RESUMEN
Processive hydrolysis of crystalline cellulose by cellulases is a critical step for lignocellulose deconstruction. The classic Trichoderma reesei exoglucanase TrCel7A, which has a closed active-site tunnel, starts each processive run by threading the tunnel with a cellulose chain. Loop regions are necessary for tunnel conformation, resulting in weak thermostability of fungal exoglucanases. However, endoglucanase CcCel9A, from the thermophilic bacterium Clostridium cellulosi, comprises a glycoside hydrolase (GH) family 9 module with an open cleft and five carbohydrate-binding modules (CBMs) and hydrolyzes crystalline cellulose processively. How CcCel9A and other similar GH9 enzymes bind to the smooth surface of crystalline cellulose to achieve processivity is still unknown. Our results demonstrate that the C-terminal CBM3b and three CBMX2s enhance productive adsorption to cellulose, while the CBM3c adjacent to the GH9 is tightly bound to 11 glucosyl units, thereby extending the catalytic cleft to 17 subsites, which facilitates decrystallization by forming a supramodular binding surface. In the open cleft, the strong interaction forces between substrate-binding subsites and glucosyl rings enable cleavage of the hydrogen bonds and extraction of a single cellulose chain. In addition, subsite -4 is capable of drawing the chain to its favored location. Cellotetraose is released from the open cleft as the initial product to achieve high processivity, which is further hydrolyzed to cellotriose, cellobiose and glucose by the catalytic cleft of the endoglucanase. On this basis, we propose a wirewalking mode for processive degradation of crystalline cellulose by an endoglucanase, which provides insights for rational design of industrial cellulases.
Asunto(s)
Proteínas Bacterianas/química , Celulasa/química , Celulosa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Celulasa/genética , Celulasa/metabolismo , Clostridium/enzimología , Clostridium/genética , Hidrólisis , Unión ProteicaRESUMEN
The budding yeast Saccharomyces cerevisiae is a platform organism for bioethanol production from various feedstocks and robust strains are desirable for efficient fermentation because yeast cells inevitably encounter stressors during the process. Recently, diverse S. cerevisiae lineages were identified, which provided novel resources for understanding stress tolerance variations and related shaping factors in the yeast. This study characterized the tolerance of diverse S. cerevisiae strains to the stressors of high ethanol concentrations, temperature shocks, and osmotic stress. The results showed that the isolates from human-associated environments overall presented a higher level of stress tolerance compared with those from forests spared anthropogenic influences. Statistical analyses indicated that the variations of stress tolerance were significantly correlated with both ecological sources and geographical locations of the strains. This study provides guidelines for selection of robust S. cerevisiae strains for bioethanol production from nature.
Asunto(s)
Adaptación Fisiológica/genética , Variación Genética , Saccharomyces cerevisiae/genética , Estrés Fisiológico/genética , Biomasa , China , Ecosistema , Etanol/metabolismo , Etanol/farmacología , Fermentación , Geografía , Microbiología Industrial/métodos , Presión Osmótica , Filogenia , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad de la Especie , TemperaturaRESUMEN
PURPOSE: To evaluate the usefulness of contrast-enhanced sonography (CEUS) in the diagnosis of small hepatocellular carcinoma (HCC) measuring < or =2 cm in diameter. METHODS: We identified 104 focal liver lesions measuring < or =2 cm in 104 consecutive patients who were enrolled for baseline sonography (BUS) and CEUS examination (49 HCCs, 55 non-HCCs). A real-time, contrast-specific mode of contrast pulse sequencing and a sulphur hexafluoride-filled microbubble contrast agent were used for CEUS. The diagnostic performances of BUS and CEUS in differentiating focal liver lesions (HCC or non-HCC) were analyzed and compared. RESULTS: On CEUS, 43 (87.8%) of the 49 HCC lesions were hyperenhanced, 5 (10.2%) were isoenhanced, and 1 (2%) was hypoenhanced during the arterial phase when compared with adjacent liver tissue. Thirty-nine (79.6%) HCCs exhibited washout from the portal phase to the late phase. The sensitivity, specificity, positive predictive value, negative predictive value, and overall accuracy before and after contrast agent administration were 28.6% (14/49) versus 79.6% (39/49) (p < 0.001), 94.5% (52/55) versus 92.7% (51/55) (p > 0.05), 82.4% (14/17) versus 90.7% (39/43) (p > 0.05), 59.8% (52/87) versus 90.7% (39/43) (p < 0.01), and 63.5% (66/104) versus 86.5% (90/104) (p < 0.001), respectively. No significant difference in diagnostic performance of CEUS was found between lesions measuring < or =1.5 cm and those 1.6-2 cm and between lesions located at a depth of < or =6 cm from the skin and those located deeper. CONCLUSIONS: CEUS significantly improved the diagnostic performance in characterization of small HCCs < or =2 cm compared with BUS.
Asunto(s)
Carcinoma Hepatocelular/diagnóstico por imagen , Neoplasias Hepáticas/diagnóstico por imagen , Hexafluoruro de Azufre , Ultrasonografía Doppler en Color/métodos , Adulto , Anciano , Biopsia , Carcinoma Hepatocelular/patología , Diagnóstico Diferencial , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Hepáticas/patología , Imagen por Resonancia Magnética , Masculino , Microburbujas , Persona de Mediana Edad , Estadificación de Neoplasias/métodos , Reproducibilidad de los Resultados , Estudios Retrospectivos , Tomografía Computarizada por Rayos XRESUMEN
BACKGROUND: Percutaneous microwave ablation and radiofrequency ablation are two commonly used modalities for the treatment of hepatocellular carcinoma; however, comparisons of them have not been documented adequately. METHODS: Of 102 patients with biopsy-proved hepatocellular carcinoma, 49 (98 nodules) were treated percutaneously with microwave ablation and 53 (72 nodules) with radiofrequency ablation. The local tumor control, complications related to treatment, and long-term results of the two modalities were compared retrospectively. RESULTS: The complete ablation rates were 94.9% (93/98) using microwave ablation vs 93.1% (67/72) using radiofrequency ablation (P = 0.75), and no significant differences were found either in the ablation of tumors of 3.0 cm or less (P = 1.00) or in those of more than 3.0 cm (P = 1.00) between the two modalities. The local recurrence rates were 11.8% (11/93) using microwave ablation vs 20.9% (14/67) using radiofrequency ablation (P = 0.12), and there were no significant differences between the two modalities either in tumors of 3.0 cm or less (P = 0.36) or in those of more than 3.0 cm (P = 0.82). The rates of major complications associated with microwave ablation and radiofrequency ablation were 8.2% (4/49) vs 5.7% (3/53; P = 0.71). The disease-free survival rates in the microwave ablation group were 45.9%, 26.9%, 26.9%, and 13.4% at 1, 2, 3, and 4 years, respectively, and those in the radiofrequency ablation group were 37.2%, 20.7%, and 15.5% at 1, 2 and 3 years, respectively (P = 0.53). The 1-, 2-, 3-, and 4-year cumulative survival rates for patients who underwent microwave ablation were 81.6%, 61.2%, 50.5%, and 36.8%, respectively, and for patients who underwent radiofrequency ablation the rates were 71.7%, 47.2%, 37.6%, and 24.2%, respectively (P = 0.12). CONCLUSIONS: Percutaneous microwave ablation and radiofrequency ablation are both effective methods in treating hepatocellular carcinomas. The local tumor control, complications related to treatment, and long-term survivals were equivalent for the two modalities.