Functional analysis of MMR gene VUS from potential Lynch syndrome patients.

Lynch syndrome is caused by inactivating variants in DNA mismatch repair genes, namely MLH1, MSH2, MSH6 and PMS2. We have investigated five MLH1 and one MSH2 variants that we have identified in Turkish and Tunisian colorectal cancer patients. These variants comprised two small deletions causing frameshifts resulting in premature stops which could be classified pathogenic (MLH1 p.(His727Profs*57) and MSH2 p.(Thr788Asnfs*11)), but also two missense variants (MLH1 p.(Asn338Ser) and p.(Gly181Ser)) and two small, in-frame deletion variants (p.(Val647-Leu650del) and p.(Lys678_Cys680del)). For such small coding genetic variants, it is unclear if they are inactivating or not. We here provide clinical description of the variant carriers and their families, and we performed biochemical laboratory testing on the variant proteins to test if their stability or their MMR activity are compromised. Subsequently, we compared the results to in-silico predictions on structure and conservation. We demonstrate that neither missense alteration affected function, while both deletion variants caused a dramatic instability of the MLH1 protein, resulting in MMR deficiency. These results were consistent with the structural analyses that were performed. The study shows that knowledge of protein function may provide molecular explanations of results obtained with functional biochemical testing and can thereby, in conjunction with clinical information, elevate the evidential value and facilitate clinical management in affected families.

Incorporation of ω3 fatty acids in the diets of Nile tilapia juvenile (Oreochromis niloticus L.): effects on growth performance, fatty acid composition, and tolerance to low temperature.

In order to increase the low-temperature resistance of Nile tilapia, the purpose of this study was to determine the potential effect of ω3 fatty acid incorporation in Oreochromis niloticus diet. To perform this, two experimental diets containing soybean oil (D1) and cod liver oil (D2) have been supplied to juvenile tilapia for 30 days. According to our results, similar improvements in the two diets have been recorded for growth performance of O. niloticus including the final body mass, specific growth rate, and feed conversion ratio. Our results showed that fish fed with diet D2 promoted high polyunsaturated fatty acids mainly n-3 series (PUFA (n-3)) percent, highlighting the increased levels of docosahexaenoic (DHA) and eicosapentaenoic (EPA) as well as the activation of their conversion enzyme ratios D5D and D6D desaturases. The second objective was to assess the effect of the two experimental diets on low water temperature tolerance. This was done by exposing juvenile fish at the end of the first experiment to 16, 14, 12, 10, and 8 °C for 12 h, 24 h, and 48 h. The sub-lethal LT50 of O. niloticus fed with diet D1 was 10.6, 11.4, and 13 °C respectively, after 12 h, 24 h, and 48 h. This pattern was commonly observed for O. niloticus fed with D2, showing that the subLT50 were 10.3, 11.1, and 12 °C during the same period. These results demonstrate that O. niloticus juveniles fed with diet D2 are more tolerant to low temperatures than those fed with diet D1.

Growth Performance, Nutrient Digestibility, Hematological Parameters, and Hepatic Oxidative Stress Response in Juvenile Nile Tilapia, Oreochromis niloticus, Fed Carbohydrates of Different Complexities.

A 45-day feeding trial was conducted to assess the capacity of juvenile Nile tilapia (2.12 ± 0.02 g) to utilize different sources of carbohydrate in their diets. Growth performance, nutrient digestibility, hematological parameters, and hepatic oxidative stress were evaluated. Four experimental diets were formulated to be isonitrogenous (25% crude protein) and isolipidic (10% crude lipid), each containing 20% glucose (GLU-diet), maltose (MAL-diet), dextrin (DEX-diet), and corn starch (CST-diet), respectively. At the end of feeding trial, survival in all groups was above 90% and was not significantly different among groups. The results indicated that fish fed the DEX-diet and CSTA-diet showed significantly (p < 0.05) better specific growth rate (SGR), feed conversion ratio (FCR), and protein efficiency ratio (PER) compared with those fed the other diets. The dry matter and carbohydrate digestibility were significantly higher (p < 0.05) in groups fed on dextrin and corn starch diets. However, the digestibility of crude protein and energy in diets did not differ significantly (p > 0.05) among groups fed on experimental diets. The activities of analyzed antioxidant enzymes in the liver were significantly (p < 0.01) higher in groups fed on glucose and maltose diets when compared to other groups. Hematological parameters were affected by the dietary carbohydrate sources; there was a significant increase in hematocrit (Ht), hemoglobin (Hb), and mean corpuscular volume (CMV) in the blood of fish fed on dextrin and cornstarch diets compared to other experimental diets. These results indicated that low complexity carbohydrate sources induced oxidative stress and depressed growth performance. Overall, these results indicate that dietary dextrin and starch were more efficiently utilized than glucose as an energy source by juvenile Nile tilapia. This information is of increasing interest in fish nutrition to provide healthy and economically feed formulations.

Evaluation of the oxidative status in Oreochromis niloticus fed with tuna by-product meal: possible human health impact.

The influence of tuna by-product meal (TBM) diets on the activities of antioxidant enzymes in the muscles, gill and kidney of tilapia was evaluated. The control diet (A0) used fish meal (FM) as the sole source of animal protein and the other three diets (A10-A30), 33%-100% of FM was substituted by TBM at 10% increments. The cytotoxicity of liver and muscles cytosol extracts in human liver cell HepG2 was undertaken. The activities of glutathione S-transferase, superoxide dismutase, catalase and TBARS values in the kidney of fish fed with diet A30 were significantly different (p<0.05) and no alterations were observed in muscle and gill anti-oxidant activities. Our study has confirmed that the cytosol extract does not exhibit cytotoxic effects on the HepG2 cells. These results indicate that the use of TBM as a total replacement for FM into tilapia diets seems to have oxidative stress, thus generating effect on fish metabolism.

Cytotoxicity evaluation and antioxidant enzyme expression related to heavy metals found in tuna by-products meal: An in vitro study in human and rat liver cell lines.

Heavy metals can accumulate in organisms via various pathways, including respiration, adsorption and ingestion. They are known to generate free radicals and induce oxidative and/or nitrosative stress with depletion of anti-oxidants. Tuna by-product meal (TBM) is rich in proteins and can, therefore, offer an attractive protein source for animals. This study was undertaken to assess the effects of metals present in TBM, namely cadmium (Cd), lead (Pb), and mercury (Hg), separately or in combination with oxidative stress, on cell viability. Three cell models: rat liver FTO2B, human hepatoma HepG2, and human hepatic WRL-68, were used. Cell viability was determined following exposure to various concentrations of the metals. Two antioxidant genes, catalase (CAT) and superoxide dismutase (SOD), were measured to obtain a better understanding of oxidative stress-associated gene expression. Among the metals present in TBM, only Cd at a concentration of 30µM was noted to exhibit cytotoxic effects. This cytotoxicity was even more pronounced after co-stimulation with H2O2, used to mimic systemic oxidative stress. At non-toxic concentrations, Hg and Pb were noted to aggravate oxidative stress toxicity. The results further revealed that exposure to Cd, Pb, and a co-stimulation of H2O2 with Hg resulted in the increased expression of antioxidant gene SOD. A risk assessment of toxic contaminants in TBM indicated that food safety objectives should consider the human health impacts of foods derived from animals fed on contaminated meal and that much care should be taken when TBM is used in animal diet.