Structural heat treatments against Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae): effect of flour depth, life stage and floor.

The effect of high temperatures (50-60°C) and two levels of sanitation (~0.5 and 43 g of flour), on mortality of eggs, young larvae, old larvae, pupae, and adults of the red flour beetle, Tribolium castaneum, were evaluated during heat treatment of a pilot flour mill at Kansas State University. The mill was heated once during 13-14 May 2009 and once during 25-26 August 2009. Each of the heat treatments lasted 24 h. Bioassay boxes, with life stages of T. castaneum and temperature sensors confined in small compartments, were placed in 25 locations across all five mill floors. Temperature data showed that the mean time to 50°C based on the two treatments ranged from 10.39 to 17.18 h, and the mean time above 50°C ranged from 6.01 to 13.63 h. The mean maximum temperatures attained ranged from 50.7 to 61.4°C. In general, temperatures were lower in compartments with 43 g of flour when compared with compartments with 0.5 g of flour. Temperatures were also lower on the first floor than on the remaining floors. In box bioassays, essentially none of the life stages survived the 24 h heat treatment (99-100% mortality), except on the first floor. The survival of insects, especially on the first floor, is related to how quickly temperatures reached 50°C and how long temperatures were held between 50 and 60°C, and the maximum temperatures attained at a given location. There were only small differences in mortality between the two levels of sanitation. These results show that heat treatment of flour mills can control all life stages of T. castaneum in 24 h. Keywords: Tribolium castaneum, Heat treatment, Sanitation, Life stages, Methyl bromide alternative

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

Impact of varying levels of sanitation on mortality of Tribolium castaneum eggs and adults during heat treatment of a pilot flour mill

The influence of sanitation on responses of life stages of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), an economically important pest in flour mills, was investigated in a pilot flour mill subjected to two, 24-h heat treatments. One hundred eggs or 100 adults of T. castaneum were exposed inside each 20-cm diameter by 15-cm high PVC rings holding 0.1-, 0.2-, 1.0-, 3.0-, 6.0-, and 10.0-cm-deep wheat flour to simulate different sanitation levels that may exist in a flour mill. These rings were placed on the first and third floors of a pilot flour mill. On the first floor, temperatures inside rings with eggs reached 50 C in 7-11 h only in 0.1- and 0.2-cm-deep flour treatments. In all other treatments the maximum temperatures attained generally were below 50 C and inversely related to flour depth. Adults of T. castaneum on this floor were less susceptible than eggs. The egg mortality decreased linearly with an increase in flour depth, whereas that of adults decreased exponentially. All eggs and adults in rings on the third floor were killed irrespective of flour depth, because temperatures inside rings reached 50 C in 15-17 h and were held above 50 C for 6-8 h with the maximum temperatures ranging between 55.0 and 57.0 C. Although the protective effects of flour on survival of T. castaneum eggs and adults were evident only if temperatures did not reach 50 C, removal of flour accumulations is essential to improve heat treatment effectiveness