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BACKGROUND: Because of the heterogeneity of metastatic colorectal cancer (mCRC), a genome-wide analysis was performed to characterize the tumor immune microenvironment (TIME). METHODS: RNA-seq analysis of 62 primary CRCs without and 63 with systemic metastasis (SM- and SM+ groups) was conducted, and the data were used in a training set after adjustment by propensity score matching. Samples were further subdivided into those with hepatic metastasis (CHM subgroup), pulmonary metastasis (CPM subgroup), or concurrent CHM and CPM (concurrent group). Validation was done by quantitative reverse-transcription polymerase chain reaction using another 40 primary CRC samples. RESULTS: Compared with the CHM or CPM subgroups, the concurrent group showed upregulated in inflammatory or immune processes, cytokine secretion, and myeloid leukocyte migration. Nine candidate genes were selected: SM-specific IDO1, JAM3, and PDE2A; CHM- or CPM-specific BIRC7; CPM-specific HISI1H2BK, and both SM-specific and CHM- or CPM-specific EPHB6, LPL, THBD, and PPBP. In a validation set of primary CRCs, JAM3 and IDO1 (p = 0.044 and p = 0.036, respectively) were confirmed to show significant upregulation and downregulation, respectively, in the SM+ group, whereas HIST1H2BK (p = 0.017) was significantly upregulated in the CPM subgroup. CONCLUSIONS: Our findings indicate that a host-suppressive TIME is established in the primary tumor of mCRC and identify immune-related site-specific markers of mCRC.
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Adenocarcinoma/patología , Neoplasias Colorrectales/patología , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/secundario , Microambiente Tumoral/genética , Estudios de Casos y Controles , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Regulación hacia Abajo , Femenino , Estudio de Asociación del Genoma Completo , Histonas/genética , Histonas/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Regulación hacia ArribaRESUMEN
Carbon dioxide (CO2) rich chlorine-bearing ices were exposed to energetic electrons in laboratory simulation experiments to investigate the formation of chlorine oxides (ClxOy) in the condensed phase on Mars. The radiolysis-induced synthesis of chlorine oxides (ClxOy) was complementarily monitored online and in situ via infrared spectroscopy (IR) and quadrupole mass spectrometry (QMS). Three discrete chlorine oxides were identified: chorine dioxide (OClO), dichlorine monoxide (ClOCl), and chloryl chloride (ClClO2). Higher irradiation doses support the facile production of ClO3- and ClO2-bearing high-order chlorine oxides. We attribute manifolds of chlorine oxides, as invoked herein, to the potential origin of perchlorates as found on Mars.
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BACKGROUND AND OBJECTIVE: Rats which have undergone spinal nerve ligation (SNL) display increases in the expression of extracellular signal-regulated kinase (ERK 1/2) and cyclic AMP response element-binding (CREB) protein. The present study was designed to determine whether lidocaine has a beneficial effect on the treatment of neuropathic pain by analysing related proteins. METHODS: Twenty-four male SpragueDawley rats were randomly allocated to three groups (eight per group): shamoperated (control) group, a neuropathic pain and normal saline group (NP+NS), a neuropathic pain and lidocaine group (NP+Lido, 2mgkg(-1) h(-1)). Anaesthetised rats received left L5 and L6 SNL. The mechanical withdrawal threshold test was performed 7 days after SNL and for 7 days with the pump implanted (saline or lidocaine). At post-implanted pump day 7, their brains and spinal cords were harvested. ERK 1/2, CREB proteins and mRNA amounts of pro-inflammatory cytokines (tumour necrosis factor a, intercellular adhesion molecule 1, monocyte chemo-attractive protein 1 and macrophage inflammatory protein 2) were assessed by immunoblotting or reverse transcriptase-PCR on samples collected from the three groups. RESULTS: Lidocaine increased the mechanical withdrawal threshold of a neuropathic rats. In only spinal tissues, ERK 1/2 and CREB proteins in the NP+Lido group was significantly reduced to 39%, and 48% in comparison with the NP+NS group. The NP+Lido group showed a significant reduction in mRNA amounts of pro-inflammatory cytokines compared with the NP+NS group (P<0.05). CONCLUSION: These results suggest that lidocaine therapy may be effective in treating neuropathic pain after spinal nerve injury, and that these effects may occur via suppression of ERK 1/2 and CREB signalling proteins and anti-inflammatory effects.
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Anestésicos Locales/farmacología , Lidocaína/farmacología , Neuralgia/tratamiento farmacológico , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Masculino , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
For time-to-event data, the power of the two sample logrank test for the comparison of two treatment groups can be greatly influenced by the ratio of the number of patients in each of the treatment groups. Despite the possible loss of power, unequal allocations may be of interest due to a need to collect more data on one of the groups or to considerations related to the acceptability of the treatments to patients. Investigators pursuing such designs may be interested in the cost of the unbalanced design relative to a balanced design with respect to the total number of patients required for the study. We present graphical displays to illustrate the sample size adjustment factor, or ratio of the sample size required by an unequal allocation compared to the sample size required by a balanced allocation, for various survival rates, treatment hazards ratios, and sample size allocation ratios. These graphical displays conveniently summarize information in the literature and provide a useful tool for planning sample sizes for the two sample logrank test.
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Ensayos Clínicos como Asunto/estadística & datos numéricos , Presentación de Datos , Tamaño de la Muestra , Humanos , Modelos de Riesgos ProporcionalesRESUMEN
Improved methods for predicting chemoresponsiveness involving the identification of polymorphic markers is highly desirable, considering narrow therapeutic index and frequent resistance to anti-cancer regimens. The genome-wide screening of chemosensitive single nucleotide polymorphisms (SNPs) was undertaken in association with in vitro chemosensitivity assays in 104 colorectal cancer patients for the initial screening step. Allele frequency, linkage disequilibrium, potential function, and Hardy-Weinberg equilibrium of the candidate SNPs were then determined for the identifying step. Finally, clinical association analysis in the other 260 evaluable patients or cell viability assays of transfected RKO cells was used to verify candidate SNPs for the validation step. In total, 12 SNPs to six regimens were initially chosen during the screening and identifying steps. In patients receiving fluoropyrimidine-based adjuvant chemotherapy, the substitution alleles of GPC5 rs553717 (AA) correlated significantly with tumor recurrence and shorter disease-free survival (P = 0.019 and 0.023, respectively). Interestingly, RKO cells expressing mutant GPC5 showed enhanced cell death in response to 5-FU in cytotoxicity assays. Patients that were homozygous for the reference alleles SSTR4 rs2567608 (AA) and EPHA7 rs2278107 (TT) showed lower disease control rates in response to irinotecan and oxaliplatin regimens, respectively, than those with substitution alleles (P = 0.022 and 0.014, respectively). Thus, we identified chemosensitive SNP markers using a novel three step process of genome-wide analysis consisting of in vitro screening, identification, and validation. The candidate chemosensitive SNP markers identified in our study, including those identified in vitro, can now be further verified in a large cohort study.
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Antineoplásicos/uso terapéutico , Biomarcadores , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Polimorfismo de Nucleótido Simple , Anciano , Quimioterapia Adyuvante , Estudios de Cohortes , Supervivencia sin Enfermedad , Femenino , Fluorouracilo/uso terapéutico , Frecuencia de los Genes , Humanos , Desequilibrio de Ligamiento , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Promoter methylation of colorectal cancer-related genes were examined with respect to phenotype and tumor progression. MATERIALS AND METHODS: We assayed promoter methylation of 11 genes including established CpG island methylator phenotype (CIMP) markers (MLH1, MINT1, MINT2, MINT31, p16 ( INK4a ), p14 ( ARF ), and CACNA1G) and four genes (COX2, DAPK, MGMT, and APC) frequently methylated in colorectal cancer in 285 patients with sporadic colorectal cancer. RESULTS: CIMP+ tumors were more than two times more frequent among high-frequency microsatellite instability tumors (MSI-H) than in tumors without MSI (P < or = .0001-.002). COX2 and DAPK methylation were significantly associated with CIMP+ and MSI. KRAS showed tendency toward more frequent codon 12-13 mutations identified in tumors with APC and p16 ( INK4a ) methylation than in those with unmethylation (P = .033 and .05, respectively). Additionally, tumors with synchronous adenoma were associated with p16 ( INK4a ) methylation (P = .004). The p16 ( INK4a ) methylation was significantly associated with poor overall and disease-free survival in 131 rectal cancer patients who underwent curative operation, according to multivariate analyses (relative risk [RR] = 0.317 and 0.349; P = .033 and .024, respectively). Specifically, in 175 stage II and III patients receiving adjuvant-based fluoropyrimidine chemotherapy, p16 ( INK4a ) methylation and MINT31 unmethylation showed a significant or tendency toward an association with recurrence and DFS (P = .007-.032). CONCLUSIONS: The study suggests that specific CIMP markers, such as p16 ( INK4a ) and MINT31, should be further verified as potential epigenetic targets for the design of efficient chemotherapy regimens. We also identified a subset of colorectal cancer, possibly comprising APC methylation-KRAS mutation-p16 ( INK4a ) methylation.
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Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Islas de CpG/genética , Metilación de ADN , Genes Supresores de Tumor , Regiones Promotoras Genéticas/genética , Adenocarcinoma/patología , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Inestabilidad de Microsatélites , Persona de Mediana Edad , Mutación/genética , Fenotipo , Pronóstico , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Proteínas ras/genéticaRESUMEN
The objective of this study was to examine the expression profiles of follistatin (FST) and its associated molecules (MSTN, INHA, INHBB, INHBA, ACVR2A, and ACVR2B) in the oviduct of laying hens at 3 hr and 20 hr post-ovulation (p.o., n = 5; 35 weeks old), molting (n = 5; 60 weeks old), and non-laying (n = 4; 35-60 weeks old) hens and also to localize the FST by using immunohistochemistry assay. Expression of FST was significantly higher (p < .05), and MSTN was lower in the uterus of laying hens around 15-20 hr p.o. (during eggshell formation), however, their expressions in the magnum remain unchanged across different physiological stages of hens. FST was mainly expressed in the luminal and glandular epithelium of the uterine tissues, and their expression intensity was highest in laying hens during the eggshell mineralization. There was a relatively increased expression of INHA in the magnum of laying hens around 3 hr p.o. as compared to non-laying and molting hens. At the same time (3 hr p.o.), there was a significant (p < .05) decrease in the expression of the INHBB, ACVR2A, and ACV2B. These results indicate that follistatin may regulate the differentiation of uterine luminal and glandular epithelium during eggshell biomineralization.
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Biomineralización/genética , Pollos/genética , Pollos/fisiología , Cáscara de Huevo/embriología , Folistatina/genética , Folistatina/metabolismo , Expresión Génica/genética , Oviductos/metabolismo , Oviposición/genética , Oviposición/fisiología , Ovulación/genética , Ovulación/fisiología , Transcriptoma , Animales , Biomineralización/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Cáscara de Huevo/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Femenino , Oviductos/fisiología , Útero/citología , Útero/metabolismoRESUMEN
BACKGROUND AND AIMS: This study was to evaluate the efficacy of histone deacetylase (HDAC) inhibitors in colorectal cancer together with other established regimens. MATERIALS AND METHODS: Chemosensitivities of 114 colorectal cancer patients to established regimens (fluorouracil (5-FU with leucovorin (FL), capecitabine, FL with irinotecan (FLIRI), and FL with oxaliplatin (FLOX)) as well as five hydroxamic acid derivatives (suberoylanilide hydroxamic acid, PXD101, and three novel candidates of CG-1, CG-2, and CG-3) were comparatively evaluated using the histoculture drug response assay. RESULTS: The chemosensitivity with established regimens was between 34.2% and 52.6%, when the cutoff value of the inhibition ratio was set at 30%, and between 54.5% and 84.1% with HDAC inhibitors. All HDAC inhibitors displayed synergistic effects in combination with established regimens of FLOX and FLIRI (P < or = 0.0001-0.002). Advanced T- and N-category tumors and patients with synchronous adenoma displayed higher chemosensitivity to CG-3, CG-2, and CG-1, respectively, on a multivariate analysis (P = 0.023, 0.044, and 0.045, respectively). Tumors with mismatch repair defects were closely correlated with chemosensitivities to combined regimens of PDX101 with FLOX and FLIRI (P = 0.044 and 0.048, respectively). CONCLUSIONS: Our findings firstly demonstrated the chemo-responsiveness of colorectal cancers to HDAC inhibitors with therapeutic efficacy comparable to the established regimens. Additionally, tumor growth and heredity were significantly associated with specific regimens, supporting their possible role as chemosensitive predictors.
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Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/enzimología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/enzimología , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Inhibidores de Histona Desacetilasas , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis MultivarianteRESUMEN
Clinicopathologic features of sporadic colorectal adenocarcinomas were compared using integrated data from 224 [corrected] patients subjected to curative resection. Individual steps in the tumorigenesis pathway, that is, adenomatosis polyposis coli (APC), Wnt-activated, base excision repair mutations, mismatch repair defects, RAF-mediated, transforming growth factor (TGF)-beta-suppressed, bone morphogenic protein (BMP)-suppressed, and p53 alterations, were examined in terms of genetic and epigenetic changes, as well as protein expression. Genetic and molecular alterations of right colon cancers were distinct from those of left colon and rectal cancers. Rectal cancers showed the attenuated phenotype of left colon cancers. Tumors most frequently displayed either TGF-beta- or BMP-suppressed alterations (81.2%), followed by RAF-mediated alterations (78.6%), and mismatch repair defects (38.4%), constituting a total of 24 integrated pathways. Tumors lacking APC mutations or carrying the RAF alteration (V600E) were frequently associated with lymphovascular invasion and lymph node metastasis (P < 0.05). Poorly differentiated or mucinous adenocarcinomas were generally associated with high level microsatellite instability, Axin2 suppression, TGF-beta1 or BMPR1A suppression, loss of heterozygosity of D18S46 or D18S474, and absence of base excision repair mutations (P < 0.0001-0.05). Early tumor recurrence was significantly correlated with lack of APC mutations (P = 0.036). Moreover, tumors that concurrently displayed APC/Wnt-activated, TGF-beta/BMP-suppressed, and p53 alterations were significantly predisposed to early recurrence (P = 0.026). Our data clearly indicate that particular steps or pathways of colorectal tumorigenesis are closely associated with characteristic clinicopathologic features that, in turn, determine biological behavior, such as tumor growth, invasion, and recurrence.
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Adenocarcinoma/etiología , Neoplasias Colorrectales/etiología , Adenocarcinoma/genética , Adenocarcinoma/patología , Adulto , Anciano , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Reparación de la Incompatibilidad de ADN , Femenino , Genes APC , Humanos , Pérdida de Heterocigocidad , Masculino , Inestabilidad de Microsatélites , Persona de Mediana Edad , Quinasas raf/fisiologíaRESUMEN
BACKGROUND AND AIM: Increased colonic motility is a well-known stress response and corticotropin releasing hormone plays an important role in this response, but sequential change of bowel habit and adrenal function during chronic stress has not been reported. The objective of this study was to evaluate the effect of chronic stress on bowel habit and adrenal function. METHODS: Male Sprague-Dawley rats were exposed to chronic variable stress (CVS) for 6 weeks. We measured daily the number and weight of pellets and weekly urinary corticosterone. After 6 weeks of experiment, visceromotor response (VMR) to colorectal distention (CRD), serum corticosterone and adrenal glands weight were measured. RESULTS: The number and weight of pellets in CVS rats was greater than those of the control rats initially and decreased during the later period. However, CVS rats showed continuously exaggerated daily variation of pellet number than control rats to the end of experimental period. Urinary corticosterone was increased in CVS rat until the fifth week, but urine and serum corticosterone were not statistically different between groups at the sixth week. However, the relative weight of adrenal glands was higher in CVS rats at the sixth week. CVS rats showed exaggerated VMR to CRD than the control rats. CONCLUSIONS: The prolonged and variable stress to rats induced sustained bowel habit dysfunction and visceral hypersensitivity without adaptation. Chronic stress also increased adrenal activity from the early phase and finally caused adrenal hypertrophy with relatively decreased activity. But adrenal change was not parallel to bowel habit change and it remains to be seen whether adrenal dysfunction is directly related to bowel habit dysfunction.
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Glándulas Suprarrenales/fisiopatología , Colon/fisiopatología , Defecación , Estrés Psicológico/fisiopatología , Glándulas Suprarrenales/metabolismo , Glándulas Suprarrenales/patología , Animales , Peso Corporal , Enfermedad Crónica , Corticosterona/sangre , Corticosterona/orina , Modelos Animales de Enfermedad , Hipertrofia , Masculino , Ratas , Ratas Sprague-Dawley , Sensación , Umbral Sensorial , Estrés Psicológico/metabolismo , Estrés Psicológico/patología , Factores de TiempoRESUMEN
Alzheimer's disease is a neurodegenerative disease characterized by the impairment of cognitive function and loss of memory, affecting millions of individuals worldwide. With the dramatic increase in the prevalence of Alzheimer's disease, it is expected to impose extensive public health and economic burden. However, this burden is particularly heavy on the caregivers of Alzheimer's disease patients eliciting neuropsychiatric symptoms that include mood swings, hallucinations, and depression. Interestingly, these neuropsychiatric symptoms are shared across symptoms of bipolar disorder, schizophrenia, and major depression disorder. Despite the similarities in symptomatology, comorbidities of Alzheimer's disease and these neuropsychiatric disorders have not been studied in the Alzheimer's disease model. Here, we explore the comprehensive changes in gene expression of genes that are associated with bipolar disorder, schizophrenia, and major depression disorder through the microarray of an Alzheimer's disease animal model, the forebrain specific PSEN double knockout mouse. To analyze the genes related with these three neuropsychiatric disorders within the scope of our microarray data, we used selected 1207 of a total of 45,037 genes that satisfied our selection criteria. These genes were selected on the basis of 14 Gene Ontology terms significantly relevant with the three disorders which were identified by previous research conducted by the Psychiatric Genomics Consortium. Our study revealed that the forebrain specific deletion of Alzheimer's disease genes can significantly alter neuropsychiatric disorder associated genes. Most importantly, most of these significantly altered genes were found to be involved with schizophrenia. Taken together, we suggest that the synaptic dysfunction by mutation of Alzheimer's disease genes can lead to the manifestation of not only memory loss and impairments in cognition, but also neuropsychiatric symptoms.
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This study was done to characterize base excision repair (BER) genes and adenomatous polyposis coli (APC) alterations in the tumorigenesis of multiple colorectal adenomas in Korean patients. In total, 217 adenomas (mean number = 10) and 117 cancers were available from 143 patients. The heterozygous genotype of OGG1 c.1-18G>T was closely associated with multiple adenoma families (P < 0.001), while MYH A359V mutation exhibited a tendency (P = 0.053). MYH R170G mutation was exclusively identified in one patient. The G:C>T:A transversion or attenuated familial adenomatous polyposis (AFAP) mutations of APC was identified in the specific genotypes of BER variants. Tubular adenomas or adenomas with none-to-mild dysplasia were significantly associated with polymorphic genotypes of OGG1 IVS4-15 and S326C. In addition, large and pedunculated adenomas were more frequent in patients with G:C>T:A transversion and AFAP mutations of APC, respectively. However, BER variants were not associated with mismatch repair or altered p53 protein expression. Conclusively, two novel mutations of MYH and a novel OGG1 polymorphism seemed to be associated with multiple colorectal adenomas in Korean families, differing from those in other ethnic groups. Some BER variants involved in specific APC mutations are associated with characteristics of histogenesis other than altered mismatch repair or p53 pathway.
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Adenocarcinoma/genética , Adenoma/genética , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Mutación , Neoplasias Primarias Múltiples/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenoma/metabolismo , Adenoma/patología , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Reparación de la Incompatibilidad de ADN , Análisis Mutacional de ADN , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/metabolismo , Estadificación de Neoplasias , Neoplasias Primarias Múltiples/metabolismo , Neoplasias Primarias Múltiples/patología , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Polimorfismo de Nucleótido Simple , Estudios ProspectivosRESUMEN
BACKGROUND: To elucidate biogas microbial communities and processes, the application of high-throughput DNA analysis approaches is becoming increasingly important. Unfortunately, generated data can only partialy be interpreted rudimentary since databases lack reference sequences. RESULTS: Novel cellulolytic, hydrolytic, and acidogenic/acetogenic Bacteria as well as methanogenic Archaea originating from different anaerobic digestion communities were analyzed on the genomic level to assess their role in biomass decomposition and biogas production. Some of the analyzed bacterial strains were recently described as new species and even genera, namely Herbinix hemicellulosilytica T3/55T, Herbinix luporum SD1DT, Clostridium bornimense M2/40T, Proteiniphilum saccharofermentans M3/6T, Fermentimonas caenicola ING2-E5BT, and Petrimonas mucosa ING2-E5AT. High-throughput genome sequencing of 22 anaerobic digestion isolates enabled functional genome interpretation, metabolic reconstruction, and prediction of microbial traits regarding their abilities to utilize complex bio-polymers and to perform specific fermentation pathways. To determine the prevalence of the isolates included in this study in different biogas systems, corresponding metagenome fragment mappings were done. Methanoculleus bourgensis was found to be abundant in three mesophilic biogas plants studied and slightly less abundant in a thermophilic biogas plant, whereas Defluviitoga tunisiensis was only prominent in the thermophilic system. Moreover, several of the analyzed species were clearly detectable in the mesophilic biogas plants, but appeared to be only moderately abundant. Among the species for which genome sequence information was publicly available prior to this study, only the species Amphibacillus xylanus, Clostridium clariflavum, and Lactobacillus acidophilus are of importance for the biogas microbiomes analyzed, but did not reach the level of abundance as determined for M. bourgensis and D. tunisiensis. CONCLUSIONS: Isolation of key anaerobic digestion microorganisms and their functional interpretation was achieved by application of elaborated cultivation techniques and subsequent genome analyses. New isolates and their genome information extend the repository covering anaerobic digestion community members.
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BACKGROUND: One of the most promising technologies to sustainably produce energy and to mitigate greenhouse gas emissions from combustion of fossil energy carriers is the anaerobic digestion and biomethanation of organic raw material and waste towards biogas by highly diverse microbial consortia. In this context, the microbial systems ecology of thermophilic industrial-scale biogas plants is poorly understood. RESULTS: The microbial community structure of an exemplary thermophilic biogas plant was analyzed by a comprehensive approach comprising the analysis of the microbial metagenome and metatranscriptome complemented by the cultivation of hydrolytic and acido-/acetogenic Bacteria as well as methanogenic Archaea. Analysis of metagenome-derived 16S rRNA gene sequences revealed that the bacterial genera Defluviitoga (5.5 %), Halocella (3.5 %), Clostridium sensu stricto (1.9 %), Clostridium cluster III (1.5 %), and Tepidimicrobium (0.7 %) were most abundant. Among the Archaea, Methanoculleus (2.8 %) and Methanothermobacter (0.8 %) were predominant. As revealed by a metatranscriptomic 16S rRNA analysis, Defluviitoga (9.2 %), Clostridium cluster III (4.8 %), and Tepidanaerobacter (1.1 %) as well as Methanoculleus (5.7 %) mainly contributed to these sequence tags indicating their metabolic activity, whereas Hallocella (1.8 %), Tepidimicrobium (0.5 %), and Methanothermobacter (<0.1 %) were transcriptionally less active. By applying 11 different cultivation strategies, 52 taxonomically different microbial isolates representing the classes Clostridia, Bacilli, Thermotogae, Methanomicrobia and Methanobacteria were obtained. Genome analyses of isolates support the finding that, besides Clostridium thermocellum and Clostridium stercorarium, Defluviitoga tunisiensis participated in the hydrolysis of hemicellulose producing ethanol, acetate, and H2/CO2. The latter three metabolites are substrates for hydrogentrophic and acetoclastic archaeal methanogenesis. CONCLUSIONS: Obtained results showed that high abundance of microorganisms as deduced from metagenome analysis does not necessarily indicate high transcriptional or metabolic activity, and vice versa. Additionally, it appeared that the microbiome of the investigated thermophilic biogas plant comprised a huge number of up to now unknown and insufficiently characterized species.
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PURPOSE: Prediction of individual responsiveness to preoperative chemoradiation therapy (CRT) is urgently needed in patients with poorly responsive locally advanced rectal cancer (LARC). METHODS AND MATERIALS: Candidate methylation genes associated with radiosensitivity were identified using a 3-step process. In the first step, genome-wide screening of methylation genes was performed in correlation with histopathologic tumor regression grade in 45 patients with LARC. In the second step, the methylation status of selected sites was analyzed by pyrosequencing in 67 LARC patients, including 24 patients analyzed in the first step. Finally, colorectal cancer cell clones with stable KLHL34 knockdown were generated and tested for cellular sensitivity to radiation. RESULTS: Genome-wide screening identified 7 hypermethylated CpG sites (DZIP1 cg24107021, DZIP1 cg26886381, ZEB1 cg04430381, DKK3 cg041006961, STL cg00991794, KLHL34 cg01828474, and ARHGAP6 cg07828380) associated with preoperative CRT responses. Radiosensitivity in patients with hypermethylated KLHL34 cg14232291 was confirmed by pyrosequencing in additional cohorts. Knockdown of KLHL34 significantly reduced colony formation (KLHL34 sh#1: 20.1%, P=.0001 and KLHL34 sh#2: 15.8%, P=.0002), increased the cytotoxicity (KLHL34 sh#1: 14.8%, P=.019 and KLHL34 sh#2: 17.9%, P=.007) in LoVo cells, and increased radiation-induced caspase-3 activity and the sub-G1 population of cells. CONCLUSIONS: The methylation status of KLHL34 cg14232291 may be a predictive candidate of sensitivity to preoperative CRT, although further validation is needed in large cohorts using various cell types.
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Quimioradioterapia , Islas de CpG/genética , Metilación de ADN , Epigénesis Genética/fisiología , Neoplasias del Recto/genética , Neoplasias del Recto/terapia , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quimiocinas , Femenino , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Marcadores Genéticos , Estudio de Asociación del Genoma Completo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Cuidados Preoperatorios , Tolerancia a Radiación/genética , Neoplasias del Recto/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Resultado del Tratamiento , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
PURPOSE AND METHODS: Docetaxel second-line chemotherapy for non-small-cell lung cancer (NSCLC) has previously been shown to improve survival significantly compared with best supportive care (BSC). This multicenter phase III trial prospectively investigated quality of life (QOL) in NSCLC patients treated with either second-line docetaxel or BSC. Patients with stage IIIB/IV NSCLC, performance status < or =2, and adequate biochemistry and hematology were eligible if they had received > or =1 platinum-based chemotherapy regimens. Patients were randomized to docetaxel 100 mg/m2 (n=49) or, after protocol amendment, to docetaxel 75 mg/m2 (n=55), or to BSC (n=100), with Lung Cancer Symptom Scale (LCSS) and/or QLQ-C30 (with LC13 module) assessment every 3 weeks. RESULTS: Longitudinal analysis including all available assessments over time showed statistically significant differences in patient-rated pain scores in favor of docetaxel overall (P=0.005) or docetaxel 100 mg/m2 (P=0.003) compared to BSC. Trends in favor of docetaxel were noted on observer-rated scales for fatigue and pain for all docetaxel patients, and for total LCSS score, appetite and fatigue with docetaxel 100 mg/m2. Changes from baseline to the last available assessment (end point) showed significantly (P<0.05) less deterioration in LCSS pain score in patients with docetaxel (75 and 100 mg/m2 combined) than with BSC. An improved mean pain score with docetaxel 100 mg/m2 was contrasted with a deterioration in mean pain score with BSC (P<0.01). There was also significantly less deterioration in the global QOL today score with docetaxel 100 mg/m2 (P<0.01). Similar trends were recorded with QLQ-C30 assessments. CONCLUSIONS: Second-line docetaxel therapy for advanced NSCLC significantly improves survival with a trend towards less deterioration in QOL compared with BSC.
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Antineoplásicos Fitogénicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/psicología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/psicología , Calidad de Vida , Taxoides/uso terapéutico , Adulto , Anciano , Carcinoma de Pulmón de Células no Pequeñas/patología , Docetaxel , Fatiga , Femenino , Humanos , Infusiones Intravenosas , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Dolor , Terapia Recuperativa , Análisis de SupervivenciaRESUMEN
PURPOSE: Studies aimed at predicting individual responsiveness to preoperative chemoradiation therapy (CRT) are urgently needed, especially considering the risks associated with poorly responsive patients. METHODS AND MATERIALS: A 3-step strategy for the determination of CRT sensitivity is proposed based on (1) the screening of a human genome-wide single-nucleotide polymorphism (SNP) array in correlation with histopathologic tumor regression grade (TRG); (2) clinical association analysis of 113 patients treated with preoperative CRT; and (3) a cell-based functional assay for biological validation. RESULTS: Genome-wide screening identified 9 SNPs associated with preoperative CRT responses. Positive responses (TRG 1-3) were obtained more frequently in patients carrying the reference allele (C) of the SNP CORO2A rs1985859 than in those with the substitution allele (T) (P=.01). Downregulation of CORO2A was significantly associated with reduced early apoptosis by 27% (P=.048) and 39% (P=.023) in RKO and COLO320DM colorectal cancer cells, respectively, as determined by flow cytometry. Reduced radiosensitivity was confirmed by colony-forming assays in the 2 colorectal cancer cells (P=.034 and .015, respectively). The SNP FAM101A rs7955740 was not associated with radiosensitivity in the clinical association analysis. However, downregulation of FAM101A significantly reduced early apoptosis by 29% in RKO cells (P=.047), and it enhanced colony formation in RKO cells (P=.001) and COLO320DM cells (P=.002). CONCLUSION: CRT-sensitive SNP markers were identified using a novel 3-step process. The candidate marker CORO2A rs1985859 and the putative marker FAM101A rs7955740 may be of value for the prediction of radiosensitivity to preoperative CRT, although further validation is needed in large cohorts.
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Biomarcadores de Tumor/genética , Quimioradioterapia/métodos , Proteínas de Microfilamentos/genética , Tolerancia a Radiación/genética , Neoplasias del Recto/genética , Neoplasias del Recto/terapia , Alelos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis , Capecitabina , Línea Celular Tumoral , Quimioradioterapia/efectos adversos , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/análogos & derivados , Marcadores Genéticos/genética , Estudio de Asociación del Genoma Completo/métodos , Técnicas de Genotipaje , Humanos , Leucovorina/administración & dosificación , Masculino , Persona de Mediana Edad , Cuidados Preoperatorios , Neoplasias del Recto/patología , Neoplasias del Recto/cirugíaRESUMEN
PURPOSE: Few efficient methylation markers of chemosensitivity have been discovered. The genome-wide analysis of methylation markers is needed to identify chemosensitive candidates to targeted therapy. METHODS: This study describes a two-step process to select chemosensitive candidates of methylation genes. A genome-wide screening of methylation genes was performed using a Beadarray and an in vitro chemosensitivity assay of 119 colorectal cancers (CRCs). Ten candidate genes identified during the initial screening were verified by biological utility assessment using cell viability assays of transfected CRC cells. RESULTS: Five methylation genes related to sensitivity to bevacizumab regimens (RASSF1, MMP25, KCNQ1, ESR1, and GALR2) or cetuximab regimens (SCL18A2, GPX7, NID2, IGFBP3, and ALX4) were chosen during the first step. A viability assay revealed that GALR2-overexpressing HCT116 cells were significantly more chemosensitive to bevacizumab regimens than control cells (P = 0.022 and 0.019 for bevacizumab with FOLFIRI and FOLFOX, respectively), concurrently verified on a caspase-3 activity assay. GPX7- or ALX4-overexpressed RKO cells were significantly less viable to cetuximab regimens compared to control cells (GPX7: P = 0.027 each for cetuximab with FOLFIRI and FOLFOX; ALX4: P = 0.049 and 0.003 for cetuximab with FOLFIRI and FOLFOX, respectively), but caspase-3 activity was not prominent in GPX7-overexpressed RKO cells. CONCLUSIONS: Two novel genes, GALR2 and ALX4, have been identified as chemosensitive methylation candidates to bevacizumab and cetuximab regimens, respectively. As our study did not include a clinical association study, the two candidates should be validated in large clinical cohorts, hopefully predicting responsive patients to targeted regimens.
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Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Metilación de ADN , Anciano , Anticuerpos Monoclonales Humanizados , Bevacizumab , Cetuximab , Proteínas de Unión al ADN/genética , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Persona de Mediana Edad , Receptor de Galanina Tipo 2/genética , Factores de Transcripción/genética , Proteínas Supresoras de TumorRESUMEN
PURPOSE: Methods for predicting individual responsiveness to targeted chemotherapy are urgently needed, considering the frequent resistance and extremely high cost. EXPERIMENTAL DESIGN: A chemosensitive single-nucleotide polymorphism (SNP) discovery schema is presented that utilizes (i) genome-wide SNP screening with a human SNP array and an in vitro chemosensitivity assay in 118 colorectal cancers, (ii) clinical association analysis in the other 98 patients who had received chemotherapy for metastatic cancer, and (iii) biological utility assessment using cell viability assays of transfected colorectal cancer (CRC) cells. RESULTS: Nine SNPs related to bevacizumab and cetuximab regimen sensitivity were chosen during screening. Overall responses for bevacizumab regimens revealed that patients carrying the TT genotype at ANXA11 rs1049550 or at least one G allele at LINS1 rs11247226 seemed greater chemosensitive than those carrying at least one C allele or the AA genotype, respectively (P < 0.05). For cetuximab regimens, patients carrying the GG genotype at DFNB31 rs2274159 or LIFR rs3729740 seemed greater chemosensitive than those carrying at least one A allele (P = 0.025 and P = 0.07). Cytotoxicity analyses showed that all RKO and HCT116 CRC clones transfected with the G allele at LIFR rs3729740 and the C allele at ISX rs361863 were more sensitive to cetuximab regimens than those with the A and T allele, respectively (P ≤ 0.001-0.024). CONCLUSIONS: Chemosensitive SNP markers were identified using a novel three-step process. The candidate marker LIFR rs3729740 and possibly ISX rs361863 will hopefully predict responsive patients to cetuximab regimens, although further validation is needed in large cohorts.
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Anticuerpos Monoclonales/uso terapéutico , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Resistencia a Antineoplásicos/genética , Polimorfismo de Nucleótido Simple , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales Humanizados , Antineoplásicos/uso terapéutico , Bevacizumab , Cetuximab , Neoplasias Colorrectales/patología , Femenino , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Metástasis de la Neoplasia , Pronóstico , Resultado del TratamientoRESUMEN
BACKGROUND: The canonical molecular changes in colorectal tumorigenesis were assessed for correlation with response to chemotherapy, in order to identify candidate markers. PATIENTS AND METHODS: In total, 156 patients received adjuvant postoperative fluoropyrimidine-based chemotherapy and 32 patients received oxaliplatin- or irinotecan-based chemotherapy following palliative surgery or for metastatic or recurrent colorectal tumors. Representative molecular changes in tumor tissues, including adenomatous polyposis coli (APC) gene, wingless-type MMTV integration site family (Wnt), mismatch repair (MMR), RAF, transforming growth factor (TGF)-beta, bone morphogenetic protein, and p53, had been previously determined, with an additional 42 patients included in this analysis. RESULTS: The disease-free survival period (mean+/-SEM) was significantly longer after fluoropyrimidine-based adjuvant chemotherapy in tumors with TGF-beta2 expression (42+/-1.4 vs. 21+/-4.7 months; p=0.005) and D18S46 loss of heterozygosity or microsatellite instability (45.7+/-1.5 vs. 40.5+/-1.4 months; p=0.048). In the metastatic settings, the high disease-control rate of oxaliplatin and irinotecan regimens correlated significantly with wild-type APC and intact MMR, respectively, relative to mutant APC and defective MMR (p=0.013, respectively). Interestingly, specific molecular steps of tumorigenensis were closely associated with particular toxicities. CONCLUSION: A subset of molecular changes occurring during colorectal tumorigenesis showed significant associations with therapeutic responses and toxicities to chemotherapy regimens, suggesting that these changes may be candidate predictors of chemoresponsiveness with further validation.