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BACKGROUND: To evaluate the chemosensitivity to doxorubicin (DOX) in two primary cells derived from a tumor of FVB/N-Trp53tm1Hw1 knockout (KO) mice with TALEN-mediated Trp53 mutant gene, we evaluated the cell survivability, cell cycle distribution, apoptotic cell numbers and apoptotic protein expression in solid tumor cells and ascetic tumor cells treated with DOX. RESULTS: The primary tumor cells showed a significant (P < 0.05) defect for UV-induced upregulation of the Trp53 protein, and consisted of different ratios of leukocytes, fibroblasts, epithelial cells and mesenchymal cells. The IC50 level to DOX was lower in both primary cells (IC50 = 0.12 µM and 0.20 µM) as compared to the CT26 cells (IC50 = 0.32 µM), although the solid tumor was more sensitive. Also, the number of cells arrested at the G0/G1 stage was significantly decreased (24.7-23.1% in primary tumor cells treated with DOX, P < 0.05) while arrest at the G2 stage was enhanced to 296.8-254.3% in DOX-treated primary tumor cells compared with DOX-treated CT26 cells. Furthermore, apoptotic cells of early and late stage were greatly increased in the two primary cell-lines treated with DOX when compared to same conditions for CT26 cells. However, the Bax/Bcl-2 expression level was maintained constant in the primary tumor and CT26 cells. CONCLUSIONS: To the best of our knowledge, these results are the first to successfully detect an alteration in chemosensitivity to DOX in solid tumor cells and ascetic tumor cells derived from tumor of FVB/N-Trp53tm1Hw1 mice TALEN-mediated Trp53 mutant gene.
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Background: The incidence of early- and late-onset sepsis and meningitis in neonates due to maternal rectovaginal group B Streptococcus (GBS) colonization may differ with serotype distribution and clonal complex (CC). CC17 strains are associated with hypervirulence and poor disease outcomes. GBS serotypes are distinguished based on the polysaccharide capsule, the most important virulence factor. We determined the sequence type distribution of GBS isolates from pregnant women in Korea and validated whole-genome sequencing (WGS)-based prediction of antimicrobial susceptibility and capsular serotypes in GBS isolates. Methods: Seventy-five GBS isolates collected from pregnant Korean women visiting Wonju Severance Christian Hospital, Wonju, Korea between 2017 and 2019 were subjected to WGS using the NovaSeq 6000 system (Illumina, San Diego, CA, USA). Multilocus sequence types, serotypes, antimicrobial resistance genes, and hemolysin operon mutations were determined by WGS, and the latter three were compared with the results of conventional phenotypic methods. Results: The predominant lineage was CC1 (37.3%), followed by CC19 (32.0%), CC12 (17.3%), and CC17 (4.0%). All isolates were cps typeable (100%, (75/75), and 89.3% of cps genotypes (67/75) were concordant with serotypes obtained using latex agglutination. The cps genotypes of the 75 isolates were serotypes III (24.0%), V (22.7%), and VIII (17.3%). All isolates harboring intact ermB and tet were non-susceptible to erythromycin and tetracycline, respectively. Three non-hemolytic strains had 1-bp frameshift insertions in cylE. Conclusions: The low prevalence of CC17 GBS colonization may explain the low frequency of neonatal GBS infections. WGS is a useful tool for simultaneous genotyping and antimicrobial resistance determination.
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Mujeres Embarazadas , Infecciones Estreptocócicas , Recién Nacido , Femenino , Humanos , Embarazo , Proteínas Hemolisinas/genética , Tipificación de Secuencias Multilocus , Streptococcus agalactiae , Infecciones Estreptocócicas/diagnóstico , Infecciones Estreptocócicas/epidemiología , Infecciones Estreptocócicas/tratamiento farmacológico , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Eritromicina , Tetraciclina , Factores de Virulencia/genéticaRESUMEN
BACKGROUND: Ultra-deep sequencing to detect low-frequency mutations in circulating tumor-derived DNA (ctDNA) increases the diagnostic value of liquid biopsy. The demand for large ctDNA panels for comprehensive genomic profiling and tumor mutational burden (TMB) estimation is increasing; however, few ctDNA panels for TMB have been validated. Here, we designed a ctDNA panel with 531 genes, named TMB500, along with a technical and clinical validation. METHODS: Synthetic reference cell-free DNA materials with predefined allele frequencies were sequenced in a total of 92 tests in 6 batches to evaluate the precision, linearity, and limit of detection of the assay. We used clinical samples from 50 patients with various cancers, 11 healthy individuals, and paired tissue samples. Molecular barcoding and data analysis were performed using customized pipelines. RESULTS: The assay showed high precision and linearity (coefficient of determination, r2 =0.87) for all single nucleotide variants, with a limit of detection of 0.24%. In clinical samples, the TMB500 ctDNA assay detected most variants present and absent in tissues, showing that ctDNA could assess tumor heterogeneity in different tissues and metastasis sites. The estimated TMBs correlated well between tissue and blood, except in 4 cases with extreme heterogeneity that showed very high blood TMBs compared to tissue TMBs. A pilot evaluation showed that the TMB500 assay could be used for disease monitoring. CONCLUSIONS: The TMB500 assay is an accurate and reliable ctDNA assay for many clinical purposes. It may be useful for guiding the treatment of cancers with diverse genomic profiles, estimating TMB in immune therapy, and disease monitoring.
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ADN Tumoral Circulante , Humanos , ADN Tumoral Circulante/genética , Biomarcadores de Tumor/genética , Biopsia Líquida , Mutación , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
BACKGROUND: Short tandem repeat (STR)-based chimerism analysis has been widely used for chimerism monitoring after hematopoietic stem-cell transplantation (HSCT), but technical artifacts can be problematic. We designed a chimerism assay using single nucleotide polymorphisms (SNPs) adjacent and in linkage-disequilibrium (CASAL), which doubly checked for SNP pairs, and thus could reduce background errors and increase analytical sensitivity. METHODS: CASAL targeted 84 SNP pairs within 10 bp distance and in perfect linkage-disequilibrium. Using undiluted and serially diluted samples, baseline error rates, and linearity was calculated. Clinical performance of CASAL was evaluated in comparison with a conventional STR assay, using 191 posttransplant samples from 42 patients with HSCT. RESULTS: CASAL had â¼10 times lower baseline error rates compared to that of ordinary next-generation sequencing. Limit of detection and quantification of CASAL were estimated to be 0.09 and 0.39%, respectively, with a linear range of 0.1-100%. CASAL correlated well with STR assay (r2 = 0.99) and the higher sensitivity enabled detection of low-level recipient chimerism and earlier prediction of relapse. CONCLUSIONS: CASAL is a simple, analytically sensitive and accurate assay that can be used in clinical samples after HSCT with a higher performance compared to that of traditional assays. It should also be useful in other forensic and archeological testing.
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Trasplante de Células Madre Hematopoyéticas , Polimorfismo de Nucleótido Simple , Quimerismo , Humanos , Desequilibrio de Ligamiento , RecurrenciaRESUMEN
Acute myeloid leukemia (AML) is one of the most common types of leukemia. With the recent advances in sequencing technology and the growing body of knowledge on the genetics of AML, there is increasing concern about cancer predisposing germline mutations as well as somatic mutations. As familial cases sharing germline mutations are constantly reported, germline predisposition gene mutations in patients with AML are gaining attention. We performed genomic sequencing of Korean patients diagnosed with AML to identify the prevalence and characteristics of germline predisposition mutations. Among 180 patients, germline predisposition mutations were identified in 13 patients (13/180, 7.2%, eight adults and five children). Germline mutations of BLM, BRCA1, BRCA2, CTC1, DDX41, ERCC4, ERCC6, FANCI, FANCM, PALB2, and SBDS were identified. Most of the mutations are in genes involved in DNA repair and genomic stability maintenance. Patients harboring germline mutations tended to have earlier onset of AML (p = 0.005), however, the presence of germline mutations did not showed significant association with other clinical characteristics or treatment outcome. Since each mutation was rare, further study with a larger number of cases would be needed to establish the effect of the mutations.
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Predisposición Genética a la Enfermedad/genética , Mutación de Línea Germinal/genética , Leucemia Mieloide Aguda/genética , Adolescente , Adulto , Edad de Inicio , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Reparación del ADN/genética , ADN de Neoplasias/genética , Genes Relacionados con las Neoplasias/genética , Predisposición Genética a la Enfermedad/epidemiología , Inestabilidad Genómica/genética , Humanos , Lactante , Recién Nacido , Persona de Mediana Edad , Prevalencia , Secuenciación del Exoma , Adulto JovenRESUMEN
The molecular features of mantle cell lymphoma (MCL), including its increased incidence, and complex therapies have not been investigated in detail, particularly in East Asian populations. In this study, we performed targeted panel sequencing (TPS) and whole-exome sequencing (WES) to investigate the genetic alterations in Korean MCL patients. We obtained a total of 53 samples from MCL patients from five Korean university hospitals between 2009 and 2016. We identified the recurrently mutated genes such as SYNE1, ATM, KMT2D, CARD11, ANK2, KMT2C, and TP53, which included some known drivers of MCL. The mutational profiles of our cohort indicated genetic heterogeneity. The significantly enriched pathways were mainly involved in gene expression, cell cycle, and programmed cell death. Multivariate analysis revealed that ANK2 mutations impacted the unfavourable overall survival (hazard ratio [HR] 3.126; P = 0.032). Furthermore, TP53 mutations were related to worse progression-free survival (HR 7.813; P = 0.043). Among the recurrently mutated genes with more than 15.0% frequency, discrepancies were found in only 5 genes from 4 patients, suggesting comparability of the TPS to WES in practical laboratory settings. We provide the unbiased genetic landscape that might contribute to MCL pathogenesis and recurrent genes conferring unfavourable outcomes.
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Ancirinas/genética , Heterogeneidad Genética , Predisposición Genética a la Enfermedad/genética , Linfoma de Células del Manto/genética , Proteína p53 Supresora de Tumor/genética , Anciano , Femenino , Humanos , Estimación de Kaplan-Meier , Linfoma de Células del Manto/diagnóstico , Linfoma de Células del Manto/mortalidad , Masculino , Persona de Mediana Edad , Mutación/genética , Pronóstico , Supervivencia sin Progresión , República de Corea/epidemiología , Análisis de Supervivencia , Secuenciación Completa del GenomaRESUMEN
From senescence and frailty that may result from various biological, mechanical, nutritional, and metabolic processes, the human body has its own antioxidant defense enzymes to remove by-products of oxygen metabolism, and if unregulated, can cause several types of cell damage. Herein, an antioxidant, artificial nanoscale enzyme, called nanozyme (NZs), is introduced that is composed of Au nanoparticles (NPs) synthesized with a mixture of two representative phytochemicals, namely, gallic acid (GA) and isoflavone (IF), referred to as GI-Au NZs. Their unique antioxidant and anti-aging effects are monitored using Cell Counting Kit-8 and senescence-associated ß-galactosidase assays on neonatal human dermal fibroblasts (nHDFs). Furthermore, alterations in epidermal thickness and SOD activity are measured under ultraviolet light to investigate the effects of the topical application of NZs on the histological structure and antioxidant activity in hairless mice skin. Then, hepatotoxicity and nephrotoxicity in the hairless mice are monitored. It is concluded that the NZs can effectively prevent serial passage-induced senescence in nHDFs, as well as oxidative stress in mice skin, suggesting a range of strategies to further develop novel therapeutics for acute frailty.
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Antioxidantes/farmacología , Fragilidad/prevención & control , Compuestos Orgánicos de Oro/farmacología , Animales , Antioxidantes/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Ácido Gálico/química , Ácido Gálico/farmacología , Humanos , Isoflavonas/química , Isoflavonas/farmacología , Masculino , Ratones , Ratones Pelados , Microscopía Fluorescente , Compuestos Orgánicos de Oro/química , Tamaño de la Partícula , Propiedades de Superficie , Rayos UltravioletaRESUMEN
Asthma is a chronic inflammatory disease characterized by T-lymphocyte and eosinophil infiltration, mucus overproduction and airway hyper-responsiveness. The present study examined the therapeutic effects and action mechanism of a saponin-enriched extract of Asparagus cochinchinensis (SEAC) on airway inflammation and remodeling in an ovalbumin (OVA)-induced asthma model. To accomplish this, alterations of the nitric oxide (NO) level, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression levels, as well as variations in immune cell numbers, immunoglobulin E (IgE) concentration, histopathological structure and inflammatory cytokine levels were measured in lipopolysaccharide (LPS)-activated RAW264.7 cells or an OVA-induced mouse model of asthma treated with SEAC. The concentration of NO and mRNA levels of COX-2 and iNOS were significantly decreased in the SEAC + LPS-treated RAW264.7 cells compared with the vehicle + LPS-treated RAW264.7 cells. Additionally, in the OVA-induced asthma model, the number of immune cells in the bronchoalveolar lavage fluid, the concentration of OVA-specific IgE, the infiltration of inflammatory cells, the bronchial thickness and the levels of the inflammatory mediators interleukin-4 (IL-4), IL-13 and COX-2 were significantly lower in the OVA + SEACtreated group compared with the OVA + vehicletreated group. In addition, a significant reduction in goblet cell hyperplasia, peribronchiolar collagen layer thickness and VEGF expression for airway remodeling was detected in the OVA + SEACtreated group compared with the OVA + vehicletreated group. These findings indicate that SEAC is a suppressor of airway inflammation and remodeling, and may therefore be useful as an anti-inflammatory drug for the treatment of asthma.
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Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Antiasmáticos/farmacología , Antiinflamatorios/farmacología , Asma/etiología , Asma/patología , Ovalbúmina/efectos adversos , Saponinas/farmacología , Animales , Antiasmáticos/química , Antiinflamatorios/química , Antioxidantes/química , Antioxidantes/farmacología , Asparagus/química , Asma/tratamiento farmacológico , Asma/metabolismo , Biomarcadores , Líquido del Lavado Bronquioalveolar , Línea Celular , Ciclooxigenasa 2/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Inmunoglobulina E/inmunología , Mediadores de Inflamación/metabolismo , Recuento de Leucocitos , Ratones , Óxido Nítrico Sintasa de Tipo II/metabolismo , Extractos Vegetales , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Saponinas/químicaRESUMEN
To investigate the beneficial effects of diosgenin (DG) on the multiple types of brain damage induced by Aß-42 peptides and neurotoxicants, alterations in the specific aspects of brain functions were measured in trimethyltin (TMT)-injected transgenic 2576 (TG) mice that had been pretreated with DG for 21 days. Multiple types of damage were successfully induced by Aß-42 accumulation and TMT injection into the brains of TG mice. However, DG treatment significantly reduced the number of Aß-stained plaques and dead cells in the granule cells layer of the dentate gyrus. Significant suppression of acetylcholinesterase (AChE) activity and Bax/Bcl-2 expression was also observed in the DG treated TG mice (TG+DG group) when compared with those of the vehicle (VC) treated TG mice (TG+VC group). Additionally, the concentration of nerve growth factor (NGF) was dramatically enhanced in TG+DG group, although it was lower in the TG+VC group than the non-transgenic (nTG) group. Furthermore, the decreased phosphorylation of downstream members in the TrkA high affinity receptor signaling pathway in the TG+VC group was significantly recovered in the TG+DG group. A similar pattern was observed in p75(NTR) expression and JNK phosphorylation in the NGF low affinity receptor signaling pathway. Moreover, superoxide dismutase (SOD) activity was enhanced in the TG+DG group, while the level of malondialdehyde (MDA), a marker of lipid peroxidation, was lower in the TG+DG group than the TG+VC group. These results suggest that DG could exert a wide range of beneficial activities for multiple types of brain damage through stimulation of NGF biosynthesis.
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A dysfunction of endoplasmic reticulum (ER) stress response can result in various diseases, including cancer, inflammation, diabetes and neurodegenerative disorders. To investigate whether ER stress response can play an essential role in the induction and treatment of chronic constipation, alterations in the key parameters for ER stress were measured in loperamide (Lop) induced constipation Sprague Dawley (SD) rats treated with aqueous extracts of Liriope platyphylla (AEtLP), which has been shown to have a laxative effect. Symptoms of chronic constipation including alteration of stool parameters and the transverse colon's structure were successfully induced by Lop treatment. Laxative effects such as enhancement of stools parameters, recovery of the mucosa thickness, increased muscle thickness and recovery of flat luminal surface were also observed in the Lop+AEtLP treated group. Furthermore, enhancement of eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation and inositol-requiring enzyme 1 beta (IRE1ß) expression, key indicators for ER stress, that were observed in the Lop+vehicle treated group were significantly recovered in the Lop+AEtLP treated group, although the phosphorylation level of c-Jun N-terminal protein kinase (JNK) remained constant. Moreover, alterations in the transcription level of the marker genes X-box binding protein 1 (XBP-1) and growth arrest and DNA damage-inducible protein (GADD34) were similar to those of eIF2α and IRE1ß. However, their level was slightly or completely recovered after AEtLP treatment. Overall, this study provides the first evidence that ER stress response may be tightly correlated with chronic constipation induced by Lop treatment, as well as the laxative effects of AEtLP.
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Asparagus cochinchinensis has been used to treat various diseases including fever, cough, kidney disease, breast cancer, inflammatory disease and brain disease, while IL-4 cytokine has been considered as key regulator on the skin homeostasis and the predisposition toward allergic skin inflammation. However, few studies have investigated its effects and IL-4 correlation on skin inflammation to date. To quantitatively evaluate the suppressive effects of ethyl acetate extracts of A. cochinchinensis (EaEAC) on phthalic anhydride (PA)-induced skin inflammation and investigate the role of IL-4 during their action mechanism, alterations in general phenotype biomarkers and luciferase-derived signals were measured in IL-4/Luc/CNS-1 transgenic (Tg) mice with PA-induced skin inflammation after treatment with EaEAC for 2 weeks. Key phenotype markers including lymph node weight, immunoglobulin E (IgE) concentration, epidermis thickness and number of infiltrated mast cells were significantly decreased in the PA+EaEAC treated group compared with the PA+Vehicle treated group. In addition, expression of IL-1ß and TNF-α was also decreased in the PA+EaEAC cotreated group, compared to PA+Vehicle treated group. Furthermore, a significant decrease in the luciferase signal derived from IL-4 promoter was detected in the abdominal region, submandibular lymph node and mesenteric lymph node of the PA+EaEAC treated group, compared to PA+Vehicle treated group. Taken together, these results suggest that EaEAC treatment could successfully improve PA-induced skin inflammation of IL-4/Luc/CNS-1 Tg mice, and that IL-4 cytokine plays a key role in the therapeutic process of EaEAC.