismo , Proliferação de Células/genética , Autofagia , Enxofre/metabolismo , Ferro/metabolismo , Regulação Neoplásica da Expressão Gênica , Movimento Celular/genéticaRESUMO
Survival from pancreatic cancer is poor because most cancers are diagnosed in the late stages and there are no therapies to prevent the progression of precancerous pancreatic intraepithelial neoplasms (PanINs). Inhibiting mutant KRASG12D, the primary driver mutation in most human pancreatic cancers, has been challenging. The cholecystokinin-B receptor (CCK-BR) is absent in the normal pancreas but becomes expressed in high grade PanIN lesions and is over-expressed in pancreatic cancer making it a prime target for therapy. We developed a biodegradable nanoparticle polyplex (NP) that binds selectively to the CCK-BR on PanINs and pancreatic cancer to deliver gene therapy. PanIN progression was halted and the pancreas extracellular matrix rendered less carcinogenic in P48-Cre/LSL-KrasG12D/+ mice treated with the CCK-BR targeted NP loaded with siRNA to mutant Kras. The targeted NP also slowed proliferation, decreased metastases and improved survival in mice bearing large orthotopic pancreatic tumors. Safety and toxicity studies were performed in immune competent mice after short or long-term exposure and showed no off-target toxicity by histological or biochemical evaluation. Precision therapy with target-specific NPs provides a novel approach to slow progression of advanced pancreatic cancer and also prevents the development of pancreatic cancer in high-risk subjects without toxicity to other tissues.
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Carcinoma in Situ , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Camundongos , Humanos , Animais , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Modelos Animais de Doenças , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/prevenção & controle , Pâncreas/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma in Situ/genética , Carcinoma Ductal Pancreático/patologia , Neoplasias PancreáticasRESUMO
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated deaths worldwide. Treatment with immune checkpoint antibodies has shown promise in advanced HCC, but the response is only 15-20%. We discovered a potential target for the treatment of HCC, the cholecystokinin-B receptor (CCK-BR). This receptor is overexpressed in murine and human HCC and not in normal liver tissue. Mice bearing syngeneic RIL-175 HCC tumors were treated with phosphate buffer saline (PBS; control), proglumide (a CCK-receptor antagonist), an antibody to programmed cell death protein 1 (PD-1Ab), or the combination of proglumide and the PD-1Ab. In vitro, RNA was extracted from untreated or proglumide-treated murine Dt81Hepa1-6 HCC cells and analyzed for expression of fibrosis-associated genes. RNA was also extracted from human HepG2 HCC cells or HepG2 cells treated with proglumide and subjected to RNA sequencing. Results showed that proglumide decreased fibrosis in the tumor microenvironment and increased the number of intratumoral CD8+ T cells in RIL-175 tumors. When proglumide was given in combination with the PD-1Ab, there was a further significant increase in intratumoral CD8+ T cells, improved survival, and alterations in genes regulating tumoral fibrosis and epithelial-to-mesenchymal transition. RNAseq results from human HepG2 HCC cells treated with proglumide showed significant changes in differentially expressed genes involved in tumorigenesis, fibrosis, and the tumor microenvironment. The use of the CCK receptor antagonist may improve efficacy of immune checkpoint antibodies and survival in those with advanced HCC.
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Carcinoma Hepatocelular , Inibidores de Checkpoint Imunológico , Neoplasias Hepáticas , Proglumida , Receptores da Colecistocinina , Animais , Camundongos , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Colecistocinina , Fibrose , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Proglumida/farmacologia , Receptores da Colecistocinina/antagonistas & inibidores , Inibidores de Checkpoint Imunológico/imunologiaRESUMO
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an accurate method for quantifying gene expression levels. Choosing appropriate reference genes to normalize the data is essential for reducing errors. Gelsemium elegans is a highly poisonous but important medicinal plant used for analgesic and anti-swelling purposes. Gelsenicine is one of the vital active ingredients, and its biosynthesis pathway remains to be determined. In this study, G. elegans leaf tissue with and without the application of one of four hormones (SA, MeJA, ETH, and ABA) known to affect gelsenicine synthesis, was analyzed using ten candidate reference genes. The gene stability was evaluated using GeNorm, NormFinder, BestKeeper, ∆CT, and RefFinder. The results showed that the optimal stable reference genes varied among the different treatments and that at least two reference genes were required for accurate quantification. The expression patterns of 15 genes related to the gelsenicine upstream biosynthesis pathway was determined by RT-qPCR using the relevant reference genes identified. Three genes 8-HGO, LAMT, and STR, were found to have a strong correlation with the amount of gelsenicine measured in the different samples. This research is the first study to examine the reference genes of G. elegans under different hormone treatments and will be useful for future molecular analyses of this medically important plant species.
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Gelsemium , Gelsemium/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Perfilação da Expressão Gênica/métodos , Padrões de Referência , Expressão Gênica , HormôniosRESUMO
Known for their regulatory roles in stem cell homeostasis, CLAVATA3/ESR-RELATED (CLE) peptides also function as mediators of external stimuli such as hormones. De novo shoot regeneration, representing the remarkable plant cellular plasticity, involves reconstitution of stem cells under control of stem-cell regulators. Yet whether and how stem cell-regulating CLE peptides are implicated in plant regeneration remains unknown. By CRISPR/Cas9-induced loss-of-function studies, peptide application, precursor overexpression, and expression analyses, the role of CLE1-CLE7 peptides and their receptors in de novo shoot regeneration was studied in Arabidopsis thaliana. CLE1-CLE7 are induced by callus-induction medium and dynamically expressed in pluripotent callus. Exogenously-applied CLE1-CLE7 peptides or precursor overexpression effectively leads to shoot regeneration suppression, whereas their simultaneous mutation results in enhanced regenerative capacity, demonstrating that CLE1-CLE7 peptides redundantly function as negative regulators of de novo shoot regeneration. CLE1-CLE7-mediated shoot regeneration suppression is impaired in loss-of-function mutants of callus-expressed CLAVATA1 (CLV1) and BARELY ANY MERISTEM1 (BAM1) genes, indicating that CLV1/BAM1 are required for CLE1-CLE7-mediated shoot regeneration signaling. CLE1-CLE7 signaling resulted in transcriptional repression of WUSCHEL (WUS), a stem cell-promoting transcription factor known as a principal regulator of plant regeneration. Our results indicate that functionally-redundant CLE1-CLE7 peptides genetically act through CLV1/BAM1 receptors and repress WUS expression to modulate shoot-regeneration capacity, establishing the mechanistic basis for CLE1-CLE7-mediated shoot regeneration and a novel role for CLE peptides in hormone-dependent developmental plasticity.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Meristema/metabolismo , Peptídeos/metabolismo , Brotos de Planta/metabolismo , Proteínas Serina-Treonina Quinases , Transdução de Sinais/genéticaRESUMO
Objective. The purpose of this meta-analysis was to investigate the effect of periprocedural myocardial injury (PPMI) on long-term all-cause mortality in patients undergoing transcatheter aortic valve replacement (TAVR) and to explore potential factors associated with mortality risk. Design. The PubMed, Embase, and Cochrane Library databases were searched up to April 2022. Studies reporting the effect of PPMI on the risk of long-term all-cause mortality were included. The summary odds ratio (OR) was calculated using a random effects model. Additionally, meta-regression and subgroup analyses were conducted according to specific research characteristics to explore sources of heterogeneity. Results. Fourteen studies involving 6,415 patients who underwent TAVR showed that the occurrence of PPMI was associated with a higher risk of long-term mortality. Subgroup analysis showed that in the group of aged ≥82 years, men accounted for less than 50%, coronary artery disease patients accounted for more than 50%, and the proportion of patients with chronic kidney disease accounted for more than 60%, the proportion of patients with atria fibrillation accounted for less than 30%, and the Society of Thoracic Surgeons predicted risk of mortality score was >8 points, patients with PPMI had higher long-term all-cause mortality than those without PPMI. Conclusions. Among the patients who underwent TAVR, those who developed PPMI had higher long-term all-cause mortality.
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Estenose da Valva Aórtica , Doença da Artéria Coronariana , Substituição da Valva Aórtica Transcateter , Masculino , Humanos , Substituição da Valva Aórtica Transcateter/métodos , Estenose da Valva Aórtica/epidemiologia , Resultado do Tratamento , Razão de Chances , Fatores de Risco , Valva Aórtica/cirurgiaRESUMO
[This corrects the article DOI: 10.1155/2020/8815195.].
RESUMO
Chaperone-mediated autophagy (CMA) is one of the three types of autophagy. In recent years, CMA has been shown to be associated with the pathogenesis of several types of cancer. However, whether CMA is involved in the pathogenesis of colorectal cancer (CRC) remains unclear. In this study, we investigated CMA activity in tissue specimens from CRC patients and mouse models of colitis-associated CRC (induced by administration of AOM plus DSS). In addition, we down-regulated CMA in CT26 colon carcinoma cells stably transfected with a vector expressing a siRNA targeting LAMP-2A, the limiting component in the CMA pathway, to explore the role of CMA in these cells. Apoptosis was detected using TUNEL assay, and the apoptosis-related proteins were detected using western blotting. Cell proliferation was assessed using MTT assay, Ki-67 labelling and western blotting for PCNA. We found that LAMP-2A expression was significantly increased in CRC patients and mouse models and varied according to the stage of the disease. Inhibition of CMA in CT26 cells facilitated apoptosis, as evidenced by increased TUNEL immunolabeling, increased expression of Bax and Bnip3, and decreased expression of Bcl-2. Cell proliferation assays showed that inhibition of CMA impeded the proliferation of CT26 cells. These data support the hypothesis that CMA is up-regulated in CRC, and inhibition of CMA may be a new therapeutic strategy for CRC patients.