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关键词： AIF/CypA complex   AIF(370-394) stapled peptide   Circular dichroism (CD)   Click chemistry   Isothermal Titration Calorimetry (ITC)   Enspire label free   NMR and molecular modeling analysis  

摘要： Background: The neuronal apoptotic process requires the nuclear translocation of Apoptosis Inducing Factor (AIF) in complex with Cyclophilin A (CypA) with consequent chromatin condensation and DNA degradation events. Targeting CypA by delivering an AIF-blocking peptide (AIF(370-394)) provides a significant neuroprotection, demonstrating the biological relevance of the AIF/CypA complex. To date pharmaceutical compounds targeting this complex are missing. Methods: We designed and synthesized a set of mono and bicyclic AIF(370-394) analogs containing both disulfide and 1,2,3-triazole bridges, in the attempt to both stabilize the peptide conformation and improve its binding affinity to CypA. Peptide structures in solution and in complex with CypA have been studied by circular dichroism (CD), Nuclear Magnetic Resonance (NMR) and molecular modeling. The ability of stapled peptides to interact with CypA was evaluated by using Epic Corning label free technique and Isothermal Titration Calorimetry experiments. Results: We identified a stapled peptide analogue of AIF(370-394) with a ten-fold improved affinity for CypA. Molecular modeling studies reveal that the new peptide acquires beta-turn/beta-fold structures and shares with the parent molecule the same binding region on CypA. Conclusions: Data obtained provide invaluable assistance in designing new ligand of CypA for therapeutic approaches in neurodegenerative diseases. General significance: Due to the crucial role of AIF/CypA complex formation in neurodegeneration, identification of selective inhibitors is of high importance for targeted therapies. We describe new bicyclic peptide inhibitors with improved affinity for CypA, investigating the kinetic, thermodynamic and structural effects of conformational constraints on the protein-ligand interaction, and their utility for drug design.

关键词： asparagine synthetase deficiency   cerebral atrophy   congenital microcephaly   dysmorphism   amino acid analysis  

摘要： Purpose Asparagine synthetase deficiency (ASNSD) is a rare neurometabolic disease. Patients may not demonstrate low asparagine levels, which highlights the advantage of molecular over biochemical testing in the initial work-up of ASNSD. We aimed to further delineate the ASNSD variant and phenotypic spectrum and determine the value of biochemical testing as a frontline investigation in ASNSD. Methods We retrospectively collected the clinical and molecular information on 13 families with ASNSD from the major metabolic clinics in Saudi Arabia. Results The major phenotypes included congenital microcephaly (100%), facial dysmorphism (100%), global developmental delay (100%), brain abnormalities (100%), spasticity (86%), and infantile-onset seizures (93%). Additional unreported phenotypes included umbilical hernia, osteopenia, eczema, lung hypoplasia, and hearing loss. Overall, seven homozygous variants accounted for ASNSD. The ***398Cys and ***75Ile variants accounted for 54% of the cases. The clinical sensitivity and specificity of the proposed biochemical analysis of cerebrospinal fluid (CSF) for the detection of patients with ASNSD were 83% and 98%, respectively. Conclusion Our study describes the largest reported ASNSD cohort with clinical, molecular, and biochemical characterization. Taking into consideration the suboptimal sensitivity of biochemical screening, the delineation of the phenotype variant spectrum is of diagnostic utility for accurate diagnosis, prognosis, counseling, and carrier screening.

关键词： Imines   Monoamine oxidase-B   Monoamine oxidase-A   Acetylcholinesterase   Kinetics   Reversibility  

摘要： A series of eleven 1-methyl, 5-phenyl substituted thiosemicarbazones (MT1-MT11) with the phenyl ring substitutions were prepared and investigated for their inhibitory activities against monoamine oxidases (MAOs) and acetylcholinesterase (AChE). [4-(dimethylamino) phenyl]methylidene}-N-methylhydrazine-1-carbothioamide (MT5) inhibited MAO-B potently with an IC50 of 8.77 mu M. Potencies for MAO-B increased in the order -N(CH3)(2) in MT5 > -OCH3 in MT3 > -Br in MT9. Most of the 11 compounds weakly inhibited AChE by < 30% at 10 mu M. MT5 competitively inhibited MAO-B and Ki value was 6.58 +/- 0.064 mu M. Reversibility experiments showed MT5 also reversibly inhibited MAO-B. MTT assays revealed that MT5 and MT3 were non-toxic to normal VERO cell lines with IC50 values of 191.96 and 187.04 mu g/mL, respectively. From the molecular docking, MT5 binding was found to be stabilized by hydrogen bonding to the non-bonding electron of the terminal N-methyl group with Cys172 (binding energy = -7.01 kcal/mol) of MAO-B. The molecular dynamics further predicted that MT5 had a major pi-pi hydrophobic interaction with Tyr326 of MAO-B, suggesting that it plays an important role in the stabilization of protein-ligand *** results documents that MT5 is a moderately selective, reversible, and competitive inhibitor of MAO-B with low cytotoxic profile.

关键词： Bothrops moojeni   Snake venom P-I metalloproteinase   Hemorrhagic metalloproteinase   Fibrinogenolytic metalloproteinase  

摘要： Snakebite envenoming is still a worrying health problem in countries under development, being recognized as a neglected disease by the World Health Organization. In Latin America, snakes from the genus Bothrops are widely spread and in Brazil, the Bothrops moojeni is a medically important species. The pharmacological effects of bothropic snake venoms include pain, blisters, bleeding, necrosis and even amputation of the affected limb. Snake venom metalloproteinases are enzymes abundantly present in venom from Bothrops snakes. These enzymes can cause hemorrhagic effects and lead to myonecrosis due to ischemia. Here, we present BmooMP-I, a new P-I class of metalloproteinase (this class only has the catalytic domain in the mature form) isolated from B. moojeni venom. This protein is able to express fibrinogenolytic and gelatinase activities, which play important roles in the prey's immobilization and digestion, and also induces weak hemorrhagic effect. The primary sequence assignment was done by a novel method, SEQUENCE SLIDER, which combines crystallographic, bioinformatics and mass spectrometry data. The high-resolution crystal structure reveals the monomeric assembly and the conserved metal binding site H(141)ExxH(145)xxG(148)xxH(151) with the natural substitution Gly148Asp that does not interfere in the zinc coordination. The presence of a structural calcium ion on the surface of the protein, which can play an important role in the stabilization of hemorrhagic toxins, was observed in the BmooMP-I structure. Due to the relevant local and systemic effects of snake venom metalloproteinases, studies involving these proteins help to better understand the pathological effects of snakebite envenoming. (C) 2020 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

关键词： Cachaca   Submandibular gland   Alcoholism   Mucin   Acid phosphatase   Alkaline phosphatase  

摘要： Objective This study aimed to evaluate the effects of chronic consumption of cachaca, a Brazilian beverage containing alcohol, on submandibular glands (SM) of rats by using histomorphometric and biochemical parameters. Materials and methods Twenty-four male rats (40 days of age) were assigned into the following four groups (n = 6): two control groups for 75 days (C75) and 105 days (C105), and two experimental groups of cachaca ingestion with ascending concentrations for consecutive 75 days (CA75) and 105 days (CA105). On the right SM glands, the striated, granular and acini ducts were processed for histomorphometric analysis. The left SM glands were weighed and stored at - 80 degrees C, to evaluate through biochemical tests carried out by spectrophotometric methods, the functional activity of total acid phosphatase (TAP), tartrate-resistant acid phosphatase (TRAP), and alkaline phosphatase (ALP) and to determine the mucin levels. Results The absolute and relative weights of the SM glands in both experimental groups were reduced in relation to the controls (p < 0.05). The histomorphometric analysis showed a significant reduction of the acini area (p < 0.05) and non-relevant reduction of striated ducts (p > 0.05). The granular ducts did not show a significant increase of the area (p > 0.05). The TAP and TRAP activities were significantly decreased in the experimental groups (p < 0.05), while the ALP functional activity decreased moderately (p > 0.05). Mucin levels also had a significant reduction when compared with the control groups (p < 0.05). Conclusions Chronic consumption of cachaca can cause morphological changes associated with glandular atrophy, loss of biochemical functionality of phosphatases, and the reduction of mucin synthesis.

关键词： Diffusion   Disintegration   Softening   Mixed Weibull function   Human gastric simulator  

摘要： The aim of this work was to investigate the role of biochemical digestion on softening and disintegration kinetics of pH 5 and pH 9 egg white gel (EWGs) during in vitro gastric digestion. EWG samples (5 mm length cubes) underwent in vitro digestion by incubation in simulated gastric fluid at different time intervals for up to 240 min. The hardness was measured using a Texture Analyser;softening kinetics was fit to the Weibull model. Results revealed that pH 9 EWG had the highest softening halftime (458 +/- 86 min), indicating the slowest softening, whereas pH 5 EWG had the lowest softening halftime (197 +/- 12 min), indicating the quickest softening. The digested samples were immediately exposed to mechanical forces generated by the human gastric simulator (HGS) for 10 min to investigate the influence of gastric juice on the breakdown behaviour of EWG cubes. The breakdown behaviour of the disintegrated samples was characterized by fitting the cumulative distributions of particle surface areas to a mixed Weibull function (R-2 > 0.99). The weight of fine particles (alpha) showed that regardless of gastric juice diffusion, the pH 5 EWG (alpha = 0.22 +/- 0.03) disintegrated into more fine particles than those resulting from pH 9 EWG disintegration (alpha = 0.07 +/- 0.02). As expected, the diffusion of gastric juice enhanced erosion of the EWG particles into fine particles. Result obtained from the particle surface area distribution is in good agreement with the softening kinetics of EWGs during simulated in vitro gastric phase.

关键词： Blood biochemistry profile   Aquaponic systems   Hydroponic plants   Aquatic plants   Carassius auratus  

摘要： Blood biochemical profile can be used to evaluate the health status of fish and ambient environmental conditions. However, it is not well known in the aquaponic systems, let alone their differences between hydroponic and aquatic plants. A 5-month trial was conducted to investigate the changes in the growth performance and blood index of Qihe crucian carpCarassius auratusin eight aquaponic systems (control,Ipomoea aquatica,Lactuca sativa,Lemna minor,Amaranthus tricolor,Ceratophyllum demersum,Vallisneria spiralis, andC. demersum-net, indicated by CK, Ia, Ls, Lm, At, Cd, Vn, and Cd-ns). Results showed that weight gain rate and specific growth rate did not significantly differ among the eight groups. However, most blood parameters significantly differed among the eight groups. The glucose level and activities of alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase were generally larger in Cd, Lm, Vn, and CK groups, and smaller in Ia, Ls, or At groups. Additionally, the triglyceride, cholesterol, albumin, and albumin/globulin were mostly elevated in Ia and At groups. Moreover, the creatinine, total proteins, and globulin levels peaked in Vn group. These findings showed that the blood biochemical profile was more sensitive than the growth performances and that hydroponic plants can induce a higher production and more healthy status of Qihe crucian carpC. auratuswhen compared with aquatic plants or no plants.

关键词： Dietary fiber fortification   Milk mineral fortification   Processed meat   Metabolomics   Nitroso compounds   Gut microbiota  

摘要： This study investigated inulin and calcium-rich milk mineral incorporation into a pork sausage in order to examine the effects on microbiome and biochemical activity in the gastrointestinal tract upon ingestion. Rats (n = 48) were fed one of four sausages;a pork sausage enriched with 1) inulin (6.0%) and milk mineral (3%), 2) inulin (6.0%), 3) milk mineral (3%) or 4) control sausages without enrichment. NMR-based metabolomics revealed that inulin-enrichment increased the fecal concentration of short-chain fatty acids (SCFAs). Milk mineral-enrichment also increased SCFA concentrations, although less pronounced. In addition, milk mineral reduced the concentration of nitroso compounds in feces and small intestinal content. Combined enrichment with both inulin and milk mineral showed no cumulative effect on SCFA formation and seemed to oppose the milk mineral-induced reduction of nitroso compound formation. 16S rRNA gene amplicon sequencing indicated that alterations of the gut microbiome contributed to the observed effects.

关键词： NaCl treatment   Phenolic acid   Flavonoids   Metabolism  

摘要： Phenolics are important secondary metabolites in plants with strong antioxidant effects. Seeds germination and exogenous stimulation could activate endogenous enzymes to enhance the content of phenolic acids and flavonoids. Barley seeds geminated under NaCl (1-20 mM) treatment to evaluate the accumulation of phenolics in this study. Results showed that NaCl treatment significantly enhanced the growth of seedlings, especially bud length. NaCl treatment up-regulated genes and proteins expression of phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL), resulting in the enhancement of their activities. As a result, phenolic acids and flavonoids contents increased by 11.19% and 32.54%, respectively, in which gallic acid, protocatechuic, fisetin, myricetin and quercetin were affected mostly. Moreover, NaCl treatment enhanced 2,2 '-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging capacity. Hence, NaCl stimulated the synthesis of phenolic components via enhancing gene, protein expression and the activity of key enzymes.

关键词： Primary hyperparathyroidism   Adenoma   Weight   Parathormone   Calcium  

摘要： BackgroundSingle parathyroid adenoma is the main cause of primary hyperparathyroidism (PHPT), with surgery remaining the gold standard for its treatment. The ability to preoperatively predict the parathyroid adenoma size and could facilitate the decision about the extent of surgical exploration. It is reasonable to hypothesize that the perioperative levels of PHPT-related variables (i.e. calcium, parathormone, phosphate) may predict the adenoma weight or/and demonstrate whether the adenoma is successfully removed or not. Aim of this study is to explore the relationship between perioperative biochemical values and adenoma weight. Secondarily, we investigated the relationship between adenoma weight and uni-/bilateral neck *** study of all patients undergone surgery for primary hyperparathyroidism due to single adenoma in a tertiary university hospital in Germany during a 6-year period. Following variables were analyzed: preoperative serum calcium, phosphorus and parathormone, intraoperative parathormone before and after adenoma excision, intraoperative PTH decrease, postoperative serum calcium and parathormone (PTHpostop-pg/ml), calcium and PTH decrease. Bivariate correlations were calculated by the Spearman's correlation test at the 95% significance *** total of 339 patients were included in the study. The median age of the patients was 60 years (range 21-90) and 77% were females. The median adenoma weight was 1 g (range 0.1-11). Adenoma weight correlated strong with maximum adenoma diameter (r=0.72, p<0.05), moderate with preoperative parathormone (r=0.44) and parathormone decrease (r=0.27), whereas there was no correlation with the intraoperative PTH decrease (r=0.02). There was also a borderline (moderate to weak) correlation with pre- and postoperative calcium levels (r=0.21 and r=0.23 respectively) and a negative borderline correlation with phosphorus (r=- 0.21). Patients who required bilateral neck exploration, had signif