Ex vivo modelling of drug efficacy in a rare metastatic urachal carcinoma

Background Ex vivo drug screening refers to the out-of-body assessment of drug efficacy in patient derived vital tumor cells. The purpose of these methods is to enable functional testing of patient specific efficacy of anti-cancer therapeutics and personalized treatment strategies. Such approaches could prove powerful especially in context of rare cancers for which demonstration of novel therapies is difficult due to the low numbers of patients. Here, we report comparison of different ex vivo drug screening methods in a metastatic urachal adenocarcinoma, a rare and aggressive non-urothelial bladder malignancy that arises from the remnant embryologic urachus in adults. Methods To compare the feasibility and results obtained with alternative ex vivo drug screening techniques, we used three different approaches; enzymatic cell viability assay of 2D cell cultures and image-based cytometry of 2D and 3D cell cultures in parallel. Vital tumor cells isolated from a biopsy obtained in context of a surgical debulking procedure were used for screening of 1160 drugs with the aim to evaluate patterns of efficacy in the urachal cancer cells. Results Dose response data from the enzymatic cell viability assay and the image-based assay of 2D cell cultures showed the best consistency. With 3D cell culture conditions, the proliferation rate of the tumor cells was slower and potency of several drugs was reduced even following growth rate normalization of the responses. MEK, mTOR, and MET inhibitors were identified as the most cytotoxic targeted drugs. Secondary validation analyses confirmed the efficacy of these drugs also with the new human urachal adenocarcinoma cell line (MISB18) established from the patient’s tumor. Conclusions All the tested ex vivo drug screening methods captured the patient’s tumor cells’ sensitivity to drugs that could be associated with the oncogenic KRASG12V mutation found in the patient’s tumor cells. Specific drug classes however resulted in differential dose response profiles dependent on the used cell culture method indicating that the choice of assay could bias results from ex vivo drug screening assays for selected drug classes

Screening of winery and olive mill wastes for lignocellulolytic enzyme production from Aspergillus species by solid-state fermentation

Wastes from olive oil and wine industries (as exhausted grape marc, vineshoot trimmings, two-phase olive mill waste, vinasses, and olive mill wastewater) were evaluated for lignocellulolytic enzyme production (as endocellulases, endoxylanases, and feruloyl esterases) by solid-state fermentation (SSF) with Aspergillus niger, Aspergillus ibericus, and Aspergillus uvarum. To study the effect of different solid medium composition and time in enzyme production, a PlackettBurman experimental design was used. Variables that had a higher positive effect in lignocellulolytic enzyme production were urea, time, and exhausted grape marc. The maximum values of enzymatic activity per unit of substrate dry mass were found with A. niger for feruloyl esterase. Enzymatic extracts from SSF with A. niger achieved maximum feruloyl esterase activity (89.53 U/g) and endoxylanase activity (3.06 U/g) and with A. uvarum for endocellulase activity (6.77 U/g). The enzyme cocktails obtained in the SSF extracts may have applications in biorefinery industries.Jose Manuel Salgado is grateful for the postdoctoral fellowship (EX-2010-0402) of the Education Ministry of Spanish Government. Luis Abrunhosa was supported by the grant SFRH/BPD/43922/2008 from Fundacao para a Ciencia e Tecnologia-FCT, Portugal

Direct use of spent mushroom substrate from Pleurotus pulmonarius as a readily delignified feedstock for cellulase production

The feasibility of spent mushroom substrate (SMS) as an alternative fermentation feedstock for cellulase production has been demonstrated in this work. Utilization of SMS as a substrate has been attempted widely due to its high cellulose content and readily available in smaller particle size. On top of that, the availability of delignified SMS by the action of Pleurotus pulmonarius during mushroom cultivation offers another benefit to its use whereby no chemical pretreatment would be required prior to fermentation. The recovery of crude laccase and manganese peroxidase from delignified SMS were found to be 3 and 1.4 U/g, respectively. Further to this, the cellulase production from SMS by Trichoderma asperellum UPM 1 under solid state fermentation was optimized by applying central composite design, resulted in increment of 1.4-fold in CMCase (171.21 U/g) and 1.5-fold in β-glucosidase (6.83 U/g), with the optimum temperature of 27.5 °C, initial moisture content 81% and initial pH of fermentation 4.5. Therefore, this study showed that the direct utilization of SMS is feasible for promising cellulase production by T. asperellum UPM 1

Effects of bacterial inoculants and an enzyme on the fermentation quality and aerobic stability of ensiled whole-crop sweet sorghum

A study was conducted to evaluate the effects of bacterial inoculation and cellulase on the fermentation quality of ensiled whole-crop sweet sorghum (WCSS, Sorghum bicolor L. Moench). The WCSS (323 g dry matter (DM)/kg, 251 g water soluble carbohydrates (WSC)/kg DM, 43 g crude protein (CP)/kg DM and 439 g neutral detergent fibre (NDF)/kg DM) was ensiled with i) no additive (control); ii) Lactobacillus buchneri (LB); iii) Lactobacillus plantarum (LP); and iv) LB+E, a combination of LB and enzyme. These treatments were ensiled in 1 L anaerobic jars for 25 days. The jars were opened on days 3, 7 and 15 to determine pH, while those of day 25 were sampled to determine nutrient composition, fermentation characteristics and aerobic stability. Inoculation reduced pH, butyric acid and ammonia-N and increased lactic acid content in sweet sorghum silage compared with the control. The aerobic stability of WCSS was improved with LB, while it was reduced with the homofermentative LP treatment compared with the control. The LB+E reduced the fibre, but increased residual WSC of silage. The aerobic stability of LB+E silage was lower than LB treated silage. Using enzymes to increase the WSC content of crops that already have high levels of WSC may result in reduced aerobic stability of silage. Further work is needed to evaluate these effects on silage produced on farm scale and on animal production performance.Keywords: Aerobic stability, enzyme, fermentation, inoculants, silag

Effects of bacterial inoculants and an enzyme on the fermentation quality and aerobic stability of ensiled whole-crop sweet sorghum

________________________________________________________________________________ Abstract A study was conducted to evaluate the effects of bacterial inoculation and cellulase on the fermentation quality of ensiled whole-crop sweet sorghum (WCSS, Sorghum bicolor L. Moench). The WCSS (323 g dry matter (DM)/kg, 251 g water soluble carbohydrates (WSC)/kg DM, 43 g crude protein (CP)/kg DM and 439 g neutral detergent fibre (NDF)/kg DM) was ensiled with i) no additive (control); ii) Lactobacillus buchneri (LB); iii) Lactobacillus plantarum (LP); and iv) LB+E, a combination of LB and enzyme. These treatments were ensiled in 1 L anaerobic jars for 25 days. The jars were opened on days 3, 7 and 15 to determine pH, while those of day 25 were sampled to determine nutrient composition, fermentation characteristics and aerobic stability. Inoculation reduced pH, butyric acid and ammonia-N and increased lactic acid content in sweet sorghum silage compared with the control. The aerobic stability of WCSS was improved with LB, while it was reduced with the homofermentative LP treatment compared with the control. The LB+E reduced the fibre, but increased residual WSC of silage. The aerobic stability of LB+E silage was lower than LB treated silage. Using enzymes to increase the WSC content of crops that already have high levels of WSC may result in reduced aerobic stability of silage. Further work is needed to evaluate these effects on silage produced on farm scale and on animal production performance. _______________________________________________________________________________