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Following recovery from the acute infection stage of the SARS-CoV-2 virus (COVID-19), survivors can experience a wide range of persistent Post-Acute Sequelae of COVID-19 (PASC), also referred to as long COVID. According to the US National Research Action Plan on Long COVID 2022, up to 23.7 million Americans suffer from long COVID, and approximately one million workers may be out of the workforce each day due to these symptoms, leading to a USD 50 billion annual loss of salary. Neurological symptoms associated with long COVID result from persistent infection with SARS-CoV-2 in the nasal neuroepithelial cells, leading to inflammation in the central nervous system (CNS). As of today, there is no evidence that vaccines or medications can clear the persistent viral infection in olfactory mucosa. Recently published clinical data demonstrate that only 5% of long COVID anosmia patients have fully recovered during the past 2 years, and 10.4% of COVID patients are still symptomatic 18 months post-infection. Our group demonstrated that epigallocatechin-3-gallate-monopalmitate (EC16m) nanoformulations possess strong antiviral activity against human coronavirus, suggesting that this green-tea-derived compound in nanoparticle formulations could be developed as an intranasally delivered new drug targeting the persistent SARS-CoV-2 infection, as well as inflammation and oxidative stress in the CNS, leading to restoration of neurologic functions. The objective of the current study was to evaluate the mucociliary safety of the EC16m nasal nanoformulations and their efficacy against human coronavirus. METHODS: Nanoparticle size and Zeta potential were measured using the ZetaView Nanoparticle Tracking Analysis system; mucociliary safety was determined using the MucilAir human nasal model; contact antiviral activity and post-infection inhibition against the OC43 viral strain were assessed by the TCID50 assay for cytopathic effect on MRC-5 cells. RESULTS: The saline-based EC16 mucoadhesive nanoformulations containing 0.005 to 0.02% w/v EC16m have no significant difference compared to saline (0.9% NaCl) with respect to tissue integrity, cytotoxicity, and cilia beat frequency. A 5 min contact resulted in 99.9% inactivation of ß-coronavirus OC43. OC43 viral replication was inhibited by >90% after infected MRC-5 cells were treated with the formulations. CONCLUSION: The saline-based novel EC16m mucoadhesive nasal nanoformulations rapidly inactivated human coronavirus with mucociliary safety properties comparable to saline, a solution widely used for nasal applications.
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With the massive release of microplastics (MPs) into the environment, research related to MPs is advancing rapidly. Effective research methods are necessary to identify the chemical composition, shape, distribution, and environmental impacts of MPs. In recent years, artificial intelligence (AI)-driven machine learning methods have demonstrated excellent performance in analyzing MPs in soil and water. This review provides a comprehensive overview of machine learning methods for the prediction of MPs for various tasks, and discusses in detail the data source, data preprocessing, algorithm principle, and algorithm limitation of applied machine learning. In addition, this review discusses the limitation of current machine learning methods for various task analysis in MPs along with future prospect. Finally, this review finds research potential in future work in building large generalized MPs datasets, designing high-performance but low-computational-complexity algorithms, and evaluating model interpretability.
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Purpose: It is necessary to establish a mouse model of keratoconus (KC) for research and therapy. We aimed to determine corneal phenotypes in 3 Ppip5k2 mouse models. Methods: Central corneal thickness (CCT) was determined using spectral domain optical coherence tomography (SD-OCT) in Ppip5k2+/K^ (n = 41 eyes), Ppip5k2K^/K^ (n = 17 eyes) and 2 knock-in mice, Ppip5k2S419A/+ (n = 54 eyes) and Ppip5k2S419A/S419A (n = 18 eyes), and Ppip5k2D843S/+ (n = 42 eyes) and Ppip5k2D843S/D843S (n = 44 eyes) at 3 and 6 months. Pachymetry maps were generated using the Mouse Corneal Analysis Program (MCAP) to process OCT images. Slit lamp biomicroscopy was used to determine any corneal abnormalities, and, last, hematoxylin and eosin (H&E) staining using corneal sections from these animals was used to examine morphological changes. Results: CCT significantly decreased from 3 to 6 months in the Ppip5k2+/K^ and Ppip5k2K^/K^ mice compared to their littermate controls. OCT-based pachymetry maps revealed abnormally localized thinning in all three models compared to their wild-type (WT) controls. Slit lamp examinations revealed corneal abnormalities in the form of bullous keratopathy, stromal edema, stromal scarring, deep corneal neovascularization, and opacities in the heterozygous/homozygous mice of the three models in comparison with their controls. Corneal histological abnormalities, such as epithelial thickening and stromal layer damage, were observed in the heterozygous/homozygous mice of the three models in comparison with the WT controls. Conclusions: We have identified phenotypic and histological changes in the corneas of three mouse lines that could be relevant in the development of animal models of KC.
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Córnea , Modelos Animais de Doenças , Ceratocone , Fenótipo , Tomografia de Coerência Óptica , Animais , Ceratocone/diagnóstico , Ceratocone/genética , Camundongos , Tomografia de Coerência Óptica/métodos , Córnea/patologia , Córnea/diagnóstico por imagem , Paquimetria Corneana , Camundongos Endogâmicos C57BL , Feminino , Masculino , Microscopia com Lâmpada de FendaRESUMO
Long noncoding RNA small nucleolar RNA host gene 1 (lncRNA SNHG1) plays a crucial role in the occurrence and progression of various tumors. This study investigates the function of lncRNA SNHG1 in hepatocellular carcinoma (HCC). We discovered that lncRNA SNHG1 is significantly upregulated in HCC and markedly enhances cell proliferation, migration, and invasion, while simultaneously suppressing apoptosis in HCC cells. Furthermore, lncRNA SNHG1 was found to downregulate miR-7-5p expression. Overexpression of lncRNA SNHG1 counteracted the suppression of HCC cell migration, proliferation, and invasion caused by miR-7-5p mimics, and reversed the miR-7-5p mimics' enhancement of apoptosis in HCC cells. Additionally, miR-7-5p was shown to negatively regulate IGF2BP2, with the silencing of IGF2BP2 diminishing the abilities of HCC cells to proliferate, migrate, and invade, and increasing their propensity for apoptosis. Overexpression of lncRNA SNHG1 negated these effects. Thus, lncRNA SNHG1 fosters HCC progression by upregulating IGF2BP2 expression through targeting miR-7-5p.
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A recent estimate indicates that up to 23.7 million Americans suffer from long COVID, and approximately one million workers may be out of the workforce each day due to associated symptoms, leading to a USD 50 billion annual loss of salary. Post-COVID (Long COVID) neurologic symptoms are due to the initial robust replication of SARS-CoV-2 in the nasal neuroepithelial cells, leading to inflammation of the olfactory epithelium (OE) and the central nervous system (CNS), and the OE becoming a persistent infection site. Previously, our group showed that Epigallocatechin-3-gallate-palmitate (EC16) nanoformulations possess strong antiviral activity against human coronavirus, suggesting this green tea-derived compound in nanoparticle formulations could be developed as an intranasally delivered new drug to eliminate the persistent SARS-CoV-2 infection, leading to restored olfactory function and reduced inflammation in the CNS. The objective of the current study was to determine the compatibility of the nanoformulations with human nasal primary epithelial cells (HNpECs). METHODS: Nanoparticle size was measured using the ZetaView Nanoparticle Tracking Analysis (NTA) system; contact antiviral activity was determined by TCID50 assay for cytopathic effect on MRC-5 cells; post-infection inhibition activity was determined in HNpECs; and cytotoxicity for these cells was determined using an MTT assay. The rapid inactivation of OC43 (a ß-coronavirus) and 229E (α-coronavirus) viruses was further characterized by transmission electron microscopy. RESULTS: A saline-based nanoformulation containing 0.1% w/v EC16 was able to inactivate 99.9999% ß-coronavirus OC43 on direct contact within 1 min. After a 10-min incubation of infected HNpECs with a formulation containing drug-grade EC16 (EGCG-4' mono-palmitate or EC16m), OC43 viral replication was inhibited by 99%. In addition, all nanoformulations tested for their effect on cell viability were comparable to normal saline, a regularly used nasal irrigation solution. A 1-min incubation of an EC16 nanoformulation with either OC43 or 229E showed an altered viral structure. CONCLUSION: Nanoformulations containing EC16 showed properties compatible with nasal application to rapidly inactivate SARS-CoV-2 residing in the olfactory mucosa and to reduce inflammation in the CNS, pending additional formulation and safety studies.
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COVID-19 , Catequina/análogos & derivados , Humanos , Estados Unidos , SARS-CoV-2 , Síndrome de COVID-19 Pós-Aguda , Antivirais/farmacologia , Estudos de Viabilidade , Solução Salina , Inflamação , LipídeosRESUMO
Skin cancer diagnosis often relies on image segmentation as a crucial aid, and a high-performance segmentation can lower misdiagnosis risks. Part of the medical devices often have limited computing power for deploying image segmentation algorithms. However, existing high-performance algorithms for image segmentation primarily rely on computationally intensive large models, making it challenging to meet the lightweight deployment requirement of medical devices. State-of-the-art lightweight models are not able to capture both local and global feature information of lesion edges due to their model structures, result in pixel loss of lesion edge. To tackle this problem, we propose LeaNet, a novel U-shaped network for high-performance yet lightweight skin cancer image segmentation. Specifically, LeaNet employs multiple attention blocks in a lightweight symmetric U-shaped design. Each blocks contains a dilated efficient channel attention (DECA) module for global and local contour information and an inverted external attention (IEA) module to improve information correlation between data samples. Additionally, LeaNet uses an attention bridge (AB) module to connect the left and right sides of the U-shaped architecture, thereby enhancing the model's multi-level feature extraction capability. We tested our model on ISIC2017 and ISIC2018 datasets. Compared with large models like ResUNet, LeaNet improved the ACC, SEN, and SPEC metrics by 1.09 %, 2.58 %, and 1.6 %, respectively, while reducing the model's parameter number and computational complexity by 570x and 1182x. Compared with lightweight models like MALUNet, LeaNet achieved improvements of 2.07 %, 4.26 %, and 3.11 % in ACC, SEN, and SPEC, respectively, reducing the parameter number and computational complexity by 1.54x and 1.04x.
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Neoplasias Cutâneas , Humanos , Pele , Algoritmos , Benchmarking , Processamento de Imagem Assistida por ComputadorRESUMO
This study aimed to explore how kinesin family member 2C (KIF2C) influences the progression of non-small cell lung cancer (NSCLC). The levels of KIF2C and microRNA-186-3p (miR-186-3p) were examined by quantitative real-time polymerase chain reaction (qRT-PCR). Through the utilization of cell counting kit-8 (CCK-8) assay, colony formation assay, wound closure assay, and Transwell assay, NSCLC cell proliferation, migration, and invasion were identified, respectively. NSCLC cell apoptosis was assessed using the TUNEL assay and flow cytometry (FCM) assay. Luciferase reporter analysis was used to investigate the relationship between KIF2C and miR-186-3p. Western blot assays were conducted to investigate the influence of KIF2C on the AKT-GSK3ß-ß-catenin pathway. The results showed that KIF2C was up-regulated in NSCLC cells, which predicted poor prognosis. KIF2C overexpression promoted the proliferation, migration, and invasion of NSCLC cells as well as inhibited NSCLC cell apoptosis. KIF2C was as a key target of miR-186-3p. High expression of KIF2C, meanwhile, increased the levels of ß-catenin, p-GSK-3ß and phosphorylated protein kinase B (p-AKT). KIF2C downregulation and miR-186-3p upregulation reversed these outcomes. As an oncogenic factor, KIF2C is negatively regulated by miR-186-3p and participates in the progression of NSCLC through the AKT-GSK3ß-ß-catenin pathway.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , MicroRNAs/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Cinesinas/genética , Cinesinas/metabolismoRESUMO
OBJECTIVES: N6-methyladenosine (m6A), a predominant RNA modification, has been recently linked to messenger RNA splicing, stability and expression, and its dysregulation may be important in the initiation as well as development of human cancers. The current study was proposed to investigate the clinico-pathological value and multiomic characteristics of m6A-linked genes in the diagnosis and prognosis of lung adenocarcinoma (LUAD). METHODS: The expression levels and mutation types of 21 previously identified m6A regulators were analyzed using the TCGA (The Cancer Genome Atlas) database. The patients were categorized into two groups, a training group (n=392) and a testing group (n=98). Next, the prognostic score of m6A regulators was determined by the Cox survival analysis and a regression model of LASSO to develop a risk profile for patients with LUAD. Moreover, features of risk signature, including chemosensitivity, tumor immune microenvironment and genetic mutation, were also explored. RESULTS: In total, 18 of 21 m6A regulators showed significantly differential expression in LUAD (P<0.05). Among them, 6 genes were observed to be associated with the Overall Survival (OS) of patients with LUAD. Three genes (IGF2BP1 and 2, and HNRNPC) were further evaluated as a prognostic signature in LUAD. Patients, grouped as high risk based on the median of risk score, had poorer OS in comparison with those in low-risk group (P<0.05). The accuracy of our prognostic signatures was high: the AUC were 0.67, 0.59, 0.64 (training set), and 0.65, 0.69, 0.64 (testing set) at survival of 1- , 3- and 5-year, respectively. The prognostic performance of IGF2BP1, IGF2BP2 and HNRNPC was successfully validated in two independent external cohorts. High-risk score was an indicator of chemoresistance, TP53 mutation and increased infiltration of immune cells, and in vitro assessment of the cellular function of HNRNPC confirmed that the gene is involved in cell proliferation and invasion. CONCLUSION: The prognostic signature based on m6A regulators might provide novel insights into prognostic assessment and individualized treatment for patients with LUAD.
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Elevated intraocular pressure (IOP) is the only modifiable risk factor for primary open-angle glaucoma (POAG). Herein we sought to prioritize a set of previously identified IOP-associated genes using novel and previously published datasets. We identified several genes for future study, including several involved in cytoskeletal/extracellular matrix reorganization, cell adhesion, angiogenesis, and TGF-ß signaling. Our differential correlation analysis of IOP-associated genes identified 295 pairs of 201 genes with differential correlation. Pathway analysis identified ß-estradiol as the top upstream regulator of these genes with ESR1 mediating 25 interactions. Several genes (i.e., EFEMP1, FOXC1, and SPTBN1) regulated by ß-estradiol/ESR1 were highly expressed in non-glaucomatous human trabecular meshwork (TM) or Schlemm's canal (SC) cells and specifically expressed in TM/SC cell clusters defined by single-cell RNA-sequencing. We confirmed ESR1 gene and protein expression in human TM cells and TM/SC tissue with quantitative real-time PCR and immunofluorescence, respectively. 17ß-estradiol was identified in bovine, porcine, and human aqueous humor (AH) using ELISA. In conclusion, we have identified estrogen receptor signaling as a key modulator of several IOP-associated genes. The expression of ESR1 and these IOP-associated genes in TM/SC tissue and the presence of 17ß-estradiol in AH supports a role for estrogen signaling in IOP regulation.
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Estrogênios/genética , Pressão Intraocular/genética , Transdução de Sinais/genética , Animais , Humor Aquoso/fisiologia , Bovinos , Linhagem Celular , Matriz Extracelular/genética , Glaucoma de Ângulo Aberto/genética , Humanos , Suínos , Malha Trabecular/fisiologiaRESUMO
Purpose: Intraocular pressure (IOP), the primary risk factor for primary open-angle glaucoma, is determined by resistance to aqueous outflow through the trabecular meshwork (TM). IOP homeostasis relies on TM responses to mechanical stretch. To model the effects of elevated IOP on the TM, this study sought to identify coding and non-coding RNAs differentially expressed in response to mechanical stretch. Methods: Monolayers of TM cells from non-glaucomatous donors (n = 5) were cultured in the presence or absence of 15% mechanical stretch, 1 cycle/second, for 24 hours using a computer-controlled Flexcell unit. We profiled mRNAs and lncRNAs with stranded total RNA sequencing and microRNA (miRNA) expression with NanoString-based miRNA assays. We used two-tailed paired t-tests for mRNAs and long non-coding RNAs (lncRNAs) and the Bioconductor limma package for miRNAs. Gene ontology and pathway analyses were performed with WebGestalt. miRNA-mRNA interactions were identified using Ingenuity Pathway Analysis Integrative miRNA Target Finder software. Validation of differential expression was conducted using droplet digital PCR. Results: We identified 219 mRNAs, 42 miRNAs, and 387 lncRNAs with differential expression in TM cells upon cyclic mechanical stretch. Pathway analysis indicated significant enrichment of genes involved in steroid biosynthesis, glycerolipid metabolism, and extracellular matrix-receptor interaction. We also identified several miRNA master regulators (miR-125a-5p, miR-30a-5p, and miR-1275) that regulate several mechanoresponsive genes. Conclusions: To our knowledge, this is the first demonstration of the differential expression of coding and non-coding RNAs in a single set of cells subjected to cyclic mechanical stretch. Our results validate previously identified, as well as novel, genes and pathways.
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MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Estresse Mecânico , Malha Trabecular/metabolismo , Células Cultivadas , Regulação para Baixo , Humanos , Regulação para CimaRESUMO
Keratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future.
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Predisposição Genética para Doença , Ceratocone/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Pró-Proteína Convertase 1/genética , Adulto , Animais , Mapeamento Cromossômico , Córnea/diagnóstico por imagem , Córnea/patologia , Topografia da Córnea/métodos , Modelos Animais de Doenças , Feminino , Ligação Genética , Genoma Humano/genética , Genótipo , Humanos , Ceratocone/patologia , Masculino , Camundongos , Mutação/genética , Linhagem , Qualidade de Vida , Sequenciamento do ExomaRESUMO
Sjogren's syndrome (SS) and type-1 diabetes are prevalent autoimmune diseases in the USA. We reported previously that epigallocatechin-3-gallate (EGCG) prevented and delayed the onset of autoimmune disease in non-obese diabetic (NOD) mice, a model for both SS and type-1 diabetes. EGCG also normalized the levels of proteins related to DNA repair and anti-oxidant activity in NOD.B10.Sn-H2 mice, a model for primary SS, prior to disease onset. The current study examined the effect of EGCG on the expression of anti-oxidant enzymes in the submandibular salivary gland and the pancreas of NOD mice and cultured human salivary gland acinar cells. NOD mice consuming 0.2% EGCG daily dissolved in water showed higher protein levels of peroxiredoxin 6 (PRDX6), a major anti-oxidant defense protein, and catalase, while the untreated NOD mice exhibited significantly lowered levels of PRDX6. Similarly, pancreas samples from water-fed NOD mice were depleted in PRDX6 and superoxide dismutase, while EGCG-fed mice showed high levels of these anti-oxidant enzymes. In cultured HSG cells EGCG increased PRDX6 levels significantly, and this was inhibited by p38 and JNK inhibitors, suggesting that the EGCG-mediated increase in protective anti-oxidant capacity is regulated in part through mitogen-activated protein kinase pathway signaling. This mechanism may explain the higher levels of PRDX6 found in EGCG-fed NOD mice. These preclinical observations warrant future preclinical and clinical studies to determine whether EGCG or green tea polyphenols could be used in novel preventive and therapeutic approaches against autoimmune diseases and salivary dysfunction involving oxidative stress.
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Antioxidantes/metabolismo , Catequina/análogos & derivados , Pâncreas Exócrino/metabolismo , Glândula Submandibular/metabolismo , Animais , Catequina/metabolismo , Catequina/farmacologia , Linhagem Celular Tumoral , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Pâncreas Exócrino/citologia , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Glândula Submandibular/citologiaRESUMO
The submandibular salivary glands of non-obese diabetic (NOD) mice, a model for Sjogren's syndrome and type-1 diabetes, show an elevated level of proliferating cell nuclear antigen (PCNA), a protein involved in cell proliferation and repair of DNA damage. We reported previously that epigallocatechin-3-gallate (EGCG), the most abundant green tea catechin, normalizes the PCNA level. PCNA's activity can be regulated by the cyclin-dependent kinase inhibitor p21, which is also important for epithelial cell differentiation. In turn, expression of p21 and PCNA are partially regulated by Rb phosphorylation levels. EGCG was found to modulate p21 expression in epithelial cells, suggesting that EGCG-induced p21 could be associated with down-regulation of PCNA in vivo. The current study examined the protein levels of p21 and p53 (which can up-regulate p21) in NOD mice fed with either water or EGCG, and the effect of EGCG on p21 and p53 in cell line models with either normal or defective Rb. In NOD mice, the p21 level was low, and EGCG normalized it. In contrast to HSG cells with functional Rb, negligible expression of p21 in NS-SVAC cells that lack Rb was not altered by EGCG treatment. Inhibition of p53 by siRNA demonstrated that p21 and p53 were induced independently in HSG cells by a physiological concentration range of EGCG, suggesting p53 could be an important but not conditional factor associated with p21 expression. In conclusion, PCNA and p21 levels are altered inversely in the NOD model for SS and in HSG cells, and warrant further study as candidate new markers for salivary dysfunction associated with xerostomia. Induction of p21 by EGCG could provide clinically useful normalization of salivary glands by promoting differentiation and reducing PCNA levels.
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Anticarcinógenos/administração & dosagem , Catequina/análogos & derivados , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Diabetes Mellitus Tipo 1/tratamento farmacológico , Células Epiteliais/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Glândulas Salivares/metabolismo , Síndrome de Sjogren/tratamento farmacológico , Proteína Supressora de Tumor p53/metabolismo , Xerostomia/tratamento farmacológico , Animais , Anticarcinógenos/efeitos adversos , Catequina/administração & dosagem , Catequina/efeitos adversos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação para Baixo , Células Epiteliais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Proteína do Retinoblastoma/metabolismoRESUMO
BACKGROUND: Mycophenolate mofetil (MMF), the prodrug of mycophenolic acid (MPA) which has been widely used for the prevention of acute graft rejection, is a potent inhibitor of inosine monophosphate dehydrogenase (IMPDH) that is up-regulated in many tumors and potentially a target for cancer therapy. MPA is known to inhibit cancer cell proliferation and induces apoptosis; however, the underlying molecular mechanisms remain elusive. METHODS: We first demonstrated MPA's antiproliferative and proapoptotic activities using in vitro studies of 13 cancer cell lines and a xenograft model. Key proteins involved in cell cycle, proliferation and apoptosis were analyzed by Western blotting. RESULTS: In vitro treatment of thirteen cancer cell lines indicated that five cell lines (AGS, NCI-N87, HCT-8, A2780 and BxPC-3) are highly sensitive to MPA (IC50 < 0.5 µg/ml), four cell lines (Hs746T, PANC-1, HepG2 and MCF-7) are very resistant to MPA (IC50 > 20 µg/ml) and the four other cell lines (KATO III, SNU-1, K562 and HeLa) have intermediate sensitivity. The anticancer activity of MPA was confirmed in vivo using xenograft model with gastric AGS cell line. Further in vitro analyses using AGS cells indicated that MPA can potently induce cell cycle arrest and apoptosis as well as inhibition of cell proliferation. Targeted proteomic analyses indicate that many critical changes responsible for MPA's activities occur at the protein expression and phosphorylation levels. MPA-induced cell cycle arrest is achieved through reduction of many cell cycle regulators such as CDK4, BUB1, BOP1, Aurora A and FOXM1. We also demonstrate that MPA can inhibit the PI3K/AKT/mTOR pathway and can induce caspase-dependent apoptosis. CONCLUSIONS: These results suggest that MPA has beneficial activities for anticancer therapy through diverse molecular pathways and biological processes.
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Antineoplásicos/farmacologia , Ácido Micofenólico/farmacologia , Neoplasias/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Células Hep G2 , Humanos , Concentração Inibidora 50 , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The autoimmune disorder primary Sjogren's syndrome (SS) is associated with xerostomia and xerophthalmia. SS pathogenesis involves both genetic/epigenetic and environmental factors. A major potential contributor is oxidative stress associated with damage to cellular components, including DNA. We reported previously that the green tea polyphenol epigallocatechin-3-gallate (EGCG) normalizes the elevated levels of proliferating cell nuclear antigen (PCNA), a key component of DNA repair, in the NOD mouse model for SS and type 1 diabetes. The current study examined levels of the antioxidant enzymes peroxiredoxin 6 (PRDX6), catalase and superoxide dismutase (SOD), as well as PCNA, in NOD.B10.Sn-H2 mice, a model for primary SS, and determined the effect of EGCG on their expression. PCNA elevation was detected in the submandibular gland and pancreas by 8 weeks of age in water-fed mice, and increased through 14 weeks of age, prior to overt onset of symptoms. This early PCNA elevation was followed by a decline of peroxiredoxin 6 protein. In contrast, EGCG-fed mice exhibited normal levels of PCNA and peroxiredoxin 6, comparable to healthy untreated BALB/c mice. Similar patterns were observed in the pancreas, even though these mice do not develop diabetes. Thus, elevated PCNA is an early biomarker for exocrine glandular dysfunction associated with SS-like autoimmune disease, accompanied subsequently by decreased PRDX6 antioxidant enzyme levels that could further contribute to oxidative stress, and these changes precede inflammatory cell infiltration. Importantly, EGCG consumption normalizes the expression of these biomarkers in this model. These observations could lead to early diagnosis and intervention of autoimmune disorders.
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Antioxidantes/metabolismo , Catequina/análogos & derivados , Reparo do DNA , Glândulas Exócrinas/metabolismo , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/metabolismo , Animais , Anticorpos Antinucleares/sangue , Anticorpos Antinucleares/imunologia , Catequina/metabolismo , Modelos Animais de Doenças , Feminino , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos NOD , Peroxirredoxina VI/metabolismo , Antígeno Nuclear de Célula em Proliferação/sangue , Antígeno Nuclear de Célula em Proliferação/imunologia , Síndrome de Sjogren/patologia , Fatores de TempoRESUMO
OBJECTIVE: To study the effect of lysophosphatidylcholine (LPC) on T-type calcium channel currents (I(Ca.T)) in cardiomyocytes, and identify the mechanism by which LPC accumulation in intracellular and/or interstitial space may uptake tachycardia and various arrhythmias during cardiac ischemia. METHODS: Neonatal rat cardiomyocytes from 1 to 3-day-old Wistar rats and hypertrophied ventricular myocytes from Wistar rats were prepared. Human cardiac T-type calcium channel α1 subunits, Ca(V3.1) and Ca(V3.2), were stably expressed in HEK293 cells. In this study, cardiomyocytes and heterologous expression of human Ca(V3.1) and Ca(V3.2) components were measureed by whole-cell patch clamp to study the up-regulation of I(Ca.T) by LPC. RESULTS: LPC markedly accelerated the spontaneous beating rates of neonatal rat cardiomyocytes from (42±8) beats/min in control to (64 ±8) beats/min after LPC application for 5 min at the physiological [Ca(2+)](i) concentration (pCa=7.2). In neonatal cardiomyocytes, I(Ca.T) was significantly increased by 10 µmol/L LPC by 21.5% when [Ca(2+)](i) was high (pCa=7). Intracellular Ca(2+)-dependent augmentation of I(Ca.T) by LPC was confirmed not only in neonatal cardiomyocytes but also in adult ventricular myocytes from the hypertrophied hearts. In this experiment, I(Ca.T) was significantly increased by 10 µmol/L LPC by 23.5% when [Ca(2+)](i) was high (pCa=7), although it was unchanged when [Ca(2+)](i) was low (pCa=11), control: (3.8±0.2) pA/pF, n=16; LPC: (3.7±0.4) pA/pF, n=10. LPC exerted no effect on the Ca(V3.1) T-type Ca(2+) channel current (I(Ca(V)3.1)) regardless of the [Ca(2+)](i) concentration at a pCa of 7 or at a pCa of 11. In contrast, LPC up-regulated the Ca(V3.2) T-type Ca(2+) channel current (I(Ca(V)3.2)), which was much larger at a pCa of 7 [LPC=10 µmol/L: (68.8±2.1) pA/pF, n=10; LPC=50 µmol/L: (78.4±4.8) pA/pF, n=9)] than that at a pCa of 11 [(38.5±2.1) pA/pF, n=11]. CONCLUSION: The present study indicates that LPC up-regulates the cardiac I(Ca.T) in physiological or higher [Ca(2+)](i) concentration, which may accelerate the pathophysiological cardiac automaticity and trigger tachyarrhythmias as novel ischemia-related mechanism.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Lisofosfatidilcolinas/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/metabolismo , Cálcio/metabolismo , Células HEK293 , Humanos , Masculino , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos WistarRESUMO
Pancreatic duodenal hemeobox-1 (PDX1) is essential for the development of the embryonic pancreas and plays a key role in pancreatic beta-cell differentiation, maturation, regeneration, and maintenance of normal pancreatic beta-cell insulin-producing function. Purified recombinant PDX1 (rPDX1) may be a useful tool for many research and clinical applications, however, using the Escherichia coli expression system has several drawbacks for producing quality PDX1 protein. To explore the yeast expression system for generating rPDX1 protein, the cDNA coding for the full-length human PDX1 gene was cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and western blotting analysis of culture medium from methanol-induced expression yeast clones demonstrated that the rPDX1 was secreted into the culture medium, had a molecular weight by SDS-PAGE of 50kDa, and was glycosylated. The predicted size of the mature unmodified PDX1 polypeptide is 31kDa, suggesting that eukaryotic post-translational modifications are the result of the increased molecular weight. The recombinant protein was purified to greater than 95% purity using a combined ammonium sulfate precipitation with heparin-agarose chromatography. Finally, 120mug of the protein was obtained in high purity from 1L of the culture supernatant. Bioactivity of the rPDX1 was confirmed by the ability to penetrate cell membranes and activation of an insulin-luciferase reporter gene. Our results suggest that the P. pastoris expression system can be used to produce a fully functional human rPDX1 for both research and clinical application.
Assuntos
Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/química , Pichia/química , Transativadores/biossíntese , Transativadores/química , Western Blotting , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Fermentação , Glicosilação , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/isolamento & purificação , Humanos , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Corantes de Rosanilina , Transativadores/genética , Transativadores/isolamento & purificaçãoRESUMO
This paper proposes a new heuristic algorithm, called Quick Method with Shared Protection (QMSP), to protect the single-link failure in survivable WDM optical networks. QMSP first computes one primary path for each connection request. If the primary path is a trap path, QMSP will compute two segment-backup paths to avoid the trap problem based on the routing policy. Compared to previous algorithms, QMSP not only has better time complexity but also can obtain higher resource utilization ratio and lower blocking probability.
RESUMO
With the maturation of the technology of Wavelength-Division-Multiplexing (WDM) in optical networks, the survivable design has become a key issue. In this paper, we propose a Segment Shared Protection Algorithm (SSPA), which is based on the reliability of the networks and the different levels of the fault tolerance requested by the users, to protect the single-link failure in WDM optical networks. The main idea of the SSPA is to provide a backup path for a segment, which is divided in accordance with the policy of the Differentiated Reliability (DiR), on the primary path of each connection request. Under the guarantee of the blocking probability and the connection's reliability, the SSPA has higher resource utilization ratio and faster recovery time than the previous algorithm PSPA-DiR. We evaluate the effectiveness of the SSPA and the results are found to be promising.
RESUMO
A routing-selection algorithm is important in survivable wavelength-division networks. A sound algorithm should carefully consider the efficiency of resource utilization and the protection-switching time. Under shared-risk link group constraints with differentiated reliability (DiR), a novel algorithm for a shared path, called a joint routing-selection algorithm (JRSA) with DiR, is proposed. The simulation results show that a JRSA with DiR not only can efficiently satisfy the specific requirements of users but also can produce nearly optimal performance and determine the appropriatetrade-offs between the resource utilization ratio and the protection-switching time.