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Stimulation of the innate immune system is crucial in both effective vaccinations and immunotherapies. This is often achieved through adjuvants, molecules that usually activate pattern recognition receptors (PRRs) and stimulate two innate immune signaling pathways: the nuclear factor kappa-light-chain-enhancer of activated B-cells pathway (NF-κB) and the interferon regulatory factors pathway (IRF). Here, we demonstrate the ability to alter and improve adjuvant activity via the addition of small molecule "immunomodulators". By modulating signaling activity instead of receptor binding, these molecules allow the customization of select innate responses. We demonstrate both inhibition and enhancement of the products of the NF-κB and IRF pathways by several orders of magnitude. Some modulators apply generally across many receptors, while others focus specifically on individual receptors. Modulators boost correlates of a protective immune responses in a commercial flu vaccine model and reduced correlates of reactogenicity in a typhoid vaccine model. These modulators have a range of applications: from adjuvanticity in prophylactics to enhancement of immunotherapy.
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Background: Mitochondrial dysfunction in retinal pigment epithelium (RPE) is a pathogenic factor in age-related macular degeneration (AMD). Improvement of mitochondrial function may ameliorate RPE bioenergetics status, which may in turn nourish the retinal photoreceptors against degenerative loss. Objective: The purpose of this study is to examine the G-protein coupled receptor (GPCR) antagonistic drug CM-20 in modulating mitochondrial function in RPE cells. Methods: Human-derived ARPE-19 cell line was differentiated to improve RPE morphology. Dose response of CM-20 was performed to examine mitochondrial membrane potential (MMP). Secondary validation with multiplexed live-cell mitochondrial imaging was performed. Protection of CM-20 to mitochondria against oxidative stress was detected under co-treatment with hydrogen peroxide. Results: Treatment with CM-20 elicited a dose-dependent increase of MMP. Multiplexed live-cell mitochondrial imaging showed consistent increase of MMP at an optimal concentration of CM-20 (12.5 µM). MMP was significantly reduced under hydrogen peroxide-induced oxidative stress and treatment with CM-20 showed rescue effects to MMP. Conclusion: CM-20 increases mitochondrial function and protects mitochondria under oxidative stress. As both GPCRs and mitochondria are potential drug targets, retinal neuroprotective testing of CM-20 is warranted in animal models of retinal degeneration.
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Axial extrusion-connection technology is one of the important connection technologies for hydraulic piping systems, with high sealing performance and mechanical strength. In this paper, the finite-element-modeling method is used to simulate the experimental process of the connection strength of the axial extrusion joint. The generation mechanism and calculation method of the connection strength are analyzed. To optimize the joint strength, orthogonal testing and grey correlation analysis are used to analyze the influencing factors of joint strength. The key factors affecting joint strength are obtained as the friction coefficient µ1, µ2 between joint components and the groove angle θ1 of the fittings body. The back-propagation (BP) neural-network algorithm is used to establish the connection-strength model of the joint and the genetic algorithm is used to optimize it. The optimal connection strength is 8.237 kN and the optimal combination of influencing factors is 0.2, 0.4 and 76.8°. Compared with the prediction results of the neural-network genetic algorithm, the relative error of the finite-element results is 3.9%, indicating that the method has high accuracy. The results show that the extrusion-based joining process offers significant advantages in the manufacture of high-strength titanium tubular joints.
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Survival in high-risk pediatric neuroblastoma has remained around 50% for the last 20 years, with immunotherapies and targeted therapies having had minimal impact. Here, we identify the small molecule CX-5461 as selectively cytotoxic to high-risk neuroblastoma and synergistic with low picomolar concentrations of topoisomerase I inhibitors in improving survival in vivo in orthotopic patient-derived xenograft neuroblastoma mouse models. CX-5461 recently progressed through phase I clinical trial as a first-in-human inhibitor of RNA-POL I. However, we also use a comprehensive panel of in vitro and in vivo assays to demonstrate that CX-5461 has been mischaracterized and that its primary target at pharmacologically relevant concentrations, is in fact topoisomerase II beta (TOP2B), not RNA-POL I. This is important because existing clinically approved chemotherapeutics have well-documented off-target interactions with TOP2B, which have previously been shown to cause both therapy-induced leukemia and cardiotoxicity-often-fatal adverse events, which can emerge several years after treatment. Thus, while we show that combination therapies involving CX-5461 have promising anti-tumor activity in vivo in neuroblastoma, our identification of TOP2B as the primary target of CX-5461 indicates unexpected safety concerns that should be examined in ongoing phase II clinical trials in adult patients before pursuing clinical studies in children.
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DNA Topoisomerases Tipo II/metabolismo , Indóis/uso terapêutico , Morfolinas/uso terapêutico , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Pirimidinas/uso terapêutico , Sulfonamidas/uso terapêutico , Animais , Benzotiazóis , Western Blotting , Linhagem Celular Tumoral , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Imunofluorescência , Camundongos , Camundongos Nus , Simulação de Dinâmica Molecular , Naftiridinas , Reação em Cadeia da Polimerase em Tempo RealRESUMO
There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1).
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Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Coronavirus Humano OC43/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , SARS-CoV-2/efeitos dos fármacos , Tiazóis/farmacologia , Células A549 , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Benzamidas , COVID-19/virologia , Domínio Catalítico , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Coronavirus Humano OC43/fisiologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Células HEK293 , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Transgênicos , Testes de Sensibilidade Microbiana , Piperidinas , Piridinas , SARS-CoV-2/enzimologia , SARS-CoV-2/fisiologia , Tiazóis/química , Tiazóis/metabolismo , Tiazóis/uso terapêutico , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
We determined differential gene expression in response to high glucose in lymphoblastoid cell lines derived from matched individuals with type 1 diabetes with and without retinopathy. Those genes exhibiting the largest difference in glucose response were assessed for association with diabetic retinopathy in a genome-wide association study meta-analysis. Expression quantitative trait loci (eQTLs) of the glucose response genes were tested for association with diabetic retinopathy. We detected an enrichment of the eQTLs from the glucose response genes among small association p-values and identified folliculin (FLCN) as a susceptibility gene for diabetic retinopathy. Expression of FLCN in response to glucose was greater in individuals with diabetic retinopathy. Independent cohorts of individuals with diabetes revealed an association of FLCN eQTLs with diabetic retinopathy. Mendelian randomization confirmed a direct positive effect of increased FLCN expression on retinopathy. Integrating genetic association with gene expression implicated FLCN as a disease gene for diabetic retinopathy.
One of the side effects of diabetes is loss of vision from diabetic retinopathy, which is caused by injury to the light sensing tissue in the eye, the retina. Almost all individuals with diabetes develop diabetic retinopathy to some extent, and it is the leading cause of irreversible vision loss in working-age adults in the United States. How long a person has been living with diabetes, the extent of increased blood sugars and genetics all contribute to the risk and severity of diabetic retinopathy. Unfortunately, virtually no genes associated with diabetic retinopathy have yet been identified. When a gene is activated, it produces messenger molecules known as mRNA that are used by cells as instructions to produce proteins. The analysis of mRNA molecules, as well as genes themselves, can reveal the role of certain genes in disease. The studies of all genes and their associated mRNAs are respectively called genomics and transcriptomics. Genomics reveals what genes are present, while transcriptomics shows how active genes are in different cells. Skol et al. developed methods to study genomics and transcriptomics together to help discover genes that cause diabetic retinopathy. Genes involved in how cells respond to high blood sugar were first identified using cells grown in the lab. By comparing the activity of these genes in people with and without retinopathy the study identified genes associated with an increased risk of retinopathy in diabetes. In people with retinopathy, the activity of the folliculin gene (FLCN) increased more in response to high blood sugar. This was further verified with independent groups of people and using computer models to estimate the effect of different versions of the folliculin gene. The methods used here could be applied to understand complex genetics in other diseases. The results provide new understanding of the effects of diabetes. They may also help in the development of new treatments for diabetic retinopathy, which are likely to improve on the current approach of using laser surgery or injections into the eye.
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Diabetes Mellitus Tipo 1/genética , Retinopatia Diabética/genética , Perfilação da Expressão Gênica , Glucose/toxicidade , Linfócitos/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas/genética , Transcriptoma , Proteínas Supressoras de Tumor/genética , Adulto , Estudos de Casos e Controles , Linhagem Celular Transformada , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/metabolismo , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/metabolismo , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Linfócitos/metabolismo , Masculino , Análise da Randomização Mendeliana , Proteínas Proto-Oncogênicas/metabolismo , Locos de Características Quantitativas , Proteínas Supressoras de Tumor/metabolismo , Adulto JovemRESUMO
There is an urgent need for anti-viral agents that treat SARS-CoV-2 infection. The shortest path to clinical use is repurposing of drugs that have an established safety profile in humans. Here, we first screened a library of 1,900 clinically safe drugs for inhibiting replication of OC43, a human beta-coronavirus that causes the common-cold and is a relative of SARS-CoV-2, and identified 108 effective drugs. We further evaluated the top 26 hits and determined their ability to inhibit SARS-CoV-2, as well as other pathogenic RNA viruses. 20 of the 26 drugs significantly inhibited SARS-CoV-2 replication in human lung cells (A549 epithelial cell line), with EC50 values ranging from 0.1 to 8 micromolar. We investigated the mechanism of action for these and found that masitinib, a drug originally developed as a tyrosine-kinase inhibitor for cancer treatment, strongly inhibited the activity of the SARS-CoV-2 main protease 3CLpro. X-ray crystallography revealed that masitinib directly binds to the active site of 3CLpro, thereby blocking its enzymatic activity. Mastinib also inhibited the related viral protease of picornaviruses and blocked picornaviruses replication. Thus, our results show that masitinib has broad anti-viral activity against two distinct beta-coronaviruses and multiple picornaviruses that cause human disease and is a strong candidate for clinical trials to treat SARS-CoV-2 infection.
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MicroRNAs (miRNAs) are short double-stranded noncoding RNAs (19-23 nucleotides) that regulate gene expression by suppressing mRNAs through RNA interference. Targeting is determined by the seed sequence (position 2-7/8) of the mature miRNA. A minimal G-rich seed of just six nucleotides is highly toxic to cells by targeting genes essential for cell survival. A screen of 215 miRNAs encoded by 17 human pathogenic viruses (v-miRNAs) now suggests that a number of v-miRNAs can kill cells through a G-rich 6mer sequence embedded in their seed. Specifically, we demonstrate that miR-K12-6-5p, an oncoviral mimic of the tumor suppressive miR-15/16 family encoded by human Kaposi sarcoma-associated herpes virus, harbors a noncanonical toxic 6mer seed (position 3-8) and that v-miRNAs are more likely than cellular miRNAs to utilize a noncanonical 6mer seed. Our data suggest that during evolution viruses evolved to use 6mer seed toxicity to kill cells.
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The tumor microenvironment (TME) is a key determinant of metastatic efficiency. We performed a quantitative high-throughput screen (qHTS) of diverse medicinal chemistry tractable scaffolds (44,420 compounds) and pharmacologically active small molecules (386 compounds) using a layered organotypic, robust assay representing the ovarian cancer metastatic TME. This 3D model contains primary human mesothelial cells, fibroblasts, and extracellular matrix, to which fluorescently labeled ovarian cancer cells are added. Initially, 100 compounds inhibiting ovarian cancer adhesion/invasion to the 3D model in a dose-dependent manner were identified. Of those, eight compounds were confirmed active in five high-grade serous ovarian cancer cell lines and were further validated in secondary in vitro and in vivo biological assays. Two tyrosine kinase inhibitors, PP-121 and milciclib, and a previously unreported compound, NCGC00117362, were selected because they had potency at 1 µmol/L in vitro Specifically, NCGC00117362 and PP-121 inhibited ovarian cancer adhesion, invasion, and proliferation, whereas milciclib inhibited ovarian cancer invasion and proliferation. Using in situ kinase profiling and immunoblotting, we found that milciclib targeted Cdk2 and Cdk6, and PP-121 targeted mTOR. In vivo, all three compounds prevented ovarian cancer adhesion/invasion and metastasis, prolonged survival, and reduced omental tumor growth in an intervention study. To evaluate the clinical potential of NCGC00117362, structure-activity relationship studies were performed. Four close analogues of NCGC00117362 efficiently inhibited cancer aggressiveness in vitro and metastasis in vivo Collectively, these data show that a complex 3D culture of the TME is effective in qHTS. The three compounds identified have promise as therapeutics for prevention and treatment of ovarian cancer metastasis.
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Ensaios de Triagem em Larga Escala/métodos , Metástase Neoplásica/prevenção & controle , Neoplasias Ovarianas/terapia , Microambiente Tumoral/genética , Animais , Feminino , Humanos , Camundongos , Camundongos NusRESUMO
Many small-interfering (si)RNAs are toxic to cancer cells through a 6mer seed sequence (positions 2-7 of the guide strand). Here we performed an siRNA screen with all 4096 6mer seeds revealing a preference for guanine in positions 1 and 2 and a high overall G or C content in the seed of the most toxic siRNAs for four tested human and mouse cell lines. Toxicity of these siRNAs stems from targeting survival genes with C-rich 3'UTRs. The master tumor suppressor miRNA miR-34a-5p is toxic through such a G-rich 6mer seed and is upregulated in cells subjected to genotoxic stress. An analysis of all mature miRNAs suggests that during evolution most miRNAs evolved to avoid guanine at the 5' end of the 6mer seed sequence of the guide strand. In contrast, for certain tumor-suppressive miRNAs the guide strand contains a G-rich toxic 6mer seed, presumably to eliminate cancer cells.
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Linhagem Celular Tumoral/efeitos dos fármacos , MicroRNAs/toxicidade , RNA Interferente Pequeno/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Marcação de Genes , Genes Essenciais/efeitos dos fármacos , Guanina , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Regiões não TraduzidasRESUMO
Trinucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington's disease (HD). Pathology is caused by protein and RNA generated from the TNR regions including small siRNA-sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR are toxic to cancer cells by targeting genes that contain long reverse complementary TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the six members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG TNR-based siRNAs induce cell death in vitro in all tested cancer cell lines and slow down tumor growth in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR-based siRNAs as a novel form of anticancer reagents.
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Proteína Huntingtina/genética , Neoplasias/genética , RNA Interferente Pequeno/farmacologia , Repetições de Trinucleotídeos/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Modelos Animais de Doenças , Humanos , Proteína Huntingtina/antagonistas & inibidores , Doença de Huntington/genética , Doença de Huntington/patologia , Camundongos , Neoplasias/patologia , Neoplasias/terapia , Fases de Leitura Aberta , RNA Interferente Pequeno/genética , Expansão das Repetições de Trinucleotídeos/genética , Repetições de Trinucleotídeos/efeitos dos fármacosRESUMO
BACKGROUND: White blood cells have been shown in animal studies to play a central role in the pathogenesis of diabetic retinopathy. Lymphoblastoid cells are immortalized EBV-transformed primary B-cell leukocytes that have been extensively used as a model for conditions in which white blood cells play a primary role. The purpose of this study was to investigate whether lymphoblastoid cell lines, by retaining many of the key features of primary leukocytes, can be induced with glucose to demonstrate relevant biological responses to those found in diabetic retinopathy. METHODS: Lymphoblastoid cell lines were obtained from twenty-three human subjects. Differences between high and standard glucose conditions were assessed for expression, endothelial adhesion, and reactive oxygen species. RESULTS: Collectively, stimulation of the lymphoblastoid cell lines with high glucose demonstrated corresponding changes on molecular, cellular and functional levels. Lymphoblastoid cell lines up-regulated expression of a panel of genes associated with the leukocyte-mediated inflammation found in diabetic retinopathy that include: a cytokine (IL-1B fold change = 2.11, p-value = 0.02), an enzyme (PKCB fold change = 2.30, p-value = 0.01), transcription factors (NFKB-p50 fold change = 2.05, p-value = 0.01), (NFKB-p65 fold change = 2.82, p-value = 0.003), and an adhesion molecule (CD18 fold change = 2.59, 0.02). Protein expression of CD18 was also increased (p-value = 2.14x10-5). The lymphoblastoid cell lines demonstrated increased adhesiveness to endothelial cells (p = 1.28x10-5). Reactive oxygen species were increased (p = 2.56x10-6). Significant inter-individual variation among the lymphoblastoid cell lines in these responses was evident (F = 18.70, p < 0.0001). CONCLUSIONS: Exposure of lymphoblastoid cell lines derived from different human subjects to high glucose demonstrated differential and heterogeneous gene expression, adhesion, and cellular effects that recapitulated features found in the diabetic state. Lymphoblastoid cells may represent a useful tool to guide an individualized understanding of the development and potential treatment of diabetic complications like retinopathy.
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Glucose/farmacologia , Regulação para Cima/efeitos dos fármacos , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Antígenos CD18/genética , Antígenos CD18/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
Degenerative loss of photoreceptors occurs in inherited and age-related retinal degenerative diseases. A chemical screen facilitates development of new testing routes for neuroprotection and mechanistic investigation. Herein, we conducted a mouse-derived photoreceptor (661W cell)-based high throughput screen of the Food and Drug Administration-approved Prestwick drug library to identify putative cytoprotective compounds against light-induced, synthetic visual chromophore-precipitated cell death. Different classes of hit compounds were identified, some of which target known genes or pathways pathologically associated with retinitis pigmentosa. Sulfaphenazole (SFZ), a selective inhibitor of human cytochrome P450 (CYP) 2C9 isozyme, was identified as a novel and leading cytoprotective compound. Expression of CYP2C proteins was induced by light. Gene-targeted knockdown of CYP2C55, the homologous gene of CYP2C9, demonstrated viability rescue to light-induced cell death, whereas stable expression of functional CYP2C9-GFP fusion protein further exacerbated light-induced cell death. Mechanistically, SFZ inhibited light-induced necrosis and mitochondrial stress-initiated apoptosis. Light elicited calcium influx, which was mitigated by SFZ. Light provoked the release of arachidonic acid from membrane phospholipids and production of non-epoxyeicosatrienoic acid metabolites. Administration of SFZ further stimulated the production of non-epoxyeicosatrienoic acid metabolites, suggesting a metabolic shift of arachidonic acid under inhibition of the CYP2C pathway. Together, our findings indicate that CYP2C genes play a direct causative role in photochemical stress-induced death of photoreceptors and suggest that the CYP monooxygenase system is a risk factor for retinal photodamage, especially in individuals with Stargardt disease and age-related macular degeneration that deposit condensation products of retinoids.
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Hidrocarboneto de Aril Hidroxilases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Citoproteção/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Sulfafenazol/farmacologia , Sequência de Aminoácidos , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Linhagem Celular , Citocromo P-450 CYP2C9 , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Família 2 do Citocromo P450 , Avaliação Pré-Clínica de Medicamentos , Expressão Gênica , Inativação Gênica , Humanos , Luz , Camundongos , Dados de Sequência Molecular , Células Fotorreceptoras de Vertebrados/enzimologia , Alinhamento de SequênciaRESUMO
OBJECTIVE: To determine the genetic contribution to leukocyte endothelial adhesion. METHODS: Leukocyte endothelial adhesion was assessed through a novel cell-based assay using human lymphoblastoid cell lines. A high-throughput screening method was developed to evaluate the inter-individual variability in leukocyte endothelial adhesion using lymphoblastoid cell lines derived from different donors. To assess heritability, ninety-two lymphoblastoid cell lines derived from twenty-three monozygotic twin pairs and twenty-three sibling pairs were compared. These lymphoblastoid cell lines were plated with the endothelial cell line EA.hy926 and labeled with Calcein AM dye. Fluorescence was assessed to determine endothelial cell adhesion to each lymphoblastoid cell line. Intra-pair similarity was determined for monozygotic twins and siblings using Pearson pairwise correlation coefficients. RESULTS: A leukocyte endothelial adhesion assay for lymphoblastoid cell lines was developed and optimized (CV = 8.68, Z'-factor = 0.67, SNR = 18.41). A higher adhesion correlation was found between the twins than that between the siblings. Intra-pair similarity for leukocyte endothelial adhesion in monozygotic twins was 0.60 compared to 0.25 in the siblings. The extent to which these differences are attributable to underlying genetic factors was quantified and the heritability of leukocyte endothelial adhesion was calculated to be 69.66% (p-value<0.0001). CONCLUSIONS: There is a heritable component to leukocyte endothelial adhesion. Underlying genetic predisposition plays a significant role in inter-individual variability of leukocyte endothelial adhesion.
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Células Endoteliais/citologia , Células Endoteliais/metabolismo , Variação Genética , Leucócitos/citologia , Leucócitos/metabolismo , Adesão Celular , Contagem de Células , Linhagem Celular , Fatores de Confusão Epidemiológicos , Humanos , Padrões de Herança/genética , Reprodutibilidade dos Testes , Irmãos , Gêmeos Monozigóticos/genéticaRESUMO
Multiple pathways have been proposed to explain how proteasome inhibition induces cell death, but mechanisms remain unclear. To approach this issue, we performed a genome-wide siRNA screen to evaluate the genetic determinants that confer sensitivity to bortezomib (Velcade (R); PS-341). This screen identified 100 genes whose knockdown affected lethality to bortezomib and to a structurally diverse set of other proteasome inhibitors. A comparison of three cell lines revealed that 39 of 100 genes were commonly linked to cell death. We causally linked bortezomib-induced cell death to the accumulation of ASF1B, Myc, ODC1, Noxa, BNIP3, Gadd45alpha, p-SMC1A, SREBF1, and p53. Our results suggest that proteasome inhibition promotes cell death primarily by dysregulating Myc and polyamines, interfering with protein translation, and disrupting essential DNA damage repair pathways, leading to programmed cell death.