Characterization of the human D-amino acid oxidase (hDAAO) - pLG72 complex involved in the onset of schizophrenia.

Schizophrenia is a chronic and severely debilitating psychiatric disorder affecting nearly 1% of the world’s population. In 2002, the new human gene G72, encoding for the pLG72 protein, and the gene encoding for DAAO have been genetically linked to the susceptibility to schizophrenia. A yeast two-hybrid screening experiment identified D-amino acid oxidase (DAAO) as a putative interacting partner of pLG72. DAAO is a FAD-containing flavooxidase that in brain is responsible for the elimination of D-serine, a co-agonist that binds to the glycine-site of the NMDA receptor. We recently demonstrated that pLG72 acts as “inactivator” of human DAAO and that the cellular concentration of D-serine depends on the expression of the active form of this flavooxidase. Based on these results, a molecular model for the onset of schizophrenia has been proposed: a decrease in pLG72 expression might yield an anomalous high level of hDAAO activity and therefore a decrease in the local concentration of D-serine, affecting glutamatergic neurotransmission mediated by NMDA receptor. The characterization of the complex is a challenging task, hardly feasible by high resolution techniques, since: 1) structural informations on pLG72 are lacking; 2) pLG72 is soluble only in the presence of mild denaturant; 3) no homologous protein has been structurally characterized so far. In this perspective, we have used low resolution strategies based on the coupling of classical biochemistry approaches (complementary proteolysis, cross-link) with mass spectrometric techniques, to characterize the pLG72-hDAAO complex. Results indicated that hDAAO exhibits different proteolysis profiles when isolated or in complex with pLG72, thus suggesting a conformational change upon binding the effector protein. Chemical cross-linking experiments will complement the proteolysis experiments providing with details the contact regions between hDAAO e pLG72

Effect of dietary supplementation with insect fats on growth performance, digestive efficiency and health of rabbits

Background: The present work aimed at evaluating the effect of the dietary replacement of soybean oil (S) by two types of insect fats extracted from black soldier fly larvae (H, Hermetia illucens L.) and yellow mealworm larvae (T, Tenebrio molitor L.) on growth performance, nutrient digestibility, blood parameters, intestinal morphology and health of growing rabbits. Methods: At weaning, 200 crossbred rabbits (36 days old) were allotted to five dietary treatments (40 rabbits/group): a control diet (C) containing 1.5% of soybean oil and four experimental diets where soybean oil was partially (50%) or totally (100%) substituted by H (H50 and H100) or T (T50 and T100) fats. Total tract digestibility was evaluated on 12 rabbits per treatment. The growth trial lasted 41 d and, at slaughtering (78 days old), blood samples were collected from 15 rabbits per treatment, morphometric analyses were performed on duodenum, jejunum and ileum mucosa, and samples of liver, spleen and kidney were submitted to histological evaluation. Results: No difference was observed between the control and the experimental groups fed insect fats in terms of performance, morbidity, mortality and blood variables. The addition of H and T fats did not influence apparent digestibility coefficients of dry matter, protein, ether extract, fibre fractions and gross energy. Gut morphometric indices and organ histopathology were not affected by dietary inclusion of H and T fats. Conclusions: H and T fats are suitable sources of lipid in rabbit diets to replace soybean oil without any detrimental effect on growth performance, apparent digestibility, gut mucosa traits and health

Evaluation of carcass and meat traits of Muscovy duck fed with black soldier fly partially defatted meal

The aim of this study was to evaluate the carcass characteristics and breast meat quality in Muscovy duck (Cairina moschata domestica) fed different inclusion levels of a partially defatted black soldier fly larva (BSF) meal. A total of 256 Muscovy ducklings (average live weight, LW: 71.32\ub12.70 g) were reared from day 3 to day 48 and randomly allotted in 32 pens (8 replicates/treatment). Four different diets were formulated with increasing substitution level of corn gluten meal with BSF larva meal (0, 3, 6 and 9%; BSF0, BSF3, BSF6 and BSF9, respectively) and divided in 3 feeding phases: starter (1-14 days), grower (14-35 days) and finisher (35-48 days). At day 48, 2 animals/replicate were slaughtered and dissected to determine their carcass yields. The weights of spleen, bursa of Fabricius, liver, heart and abdominal fat were recorded. Breast and thigh muscles were then excised from 16 ducks/treatment and weighted. Ultimate pH (pHu) and L*, a*, b* colour values were then measured on breast muscle. The collected data were tested by means of oneway ANOVA evaluating the effect of dietary BSF inclusion level by polynomial contrasts. Significance was declared at P<0.05. The inclusion of BSF did not affect final LW (2,515.68\ub192.42 g on average). Hot and cold carcass weights showed a quadratic response (P<0.05) to increasing BSF larva meal, with a minimum corresponding to BSF6; however, refrigeration losses were not affected by treatments. Weight of spleen, bursa of Fabricius, liver and heart did not differ among treatments. The weight of abdominal fat showed a quadratic response to increasing BSF meal with a minimum corresponding to BSF6 group (P<0.05). Breast and thigh yields, pHu and L*, a*, b* colour values did not differ among groups. With the exception of BSF6, the inclusion of BSF meal did not affect meat traits and carcass characteristics, confirming the potential use of BSF meal in Muscovy duck diets

A deep-learning sequence-based method to predict protein stability changes upon genetic variations

Several studies have linked disruptions of protein stability and its normal functions to disease. Therefore, during the last few decades, many tools have been developed to predict the free energy changes upon protein residue variations. Most of these methods require both sequence and structure information to obtain reliable predictions. However, the lower number of protein structures available with respect to their sequences, due to experimental issues, drastically limits the application of these tools. In addition, current methodologies ignore the antisymmetric property characterizing the thermodynamics of the protein stability: a variation from wild-type to a mutated form of the protein structure (XW→XM) and its reverse process (XM→XW) must have opposite values of the free energy difference (ΔΔGWM=−ΔΔGMW). Here we propose ACDC-NN-Seq, a deep neural network system that exploits the sequence information and is able to incorporate into its architecture the antisymmetry property. To our knowledge, this is the first convolutional neural network to predict protein stability changes relying solely on the protein sequence. We show that ACDC-NN-Seq compares favorably with the existing sequence-based methods

Quality and consumer acceptance of meat from rabbits fed diets in which soybean oil is replaced with black soldier fly and yellow mealworm fats

This trial investigated the effect of the dietary inclusion of Hermetia illucens (H) and Tenebrio molitor (T) fats as alternative lipid sources for growing rabbits, and assessed the carcass characteristics, proximate composition, lipid peroxidation, and fatty acid profile of the meat, as well as consumer acceptance. At weaning, 200 crossbred rabbits (1051\ub1138 g initial body weight) were allotted to five isolipidic (4% dry matter (DM)) dietary treatments: a control diet (C) containing 1.5% of soybean oil, and four experimental diets in which soybean oil was partially (50%) or totally (100%) substituted by H (H50 and H100) or T (T50 and T100) fats. The carcass characteristics, the meat quality traits, and the consumer acceptance of the cooked meat were not affected. The fat content of Longissimus thoracis et lumborum muscle of the rabbits was 1.1% on average. In the case of rabbit fed the H diets (average of diets H50 and H100), the same muscles revealed a higher saturated fatty acid proportion (47.1% vs. 39.7% and 40.8%, respectively) and a lower polyunsaturated fatty acid proportion than the rabbits fed the C and T diets (average of diets T50 and T100) (26.5% vs. 31.7% and 29.7%) (p<0.001). The meat of the rabbits fed the diets containing insect fat (average for H and T diets) was less susceptible to oxidation (0.24 vs. 0.39 mg malondialdehyde/kg meat in the C group; p<0.01)

W-F substitutions in apomyoglobin increase the local flexibility of the N-terminal region causing amyloid aggregation: A H/D exchange study.

Myoglobin is an α-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues impairs the productive folding of the protein making the polypeptide chain highly prone to aggregate forming amyloid fibrils at physiological pH and room temperature. The role played by tryptophanyl residues in driving the productive folding process was investigated by providing structural details at low resolution of compact intermediate of three mutated apomyoglobins, i.e., W7F, W14F and the amyloid forming mutant W7FW14F. In particular, we followed the hydrogen/deuterium exchange rate of protein segments using proteolysis with pepsin followed by mass spectrometry analysis. The results revealed significant differences in the N-terminal region, consisting in an alteration of the physico-chemical properties of the 7-11 segment for W7F and in an increase of local flexibility of the 12-29 segment for W14F. In the double trypthophanyl substituted mutant, these effects are additive and impair the formation of native-like contacts and favour inter-chain interactions leading to protein aggregation and amyloid formation at physiological pH

Screening of fungal strains for cellulolytic and xylanolytic activities production and evaluation of brewers’ spent grain as substrate for enzyme production by selected fungi

Brewer’s spent grain (BSG), the solid residue of beer production, is attracting significant attention as raw material for the production of added value substances, since until recently it was mainly used as animal feed or deposited in landfills, causing serious environmental problems. Therefore, this work aimed at developing a bioprocess using BSG as a substrate for the production of cellulases and xylanases for waste saccharification and bioenergy production. Different fungi were analyzed for their cellulolytic and xylanolytic abilities, through a first screening on solid media by assessment of fungal growth and enzyme production on agar containing carboxylmethylcellulose or xylan as the sole carbon source, respectively. The best cellulase and xylanase producers were subjected to quantitative evaluation of enzyme production in liquid cultures. Aspergillus niger LPB-334 was selected for its ability to produce cellulase and xylanase at high levels and it was cultivated on BSG by solid state fermentation. The cellulase production reached a maximum of 118.04 8.4 U/g of dry substrate after 10 days of fermentation, while a maximum xylanase production of 1315.15 37.5 U/g of dry substrate was reached after 4 days. Preliminary characterization of cellulase and xylanase activities and identification of the enzymes responsible were carried out

Genetic and epigenetic characterization of a discordant kmt2a/aff1-rearranged infant monozygotic twin pair

The KMT2A/AFF1 rearrangement is associated with an unfavorable prognosis in infant acute lymphocytic leukemia (ALL). Discordant ALL in monozygotic twins is uncommon and represents an attractive resource to evaluate intrauterine environment–genetic interplay in ALL. Mutational and epigenetic profiles were characterized for a discordant KMT2A/AFF1-rearranged infant monozygotic twin pair and their parents, and they were compared to three independent KMT2A/AFF1-positive ALL infants, in which the DNA methylation and gene expression profiles were investigated. A de novo Q61H NRAS mutation was detected in the affected twin at diagnosis and backtracked in both twins at birth. The KMT2A/AFF1 rearrangement was absent at birth in both twins. Genetic analyses conducted at birth gave more insights into the timing of the mutation hit. We identified correlations between DNA methylation and gene expression changes for 32 genes in the three independent affected versus remitted patients. The strongest correlations were observed for the RAB32, PDK4, CXCL3, RANBP17, and MACROD2 genes. This epigenetic signature could be a putative target for the development of novel epigenetic-based therapies and could help in explaining the molecular mechanisms characterizing ALL infants with KMT2A/AFF1 fusions