INDEKS DISPERZIJE BJELANČEVINA I RASTVORLJIVOST BJELANČEVINA U KALIJEVOM HIDROKSIDU KAO INDIKATORI STUPNJA TERMIČKE OBRADE PUNOMASNE SOJE

Soybean prior to oil extraction is referred to as full-fat soybean (FFSB). Raw FFSB contains anti-nutritional factors (ANFs), which can be destroyed by moderate heating. Both over- and under- heat processing will limit the level of amino acids available to the animal. There is a number of laboratory methods that can be used to estimate the degree of FFSB heat treatment. Two of them, Protein dispersibility index (PDI) and Protein solubility in potassium hydroxide (PSKOH), are based on protein solubility, which was claimed to be the most reliable indicator of the degree of FFSB heat treatment. This paper presents the results of an inter-laboratory study conducted to determine the repeatability limits of the PDI and PSKOH methods. Five samples of FFSB were processed by dry extrusion at temperatures between 110 °C and 164 °C and sent to 8 laboratories for PDI determination and to 9 laboratories for PSKOH analysis. The repeatability limit for the PDI method was found to be 2.31 index units, whereas for the PSKOH method the obtained repeatability limit was 3.77%. Both methods generated acceptable repeatability limits. Due to relatively expansive special blender required for the PDI analysis, which is not always available to laboratories, the preference in routine, every-day analysis should be given to the PSKOH method.Zrno soje prije ekstrakcije ulja naziva se punomasnom sojom. Sirova punomasna soja sadrži anti-nutritivne faktore, koji se mogu ukloniti termičkom obradom. Međutim, i nedovoljna i prekomjerna termička obrada dovodi do smanjivanja razine amino kiselina pristupačnih životinji. Postoji veći broj laboratorijskih metoda koje se koriste za utvrđivanje stupnja termičke obrade punomasne soje. Dvije od ovih metoda, indeks disperzije bjelančevina (PDI) i rastvrorljivost bjelančevina u kalijevom hidroksidu (PSKOH), temelje se na rastvorljivosti, bjelančevina za koje je utvrđeno da su jedan od najboljih indikatora razine termičke obrade punomasne soje. U ovom radu su prikazani rezultati inter-laboratorijskog ispitivanja u cilju određivanja ponovljivosti PDI i PSKOH metoda. Pet uzoraka sirove punomasne soje je termički obrađeno suhim ekstrudiranjem na temperaturama između 110 °C i 164 °C i analizirano u 8 laboratorija na PDI, a u 9 laboratorija na PSKOH. Za PDI metodu dobivena je ponovljivost od 2,31 indeks jedinica, dok je ponovljivost za PSKOH metodu bila 3,77%, te je za obje ispitivane metode dobivena prihvatljiva ponovljivost. Kako je za izvođenje PDI metode neophodan specijalni blender, koji nije uvijek dostupan laboratorijima, preporuka je da se u rutinskoj analizi kvaliteta obrađene punomasne soje koristi PSKOH metoda

Lessons Learned in Applying the U.S. EPA Proposed Cancer Guidelines to Specific Compounds

An expert panel was convened to evaluate the U.S. Environmental Protection Agency’s “Proposed Guidelines for Carcinogen Risk Assessment” through their application to data sets for chloroform (CHCl3) and dichloroacetic acid (DCA). The panel also commented on perceived strengths and limitations encountered in applying the guidelines to these specific compounds. This latter aspect of the panel’s activities is the focus of this perspective. The panel was very enthusiastic about the evolution of these proposed guidelines, which represent a major step forward from earlier EPA guidance on cancer-risk assessment. These new guidelines provide the latitude to consider diverse scientific data and allow considerable flexibility in dose-response assessments, depending on the chemical’s mode of action. They serve as a very useful template for incorporating state-of-the-art science into carcinogen risk assessments. In addition, the new guidelines promote harmonization of methodologies for cancer- and noncancer-risk assessments. While new guidance on the qualitative decisions ensuing from the determination of mode of action is relatively straightforward, the description of the quantitative implementation of various risk-assessment options requires additional development. Specific areas needing clarification include: (1) the decision criteria for judging the adequacy of the weight of evidence for any particular mode of action; (2) the role of mode of action in guiding development of toxicokinetic, biologically based or case-specific models; (3) the manner in which mode of action and other technical considerations provide guidance on margin-of-exposure calculations; (4) the relative roles of the risk manager versus the risk assessor in evaluating the margin of exposure; and (5 ) the influence of mode of action in harmonizing cancer and noncancer risk assessment methodologies. These points are elaborated as recommendations for improvements to any revisions. In general, the incorporation of examples of quantitative assessments for specific chemicals would strengthen the guidelines. Clearly, any revisions should retain the emphasis present in these draft guidelines on flexibility in the use of scientific information with individual compounds, while simultaneously improving the description of the processes by which these mode-of-action data are organized and interpreted

Polygenic architecture informs potential vulnerability to drug-induced liver injury

Drug-Induced-Liver-Injury (DILI) is a leading cause of termination in drug development programs and removal of drugs from the market, and this is partially due to the inability to identify patients who are at risk1. Here, we developed a polygenic risk score (PRS) for DILI by aggregating effects of numerous genome-wide loci identified from previous large-scale genome-wide association studies (GWAS)2. The PRS predicted the susceptibility to DILI in patients treated with fasiglifam, amoxicillin-clavulanate or flucloxacillin, and in primary hepatocytes and stem cell-derived organoids from multiple donors treated with over 10 different drugs. Pathway analysis highlighted processes previously implicated in DILI, including unfolded protein responses and oxidative stress. In silico screening identified compounds that elicit transcriptomic signatures present in hepatocytes from individuals with elevated PRS, supporting mechanistic links and suggesting a novel screen for safety of new drug candidates. This genetic-, cellular-, organoid- and human-scale evidence underscored the polygenic architecture underlying DILI vulnerability at the level of hepatocytes, thus facilitating future mechanistic studies. Moreover, the proposed “polygenicity-in-a-dish” strategy might potentially inform designs of safer, more efficient, and robust clinical trials

Differential gene expression in mouse primary hepatocytes exposed to the peroxisome proliferator-activated receptor α agonists

BACKGROUND: Fibrates are a unique hypolipidemic drugs that lower plasma triglyceride and cholesterol levels through their action as peroxisome proliferator-activated receptor alpha (PPARα) agonists. The activation of PPARα leads to a cascade of events that result in the pharmacological (hypolipidemic) and adverse (carcinogenic) effects in rodent liver. RESULTS: To understand the molecular mechanisms responsible for the pleiotropic effects of PPARα agonists, we treated mouse primary hepatocytes with three PPARα agonists (bezafibrate, fenofibrate, and WY-14,643) at multiple concentrations (0, 10, 30, and 100 μM) for 24 hours. When primary hepatocytes were exposed to these agents, transactivation of PPARα was elevated as measured by luciferase assay. Global gene expression profiles in response to PPARα agonists were obtained by microarray analysis. Among differentially expressed genes (DEGs), there were 4, 8, and 21 genes commonly regulated by bezafibrate, fenofibrate, and WY-14,643 treatments across 3 doses, respectively, in a dose-dependent manner. Treatments with 100 μM of bezafibrate, fenofibrate, and WY-14,643 resulted in 151, 149, and 145 genes altered, respectively. Among them, 121 genes were commonly regulated by at least two drugs. Many genes are involved in fatty acid metabolism including oxidative reaction. Some of the gene changes were associated with production of reactive oxygen species, cell proliferation of peroxisomes, and hepatic disorders. In addition, 11 genes related to the development of liver cancer were observed. CONCLUSION: Our results suggest that treatment of PPARα agonists results in the production of oxidative stress and increased peroxisome proliferation, thus providing a better understanding of mechanisms underlying PPARα agonist-induced hepatic disorders and hepatocarcinomas

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival