Project description:Interest in exploiting algae as a biofuel source and the role of nutrient deficiency in inducing triacylglyceride (TAG) accumulation in cells necessitates a strategy to efficiently formulate species-specific culture media that can be easily manipulated. Using the reference organism Chlamydomonas reinhardtii, we tested the hypothesis that modeling trace element supplements on the ionomes of cells would result in optimized cell growth. We characterized the ionomes of multiple wild-type Chlamydomonas strains in various culture conditions and developed a revised trace element solution to parallel our measurements. Comparison of cells growing in the revised supplement versus a traditional trace element solution revealed faster growth rates and higher maximum cell densities with the revised recipe. RNA-seq analysis of cultures growing in the traditional versus revised medium suggest that the variation in transcriptomes was smaller than that found between laboratories using the same supplement. Visual observation did not reveal defects in cell motility or mating efficiency. Ni2+-inducible expression from the CYC6 promoter remained a useful tool, albeit with an increased amount of Ni2+ supplementation due to the introduction of an EDTA buffer system in the revised medium. Equilibrium modeling of the revised supplement predicts less metal precipitation in the revised medium. Other advantages include more facile preparation of trace element stock solutions that can readily be adapted for deficiency studies, a reduction in total chemical use, a more consistent batch-to-batch formulation, and long-term stability (up to 5 years). Under the new growth regime, we analyzed cells growing under different macro- and micronutrient-deficiencies. In N and S deficiency, cells accumulate TAG as well in the new medium as previously demonstrated. Fe and Zn deficiency also induced TAG accumulation as suggested by Nile Red and Bodipy staining. This ionomic approach can be used to efficiently optimize culturing conditions for other algal species to improve growth and assay cell physiology. Sampling of Chlamydomonas CC-1021 (2137) cultivated in TAP medium supplemented with a revised trace element recipe based on ionomic data.

Project description:Interest in exploiting algae as a biofuel source and the role of nutrient deficiency in inducing triacylglyceride (TAG) accumulation in cells necessitates a strategy to efficiently formulate species-specific culture media that can be easily manipulated. Using the reference organism Chlamydomonas reinhardtii, we tested the hypothesis that modeling trace element supplements on the ionomes of cells would result in optimized cell growth. We characterized the ionomes of multiple wild-type Chlamydomonas strains in various culture conditions and developed a revised trace element solution to parallel our measurements. Comparison of cells growing in the revised supplement versus a traditional trace element solution revealed faster growth rates and higher maximum cell densities with the revised recipe. RNA-seq analysis of cultures growing in the traditional versus revised medium suggest that the variation in transcriptomes was smaller than that found between laboratories using the same supplement. Visual observation did not reveal defects in cell motility or mating efficiency. Ni2+-inducible expression from the CYC6 promoter remained a useful tool, albeit with an increased amount of Ni2+ supplementation due to the introduction of an EDTA buffer system in the revised medium. Equilibrium modeling of the revised supplement predicts less metal precipitation in the revised medium. Other advantages include more facile preparation of trace element stock solutions that can readily be adapted for deficiency studies, a reduction in total chemical use, a more consistent batch-to-batch formulation, and long-term stability (up to 5 years). Under the new growth regime, we analyzed cells growing under different macro- and micronutrient-deficiencies. In N and S deficiency, cells accumulate TAG as well in the new medium as previously demonstrated. Fe and Zn deficiency also induced TAG accumulation as suggested by Nile Red and Bodipy staining. This ionomic approach can be used to efficiently optimize culturing conditions for other algal species to improve growth and assay cell physiology.

Project description:Introduction: Several attempts have been made to expand the indications of liver transplantation for hepatocellular carcinoma (HCC) beyond Milan criteria. We aimed to define genomic features that enable the identification of patients with HCC beyond Milan criteria who have acceptable transplant outcomes. Methods: Among 770 consecutive HCC patients transplanted at Mount Sinai Hospital and Mayo Clinic between 1990 and 2013, 132 had tumors exceeding Milan criteria on pathology and were enrolled in the study. Explant tumors were analyzed to assess genomic signatures of poor prognosis and immunohistochemical markers associated with tumor recurrence and overall survival. Results: Most of the patients were males (80%) and HCV positive (71%) with a median age of 56. HCC beyond Milan criteria was defined pre-operative in 67%. On pathology, 44% of the patients satisfied the ‘up-to-7 rule’. At a median follow-up of 88 months, 64 patients had died and 45 recurred; the 5-year overall survival (OS) and recurrence rates were 57% and 35%, respectively. CK19 gene signature was independently associated with recurrence (HR=2.95, p<0.001), along with tumor size >5 cm (HR=3.37, p=0.023) and presence of satellite lesions (HR=2.98, p=0.001). S2 subclass signature, which is known to be associated with progenitor cell markers, was independently associated with poor OS (HR=3.18, p=0.001), along with tumor size >5 cm (HR=5.06, p<0.001) and outside up-to-seven rule (HR=2.50, p=0.002). Using the presence of markers of progenitor cells (either CK19 or S2 subclass signatures) patients may be classified into poor-prognosis (n=58; 5-year recurrence 53%, survival 45%) and good-prognosis subgroups (n=74; 5-year recurrence 19%, survival 67%) (HR=3.16, p<0.001 for recurrence, and HR=1.72, p=0.04 for OS). Conclusion: Gene signatures associated with progenitor cell markers (CK19 and S2 subclass signatures) define outcome of HCC patients beyond Milan criteria after transplantation. Patients without these signatures achieve survival rates similar as those patients within Milan criteria. Once prospectively validated, these markers may provide the rationale for a limited expansion of liver transplantation indications.

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Genomic validation of the revised Bethesda criteria in terms of pathogenesis and oncologic significance

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