RESUMEN
Filamentous bacteria, as one of the common bacteria types in wastewater biological treatment, are considered to be the main factor to induce sludge bulking. However, because of its special filamentous shape, it plays a crucial role in the formation of sludge particles. Taking filamentous bulking sludge as the research object, the effect of filamentous bacteria on the sludge granulation process and maintaining the stability of sludge granules was studied, and the microbial diversity of the sludge system was analyzed. Filamentous bulking sludge (SVI=241.56 mL·g-1) and flocculated sludge (SVI=64.22 mL·g-1) were respectively inoculated to carry out granulation culture. The results showed that the time of particle appearance of bulking sludge and flocculated sludge was 20 days and 40 days, respectively; the mature particle sizes were 650 µm and 700 µm, respectively; and the granulation time of bulking sludge was only half that of flocculated sludge. After adding the anoxic zone, the granules were broken to differing degrees, but the SV30/SV5 value of mature granules recovered to 1 after short-term fluctuation, and the stability of the mature granules was stronger. The analysis of microbial community structure showed that the relative abundance of norank_o__Saccharimonadales, unclassified_o__Saccharimonadales, and unclassified_f__Saccharimonadaceae increased from 0.05%, 0.01%, and 0.01% to 4.09%, 3.15%, and 1.12%. The existence and accumulations of these hydrophobic bacteria were important for rapid granulation. The removal rates of COD, NH4+-N, and TN were 94%, 99%, and 35% and 92%, 97%, and 30%, respectively, in SBR1 of bulking sludge and SBR2 of flocculated sludge, and the removal rates of TP were 60% and 30%, respectively.
Asunto(s)
Microbiota , Aguas del Alcantarillado , Bacterias , Reactores Biológicos/microbiología , Aguas del Alcantarillado/microbiología , Eliminación de Residuos Líquidos/métodosRESUMEN
Two ATP-binding cassette transporters, ABCB1/MDR1 and ABCG2/BCRP, are considered the most critical determinants for chemoresistance in hepatocellular carcinoma. However, their roles in the chemoresistance in liver cancer stem cells remain elusive. Here we explored the role of inhibition of MDR1 or ABCG2 in sensitizing liver cancer stem cells to doxorubicin, the most frequently used chemotherapeutic agent in treating liver cancer. We show that the inhibition of MDR1 or ABCG2 in Huh7 and PLC/PRF/5 cells using either pharmacological inhibitors or RNAi resulted in the elevated level of intracellular concentration of doxorubicin and the accompanied increased apoptosis as determined by confocal microscopy, high-performance liquid chromatography, flow cytometry, and annexin V assay. Notably, the inhibition of MDR1 or ABCG2 led to the reversal of the chemoresistance, as evident from the enhanced death of the chemoresistant liver cancer stem cells in tumorsphere-forming assays. Thus, the elevation of effective intracellular concentration of doxorubicin via the inhibition of MDR1 or ABCG2 represents a promising future strategy that transforms doxorubicin from a traditional chemotherapy agent into a robust killer of liver cancer stem cells for patients undergoing transarterial chemoembolization.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Carcinoma Hepatocelular/metabolismo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Carcinoma Hepatocelular/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclosporinas/farmacología , Dicetopiperazinas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Células Madre Neoplásicas/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Hematopoietic stem cells (HSCs) can survive long-term in a state of dormancy. Little is known about how histone deacetylase inhibitors (HDACi) affect HSC kinetics. Here, we use trichostatin A (TSA), a histone deacetylase inhibitor, to enforce histone acetylation and show that this suppresses cell cycle entry by dormant HSCs. Previously, we found that haploinsufficiency of PSF1, a DNA replication factor, led to attenuation of the bone marrow (BM) HSC pool size and lack of acute proliferation after 5-FU ablation. Because PSF1 protein is present in CD34(+) transiently amplifying HSCs but not in CD34(-) long-term reconstituting-HSCs which are resting in a dormant state, we analyzed the relationship between dormancy and PSF1 expression, and how a histone deacetylase inhibitor affects this. We found that CD34(+) HSCs produce long functional PSF1 (PSF1a) but CD34(-) HSCs produce a shorter possibly non-functional PSF1 (PSF1b, c, dominantly PSF1c). Using PSF1a-overexpressing NIH-3T3 cells in which the endogenous PSF1 promoter is suppressed, we found that TSA treatment promotes production of the shorter form of PSF1 possibly by inducing recruitment of E2F family factors upstream of the PSF1 transcription start site. Our data document one mechanism by which histone deacetylase inhibitors affect the dormancy of HSCs by regulating the DNA replication factor PSF1.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/biosíntesis , Animales , Ciclo Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Relación Estructura-ActividadRESUMEN
SLD5 is a member of the GINS complex composed of PSF1, PSF2, PSF3 and SLD5, playing a critical role in the formation of the DNA replication fork with CDC45 in yeast. Previously, we had isolated a PSF1 orthologue from a murine hematopoietic stem cell DNA library and were then able to identify orthologues of all the other GINS members by the yeast two hybrid approach using PSF1 as the bait. These GINS orthologues may also function in DNA replication in mammalian cells because they form tetrameric complexes as observed in yeast, and gene deletion mutants of both PSF1 and SLD5 result in a lack of epiblast proliferation and early embryonic lethality. However, we found that PSF1 is also involved in chromosomal segregation in M phase, consistent with recent suggestions that homologues of genes associated with DNA replication in lower organisms also regulate cellular events other than DNA replication in mammalian cells. Here we analyzed the function of SLD5 other than DNA replication and found that it is active in DNA damage and repair. Attenuation of SLD5 expression results in marked DNA damage in both normal cells and cancer cells, suggesting that it protects against DNA damage. Attenuation of SLD5 delays the DNA repair response and cell cycle restoration in normal cells but not in cancer cells. These findings suggest that SLD5 might represent a therapeutic target molecule acting at the level of tumor stromal cells rather than the cancerous cells themselves, because development of the tumor microenvironment could be delayed or disrupted by the suppression of its expression in the normal cell types within the tumor.
Asunto(s)
Proteínas Portadoras/metabolismo , Daño del ADN , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Puntos de Control del Ciclo Celular , Línea Celular , Reparación del ADN , Ratones , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Recombinasa Rad51/metabolismoRESUMEN
PSF1 is an evolutionarily conserved DNA replication factor, which forms the GINS complex with PSF2, PSF3, and SLD5. The mouse PSF1 homolog has been identified from a stem cell-specific cDNA library. To investigate its transcriptional regulatory mechanisms during differentiation, we studied PSF1 mRNA expression in testis and characterized its promoter. No canonical TATA or CAAT boxes could be found in the PSF1 5'-flanking region, whereas several consensus AML1, GATA, and Sry putative binding sequences are predicted within 5 kb of the putative transcription start site. In addition, binding sites for oncoproteins such as Myb and Ets were also found in the promoter. In testis, various PSF1 gene transcription initiation sites are present and short transcripts encoding two novel isoforms, PSF1b and 1c, were found. However, spermatogonium stem cells specifically express transcripts for PSF1a. These data suggest that PSF1 is tightly regulated at the transcriptional level in stem cells.