Trypsin Research Articles

A novel and intelligent molecularly imprinted enzymatic biosensor for biosensing of human serum albumin in the presence of gamma-globulin, and glucose as uncalibrated interference

Human serum albumin (HSA) is not electroactive, because the electroactive amino acids have been buried in its structure. Therefore, direct electrochemical determination of the HSA as the most abundant protein in human plasma cannot be performed. In this work, a novel electrochemical biosensor has been developed based on synthesis of dual templates molecularly imprinted polymers (DTMIPs) having trypsin (TRP) and HSA as template molecules onto the surface of a glassy carbon electrode (GCE) modified with graphene-ionic liquid (Gr-IL). By incubation of the biosensor with TRP, and HSA, tryptic hydrolysis of the HSA is occurred which breaks the HSA down into free amino acids. The HSA involves five electroactive amino acids including cysteine, tryptophan, tyrosine, methionine and histidine whose differential normal pulse voltammetric (DNPV) responses are overlapped and generated a single peak. In order to increase the sensitivity of the DTMIP/Gr-IL/GCE for determination of the HSA, hydrodynamic DNPV (HDNPV) data were generated, and used for analytical purposes. The second-order HDNPV data were generated at different pulse amplitudes and used to develop four second-order calibration models by multivariate curve resolution-alternating least squares (MCR-ALS), parallel factor analysis2 (PARAFAC2), multi-way partial least squares/residual bilinearization (N-PLS/RBL), and unfolded partial least squares/residual bilinearization (U-PLS/RBL) to select the best procedure for determination of the HSA in the presence of gamma-globulin and glucose as uncalibrated interferences. The results confirmed the best performance for the biosensor assisted by MCR-ALS for ultrasensitive and selective determination of the HSA in both synthetic and real matrices which was comparable with HPLC-UV as a reference method.

An upconversion fluorescent and colorimetric dual-mode optical probe for triazophos pesticides

A dual-mode optical probe for organophosphorus pesticides triazophos (TRP) was developed on the basis of internal filter effect (IFE) between upconversion nanoparticles NaYF4: Yb/Er @NaYF4@SiO2 (UCNPs) and gold nanoparticles (AuNPs) in the presence of protamine (PRO) and Trypsin (TRY). In this essay, AuNPs can quench significantly the fluorescence intensity of UCNPs at 550 nm with 980 nm excitation. PRO is a cationic protein that binds AuNPs effectively and aggregates AuNPs due to electrostatic attraction. The aggregation of AuNPs made the solution blue and the fluorescence intensity of UCNPs at 550 nm was recovered correspondingly. Furthermore, TRY could hydrolyze PRO, and PRO will reduce AuNPs clustering due to the addition of TRY, inducing the solution to purple and quench the fluorescence of UCNPs again. After adding the TRP, the activity of TRY was inhibited, thereby preventing PRO hydrolysis and causing AuNPs to reaggregate. Reassembled AuNPs restored the fluorescence of UCNPs again. The inhibition efficiency of fluorescence and colorimetric has a good linear relationship with the logarithmic TRP concentration in the range of 0.03∼300 ng/mL, and the detection limits are 0.0052 and 0.0026 ng/mL, respectively. In addition, the recovery of TRP in real food samples with satisfactory results ranged from 96.02 to 104.30% and 95.63 to 105.64%, respectively.

Tryptophan can alleviate the inhibition in growth and immunity of tilapia (GIFT Oreochromis spp.) induced by high dietary soybean meal level

A 85-day feeding trial was designed to verify the effects of soybean meal on growth, feed utilization, antioxidants, and immune response of tilapia (GIFT Oreochromis spp.), and evaluated the effects of dietary tryptophan on alleviating the negative responses induced by high inclusion of soybean meal. Five experimental diets, two of which substituted 15 % and 45 % of the fish meal with soybean meal, the others supplemented with 0.2 %, 0.4 % and 0.6 % L-tryptophan on the basis of 45 % soybean meal, respectively, were fed 400 juvenile fish (initial weight: 2.98 ± 0.04 g). The results showed that the crude ash content, red blood cell count (RBC), lipase (LPS) and glutathione peroxidase (GSH-Px) activities of tilapia significantly decreased when the soybean meal replaced fish meal by 45 % (p < 0.05). Although the growth performance values of replacing 45 % soybean meal were slightly lower than that of replacing 15 % soybean meal, while there was no significant difference between them (p > 0.05). However, supplementation with tryptophan in the high soybean meal replacement groups significantly improved the final weight, weight gain rate (WGR), specific growth rate (SGR), phagocytosis index (PI), RBC, trypsin (TPS), LPS and GSH-Px activities of tilapia (p < 0.05), and the apparent digestibility and feed coefficient of tilapia were significantly decreased (p < 0.05). Taken together, these results suggested that excessive soybean meal inhibited the feed utilization and immunity of tilapia to a certain extent, while the inclusion of 0.2 %−0.4 % tryptophan in the diet could alleviate the negative effects of high soybean meal diet in tilapia. This study will provide ideas and a theoretical basis for further expanding the application of soybean meal in tilapia feed.

Combined effect of salinity and hypoxia on digestive enzymes and intestinal microbiota in the oyster Crassostrea hongkongensis

Anthropologic activities caused frequent eutrophication in coastal and estuarine waters, resulting in diel-cycling hypoxia. Given global climate change, extreme weather events often occur, thus salinity fluctuation frequently breaks out in these waters. This study aimed to evaluate the combined effects of salinity and hypoxia on intestinal microbiota and digestive enzymes of Crassostrea hongkongensis. Specifically, we sequenced 16 S rRNA of intestinal microbiota and measured the digestive enzymes trypsin (TRS), lipase (LPS) and amylase (AMY) in oysters exposed for 28 days to three salinities (10, 25 and 35) and two dissolved oxygen conditions, normoxia (6 mg/L) and hypoxia (6 mg/L for 12 h, 2 mg/L for 12 h). Oysters in normoxia and salinity of 25 were treated as control. After 28-day exposure, for microbial components, Fusobacteriota, Firmicutes, Bacteroidota, Proteobacteria and Actinobacteriota comprised the majority for all experimental groups. Compared with the control group, the diversity and structure of intestinal microbiota tended to change in all treated groups. The species richness in C. hongkongensis intestine also changed. It was the most significant that high salinity increased Proteobacteria proportion while low salinity and hypoxia increased Fusobacteriota but decreased Proteobacteria, respectively. Additionally, Actinobacteriota was sensitive and changed under environmental stressor (P < 0.01). The prediction results on intestinal microbiota showed that, all functions of oysters were up-regulated to distinct degrees under low/high salinity with hypoxia. According to the KEGG prediction, cellular processes were more active and energy metabolism upregulated, indicating the adaptation of C. hongkongensis to environmental change. Periodical hypoxia and low/high salinity had complex effect on the digestive enzymes, in which the activity of TRS and LPS decreased while AMY increased. High/low salinity and periodical hypoxia can change the secretion of digestive enzymes and influence intestinal microbial diversity and species richness of C. hongkongensis, deducing the chronic adverse effects on the digestive physiology in long-term exposure.

Dietary supplementation with fermented antarctic krill shell improved the growth performance, digestive and antioxidant capability of Macrobrachium nipponense

To investigate the effects of different levels of fermented antarctic krill shell (FAKS) on the growth performance, hemolymph biochemistry, hepatopancreatic antioxidant capacity, and digestive enzyme activities of Macrobrachium nipponense, five isonitrogenous and isoenergetic experimental diets containing different proportions of FAKS (0 %, 0.25 %, 0.5 %, 1.0 %, 2.0 %) were fed to M. nipponense for 8 weeks. The results showed the weight gain rate (WGR), specific growth rate (SGR), head-to-body ratio (H/S), and head-to-tail ratio (H/T) increased first and then decreased with higher proportions of FAKS. The FAKS1.0 group exhibited the highest values, and significantly higher than that of the FAKS0 group (P < 0.05). Compared with the control group, FAKS inclusion could increase the concentration of hemolymph albumin (ALB), and reduce the concentration of alkaline phosphatase (ALP) (P < 0.05). The concentration of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly lower in the FAKS1.0 group (P < 0.05). The hepatopancreas trypsin (TPS) concentration in the FAKS1.0 group, and the lipoprotein lipase (LPL) concentration in the FAKS0.25, FAKS0.5 and FAKS1.0 groups were significantly higher than that in other groups respectively (P < 0.05). With increasing proportions of FAKS, the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and ceruloplasmin (CP) in the hepatopancreas tended to increase and then decrease, and were significantly higher in the FAKS0.5 group than that in the FAKS0 group (P < 0.05). The concentration of malondialdehyde (MDA) in the FAKS0.25, FAKS0.5 and FAKS1.0 groups was significantly lower than that in the FAKS0 group (P < 0.05). In conclusion, adding a certain level of FAKS to the feed significantly improved the growth performance, digestive enzyme, and antioxidant enzyme activity of M. nipponense. Based on the quadratic regression curve fitting, the optimal WGR and SGR can be achieved when the proportion of dietary FAKS is 1.140 % and 1.149 %, respectively.

Spectrum of PRSS1, SPINK1, CTRC, CFTR, and CPA1 Gene Variants in Chronic Pancreatitis Patients in Russia.

The study group included 105 patients with CP, with the age of the disease onset under 40 years old (the average age of onset was 26.9 years). The control group consisted of 76 persons without clinical signs of pancreatitis. The diagnosis of chronic pancreatitis in patients was made on the basis of clinical manifestations and the results of laboratory and instrumental investigations. Genetic examination of patients was conducted using the next-generation sequencing (NGS) technology and included targeted sequencing of all exons and exon-intron boundaries of the PRSS1, SPINK1, CTRC, CFTR, and CPA1 genes. The genotyping of the rs61734659 locus of the PRSS2 gene was also conducted. Genetic risk factors of the CP development were found in 61% of patients. Pathogenic and likely-pathogenic variants associated with the risk of CP development were identified in the following genes: CTRC (37.1% of patients), CFTR (18.1%), SPINK1 (8.6%), PRSS1 (8.6%), and CPA1 (6.7%). The frequent gene variants in Russian patients with CP were as follows: CTRC gene - c.180C>T (rs497078), c.760C>T (rs121909293), c.738_761del24 (rs746224507); cumulative odds ratio (OR) for all risk alleles was 1.848 (95% CI: 1.054-3.243); CFTR gene - c.3485G>T (rs1800120), c.1521_1523delCTT (p.Phe508del, rs113993960), and c.650A>G (rs121909046); OR=2.432 (95% CI: 1.066-5.553). In the SPINK1, PRSS1, and CPA1 genes, pathogenic variants were found only in the group of patients with CP. The frequent variants of the SPINK1 gene include c.101A>G (p.Asn34Ser, rs17107315) and c.194+2T>C (rs148954387); of the PRSS1 gene - c.86A>T (p.Asn29Ile, rs111033566); of the CPA1 gene - c.586-30C>T (rs782335525) and c.696+23_696+24delGG. The OR for the CP development for the c.180TT genotype (rs497078) CTRC according to the recessive model (TT vs. CT+CC) was 7.05 (95% CI: 0.86-263, p=0.011). In the CTRC gene, the variant c.493+49G>C (rs6679763) appeared to be benign, the c.493+51C>A (rs10803384) variant was frequently detected among both the diseased and healthy persons and did not demonstrate a protective effect. The protective factor c.571G>A (p.Gly191Arg, rs61734659) of the PRSS2 gene was detected only in the group of healthy individuals and confirmed its protective role. 12.4% of the patients with CP had risk factors in 2 or 3 genes. Sequencing of the coding regions of the PRSS1, SPINK1, CTRC, CFTR, and CPA1 genes allowed to identify genetic risk factors of the CP development in 61% of cases. Determining the genetic cause of CP helps to predict the disease course, perform preventive measures in the proband's relatives, and facilitate a personalized treatment of the patient in future.

Effects of Soybean Trypsin Inhibitor on Pancreatic Oxidative Damage of Mice at Different Growth Periods.

The bioactive components in soybeans have significant physiological functions. However, the intake of soybean trypsin inhibitor (STI) may cause metabolic disorders. To investigate the effect of STI intake on pancreatic injury and its mechanism of action, a five-week animal experiment was conducted, meanwhile, a weekly monitor on the degree of oxidation and antioxidant indexes in the serum and pancreas of the animals was carried out. The results showed that the intake of STI had irreversible damage to the pancreas, according to the analysis of the histological section. Malondialdehyde (MDA) in the pancreatic mitochondria of Group STI increased significantly and reached a maximum (15.7 nmol/mg prot) in the third week. Meanwhile, the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), trypsin (TPS), and somatostatin (SST) were decreased and reached minimum values (10 U/mg prot, 87 U/mg prot, 2.1 U/mg prot, 10 pg/mg prot) compared with the Group Control. The RT-PCR results of the expression of SOD, GSH-Px, TPS, and SST genes were consistent with the above. This study demonstrates that STI causes oxidative structural damage and pancreatic dysfunction by inducing oxidative stress in the pancreas, which could increase with time.

Characterization of the Interactions between Minocycline Hydrochloride and Trypsin with Spectroscopic and Molecular Docking Technology

In the current study, the interaction of minocycline hydrochloride (MC) and trypsin (TRP) was studied using fluorescence spectroscopy, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, UV-Vis spectroscopy, and molecular docking simulation techniques. The results show that the fluorescence quenching of trypsin at different degrees can be caused by minocycline hydrochloride at different temperatures. According to the Stern-Volmer equation, the fluorescence quenching type was static quenching. By calculating critical distance, we concluded that there is a possibility of non-radiative energy transfer between minocycline hydrochloride and trypsin. The effect of minocycline hydrochloride on the secondary structure of trypsin was demonstrated using ultraviolet spectroscopy. Synchronous fluorescence spectroscopy showed that minocycline hydrochloride could bind to tryptophan residues in trypsin, resulting in corresponding changes in the secondary structure of trypsin. Three-dimensional fluorescence spectroscopy showed that minocycline hydrochloride had a particular effect on the microenvironment of trypsin that led to changes in the secondary structure of trypsin. The molecular docking technique demonstrated that the binding of minocycline hydrochloride and trypsin was stable. Circular dichroism showed that the secondary structure of trypsin could be changed by minocycline hydrochloride.

A Facile Surface-Imprinting Strategy for Trypsin-Imprinted Polymeric Chemosensors Using Two-Step Spin-Coating

Surface imprinting used for protein recognition in functional cavities is highly effective in imprinting biomacromolecules to avoid template encapsulation during the formation of a molecularly imprinted polymer (MIP) matrix. Herein, we introduce a facile surface-imprinting method based on two-step spin-coating and photopolymerization to design highly efficient imprinted sites on polymeric films to detect trypsin (TRY). Well-distributed template imprinting is successfully achieved for maximized sensing responses by controlling the composition of functional monomers and crosslinkers in the precursor solution and the concentration of TRY in the imprinting solution. The MIP film exhibits higher sensitivity (−841 ± 65 Hz/(μg/mL)) with a coefficient of determination of 0.970 and a higher imprinting factor of 4.5 in a 0.24 µg/mL TRY solution compared to the nonimprinted polymer (NIP) film. Moreover, the limit of detection and limit of quantification are calculated to be 25.33 and 84.42 ng/mL, respectively. Finally, the selectivity coefficient is within the range of 3.90–6.78 for TRY against other proteins. These sensing properties are superior to those of the corresponding nonimprinted polymer matrix. Thus, the proposed facile surface-imprinting method is highly effective for protein imprinting with high sensitivity and selectivity.

Will ocean acidification affect the digestive physiology and gut microbiota of whelk Brunneifusus ternatanus?

To understand the physiological responses of the Brunneifusus ternatanus to future ocean acidification (OA), histology, enzyme activity and gut bacterial composition at different pH levels (Control: C group, pH 8.1; Exposure period: EP group, pH 7.3) for 28 days were studied under laboratory conditions. Microbiota composition was analyzed using 16S rRNA gene amplicon sequencing. Enzyme activities of trypsin (TRY), lipase (LPS), amylase (AMS), and lysozyme (LZM) were used as biochemical indicators, as well as weight gain rate (WGR), specific growth rate (SGR) as growth indicators. The stress caused by OA resulted in alterations to the intestine, including partially swollen and degranulated enterocytes and rough endoplasmic reticulum (RER). The relative abundance of the core phylum in the acidified group changed significantly, showing an increase in Tenericutes and a decrease in Proteobacteria. Firmicutes/Bacteroides ratio declined from 4.38 in the control group to 1.25 in the EP group. We found that the enzymes TRY, LPS, and AMS activities were inhibited at reduced pH, which was positively correlated with the dominant genera Mycoplasma and Bacteroides; while LZM activities showed a significant increment, but showing a strong negative correlation. Furthermore, both WG and SRG values showed a depression at low pH lever. These results suggest that if anthropogenic CO2 emissions continue to accelerate, OA could negatively impact the whelk’s health, compromising their growth performance and even survival. These findings will benefit the future risk assessments of OA or other related emerging environmental issues.

Will ocean acidification affect the digestive physiology and gut microbiota of whelk Brunneifusus ternatanus?

To understand the physiological responses of the Brunneifusus ternatanus to future ocean acidification (OA), histology, enzyme activity and gut bacterial composition at different pH levels (Control : C group, pH 8.1; Exposure period : EP group, pH 7.3) for 28 days were studied under laboratory conditions. Microbiota composition was analyzed using 16S rRNA gene amplicon sequencing. Enzyme activities of trypsin (TRY), lipase (LPS), amylase (AMS), and lysozyme (LZM) were used as biochemical indicators, as well as weight gain rate (WGR), specific growth rate (SGR) as growth indicators. The stress caused by OA resulted in alterations to the intestine, including partially swollen and degranulated enterocytes and rough endoplasmic reticulum (RER). The relative abundance of the core phylum in the acidified group changed significantly, showing an increase in Tenericutes and a decrease in Proteobacteria. Firmicutes/Bacteroides ratio declined from 4.38 in the control group to 1.25 in the EP group. We found that the enzymes TRY, LPS, and AMS activities were inhibited at reduced pH, which was positively correlated with the dominant genera Mycoplasma and Bacteroides; while LZM activities showed a significant increment, but showing a strong negative correlation. Furthermore, both WG and SRG values showed a depression at low pH lever. These results suggest that if anthropogenic CO2 emissions continue to accelerate, OA could lead to a negative impact on the whelk health, also compromising their growth performance and even survival. These findings will benefit the future risk assessments of OA or other related emerging environmental issue.

Conserved folding landscape of monomeric initiator caspases

The apoptotic caspase subfamily evolved into two subfamilies-monomeric initiators and dimeric effectors; both subfamilies share a conserved caspase-hemoglobinase fold with a protease domain containing a large subunit and a small subunit. Sequence variations in the conserved caspase-hemoglobinase fold resulted in changes in oligomerization, enzyme specificity, and regulation, making caspases an excellent model for examining the mechanisms of molecular evolution in fine-tuning structure, function, and allosteric regulation. We examined the urea-induced equilibrium folding/unfolding of two initiator caspases, monomeric caspase-8 and cFLIPL, over a broad pH range. Both proteins unfold by a three-state equilibrium mechanism that includes a partially folded intermediate. In addition, both proteins undergo a conserved pH-dependent conformational change that is controlled by an evolutionarily conserved mechanism. We show that the conformational free energy landscape of the caspase monomer is conserved in the monomeric and dimeric subfamilies. Molecular dynamics simulations in the presence or the absence of urea, coupled with limited trypsin proteolysis and mass spectrometry, show that the small subunit is unstable in the protomer and unfolds prior to the large subunit. In addition, the unfolding of helix 2 in the large subunit results in disruption of a conserved allosteric site. Because the small subunit forms the interface for dimerization, our results highlight an important driving force for the evolution of the dimeric caspase subfamily through stabilizing the small subunit.

Metal-Organic Materials (MOMs) Enhance Proteolytic Selectivity, Efficiency, and Reusability of Trypsin: A Time-Resolved Study on Proteolysis.

Proteases are involved in essential biological functions in nature and have become drug targets recently. In spite of the promising progress, two challenges, (i) the intrinsic instability and (ii) the difficulty in monitoring the catalytic process in real time, still hinder the further understanding and engineering of protease functionalities. These challenges are caused by the lack of proper materials/approaches to stabilize proteases and monitor proteolytic products (truncated polypeptides) in real time in a highly heterogeneous reaction mixture. This work combines metal-organic materials (MOMs), site-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy, and mass spectrometry (MS) to overcome both barriers. A model protease, trypsin, which cleaves the peptide bonds at lysine or arginine residues, was immobilized on a Ca-MOM via aqueous-phase, one-pot cocrystallization, which allows for trypsin protection and ease of separation from its proteolytic products. Time-resolved EPR and MS were employed to monitor the populations, rotational motion, and sequences of the cleaved peptide truncations of a model protein substrate as the reaction proceeded. Our data suggest a significant (at least 5-10 times) enhancement in the catalytic efficiency (kcat/km) of trypsin@Ca-MOM and excellent reusability as compared to free trypsin in solution. Surprisingly, entrapping trypsin in Ca-MOMs results in cleavage site/region selectivity against the protein substrate, as compared to the near nonselective cleavage of all lysine and arginine residues of the substrate in solution. Remarkably, immobilizing trypsin allows for the separation and, thus, MS study on the sequences of truncated peptides in real time, leading to a time-resolved "movie" of trypsin proteolysis. This work demonstrates the use of MOMs and cocrystallization to enhance the selectivity, catalytic efficiency, and stability of trypsin, suggesting the possibility of tuning the catalytic performance of a general protease using MOMs.

P905 Predictive pharmacogenetic risk of pancreatitis in Inflammatory Bowel Disease patients treated with thiopurines: a case-control study from the ENEIDA registry

Abstract Background Treatment with thiopurines may be associated with different adverse effects, including acute pancreatitis. Risk factors for developing pancreatitis due to thiopurines in patients with Inflammatory Bowel Disease (IBD) are not clearly identified, with underlying genetic predisposition being a possible cause. Our aim was to evaluate predictive pharmacogenetic risk of pancreatitis in IBD patients treated with thiopurines. Methods IBD patients treated with thiopurines were identified from the prospectively maintained ENEIDA registry of the Spanish Working Group on Crohn’s Disease and Ulcerative Colitis (GETECCU). We included as cases those patients who met the Atlanta diagnostic criteria for pancreatitis and had an imaging test that ruled out biliary origin. We included as controls those patients without pancreatitis after at least two years of treatment with thiopurines. Blood samples were collected, and DNA was extracted from leukocytes for all participants and, subsequently, pooled samples were sequenced. Sequencing of a panel of genes (CASR, CEL, CDLN2, CFTR, CPA1, CTRC, PRSS1, and SPINK1) was carried out in the NextSeq500 platform. Variants detected were classified as pathogenic, variants of unknown significance, and benign according to the American College of Medical Genetics and Genomics (ACMGG) guidelines. Results Ninety-five cases and 105 controls were enrolled, 57% were women. Pancreatitis was diagnosed at a median age of 39±13 years old. No patient had previous history of chronic or acute pancreatitis. There were no differences between thiopurine-treated patients with and without pancreatitis in age, sex, and age at IBD onset. We identified 81 benign variants (50 in cases and 67 in controls) and a total of 35 distinct rare pathogenic and unknown significance variants (10 in CEL, 21 in CFTR, 1 in CDLN2, and 3 in CPA1). Of these 35 variants (12 in cases and 18 in controls), 6 were classified as pathogenic (1 in cases and 5 in controls), belonging to the CEL and CFTR genes, and 30 as variants of uncertain significance (21 in cases and 22 in controls). There were no significant differences between the groups of cases and controls, both in totals and by genes. None of the cases or controls carried pancreatitis-predisposing variants within the CASR, CPA1, PRSS1, and SPINK1 genes. None of the analysed samples with pancreatitis carried a pathogenic CFTR mutation. Four different variants of unknown significance were detected in the CDLN and CPA1 genes. One of them was in the CDLN gene in a single patient with pancreatitis, and 3 variants in the CPA1 gene in 5 control individuals. Conclusion In patients with IBD, there is no predisposition to thiopurine-induced pancreatitis associated with genes known to cause pancreatitis.

Effects of Carbohydrase Supplementation on Growth Performance, Intestinal Digestive Enzymes and Flora, Glucose Metabolism Enzymes, and glut2 Gene Expression of Hybrid Grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂) Fed Different CHO/L Ratio Diets.

An optimal carbohydrate-to-lipid (CHO: L) ratio facilitates fish growth and protein conservation, and carbohydrase promotes nutrient absorption. Therefore, an 8-week feeding trial was conducted to investigate the effects of carbohydrase supplementation on growth performance, intestinal digestive enzymes and flora, glucose metabolism enzymes and glut2 gene expression in juvenile hybrid grouper (Epinephelus fuscoguttatus♀× Epinephelus lanceolatus♂) fed different CHO: L ratios diets. L, M, and H represent CHO:L ratios of 0.91, 1.92 and 3.91, respectively. LE, ME, and HE represent CHO:L ratios of 0.91, 1.92, 3.91, respectively, supplemented with the same ratio of carbohydrase. Results showed that weight gain rate (WGR) and specific growth rate (SGR) reached a maximum in group M and were significantly enhanced by carbohydrase (p < 0.05). Crude lipid content decreased significantly with an increase in the dietary CHO:L ratio (p < 0.05). Significant increases in the trypsin (TRY) and amylase (AMS) activities and significant decreases in the lipase (LPS) activity were observed with increasing dietary CHO:L ratio, and the former two were significantly promoted by carbohydrase (p < 0.05). The content of liver and muscle glycogen increased significantly with the increasing dietary CHO:L ratio but decreased significantly after carbohydrase supplementation (p < 0.05). The glucokinase (GK), pyruvate kinase (PK), Phosphate 6 fructokinase-1 (PFK-1) and phosphoenolpyruvate kinase (PEPCK) activities increased significantly with increasing dietary CHO:L ratio (p < 0.05). Glut2 mRNA expression decreased significantly in liver and increased significantly in intestine with increasing dietary CHO:L ratio (p < 0.05). By linear discriminant analysis (LDA), the abundance of Alistipe was significantly higher in Group ME than in Group M. These results suggested that hybrid grouper can only moderately utilize dietary carbohydrate and lipid in diet, and a certain amount of high glycemic lipids occurred when fed with high-carbohydrate diets. By the weight gain for basis, the supplementation of carbohydrase in Group H with amylase, glycosylase, and pullulanase in a 1:1:1 ratio effectively lowered glycemic lipids, promoted the growth of grouper, digestive enzymes activities and carbohydrate metabolic enzyme, and glut2 gene expression in intestine, effectively balancing the negative effects of high-carbohydrate diet and improving the utilization of carbohydrate.

Клиническое наблюдение наследственного панкреатита у 4 сибсов вследствие мутации в гене PRSS1

Hereditary pancreatitis in children remains a challenging problem in clinical genetics, gastroenterology, and surgery. Genetic causes account for more than 50% of all cases of acute recurrent pancreatitis and 75% of chronic pancreatitis in children. The development of chronic pancreatitis is commonly caused by mutations in the CFTR, PRSS1, SPINK1, CTRC, and CPA1 genes. This article presents a clinical case of hereditary pancreatitis with an autosomal dominant inheritance in 4 siblings, in whom a pathogenic variant in the PRSS1 gene was detected during molecular genetic testing. Key words: hereditary pancreatitis, pathogenic variant, PRSS1 gene, children, siblings

Influence of environmental factors and polymorphic loci rs6580502 of the SPINK1 gene, rs10273639 of the PRSS1 gene, rs213950 of the CFTR gene on the risk of developing acute alcoholic-alimentary pancreatitis

The study of genetic and environmental factors on the risk of acute alcoholic-alimentary pancreatitis (AАAР) is especially relevant to interpret individual links of pathogenesis, to reduce the incidence by eliminating the impact of harmful factors and improve the quality of life of the population through the introduction of optimal nutrition, and a healthy lifestyle, which is especially important for carriers of risk genotypes. The aim of the research was to study the influence of environmental factors and polymorphic loci rs6580502 of the SPINK1 gene, rs10273639 of the PRSS1 gene, rs213950 of the CFTR gene on the risk of АAР. Material and methods. Blood DNA samples obtained from 547 patients with AАAР and 573 healthy individuals were used as the material for the study. The groups were comparable by sex and age. All participants were assessed qualitatively and quantitatively for risk factors, smoking and alcohol consumption, the frequency, quantity and regularity of intake of various types of foods, as well as the size and number of portions eaten. Genomic DNA was isolated by the standard phenol-chloroform extraction method, multiplex genotyping of SNPs was performed on a MALDI-TOF MassARRAY-4 genetic analyzer. Results. It was found that the T/T genotype (p=0.0012) of the rs6580502 SPINK1 was associated with an increased risk of AAAP, and the T allele (p=0.0001) and C/T and T/T genotypes (p=0.0001) of the rs10273639 PRSS1, A allele (p=0.01) and A/G and A/A genotypes (p=0.0006) of the rs213950 CFTR were associated with an decreased risk of the disease. The revealed effects of polymorphic loci of candidate genes were enhanced by the effect of alcohol consumption. The risk of AAAP was reduced by fat intake of less than 89 g per day in carriers of the A/G-A/A CFTR genotypes (rs213950), consumption of fresh vegetables and fruits of more than 27 g per day in carriers of the T/C-T/T PRSS1 genotypes (rs10273639), protein intake of more 84 g per day in carriers of T/C-T/T PRSS1 rs10273639 and A/G-A/A CFTR rs213950. The most significant models of gene-environment interactions included risk factors: deficiency in the diet of protein, fresh vegetables and fruits, smoking, and polymorphic variants of the PRSS1 (rs10273639) and SPINK (rs6580502) genes. Conclusion. In order to prevent the development of AAAP, carriers of risk genotypes of candidate genes need not only to exclude or significantly reduce alcohol consumption (in terms of volume, frequency and duration); but also carriers of the A/G-A/A CFTR genotypes (rs213950) need to balance the diet by reducing fat intake to less than 89 g per day and increasing protein intake to more than 84 g per day; carriers of the T/C-T/T PRSS1 (rs10273639) genotypes should increase their intake of fresh vegetables and fruits over 27 g/day and protein over 84 g/day.

The combined effects of temperature and salinity on the digestion and respiration metabolism of Pinctada fucata

The combined effects of temperature and salinity on the digestion and respiration metabolism of Pinctada fucata were evaluated via response surface methodology and box-benhnken design under laboratory condition. Results indicated that the primary and secondary effects of salinity and temperature had significant effects on amylase (AMS) of P. fucata (P < 0.05)., The digestive enzyme reached the maximum activity when temperature was 26 °C. The AMS and trypsin (TRYP) increased at first, and then decreased with increasing temperature. The Lipase (LPS) was positively correlated with either salinity or temperature. Salinity had no significant effect on TRYP as a primary effect (P > 0.05), but had a significant effect on TRYP as a secondary effect (P < 0.01). These effects were completely opposite to the effect of temperature on pepsin (PEP) as primary and secondary effects. The combined effects of salinity and temperature on AMS, TRYP and PEP were significant (P < 0.01), but had no significant effect on LPS (P > 0.05). The primary, secondary and interaction effects of salinity had significant effects on NKA (Na+-K+-ATPase) of P. fucata (P < 0.05), and NKA presented a U-shaped distribution with increasing salinity. The quadratic and interactive effects of temperature had a significant effect on AKP (P < 0.05), and AKP showed a U-shaped distribution with increasing temperature. Lactate dehydrogenase (LDH) activity decreased at first, and then increased when temperature and salinity changed from 20 to 30 °C and 23–33 ‰, respectively. The expression of GPX gene affected by temperature in gills may be delayed compared with that in hepatopancreas, and its expression is tissue-specific. The appropriate digestion and respiratory metabolism index models were established under the combined temperature and salinity conditions. The optimization results showed that the optimal combination of temperature and salinity was 26.288 °C/28.272‰. The desirability was 0.832. Results from the present study will provide a theoretical reference for shellfish culture affected by environmental interactions and the establishment of related index models.

Effect of water temperature on embryonic development of Protunus trituberculatus in an off-season breeding mode

Portunus trituberculatus (swimming crab) is an important breeding crab in China. The current breeding mode of swimming crab is still the traditional “spring seedling and winter harvest” breeding mode. In recent years, researchers have begun to explore a new off-season breeding mode through autumn seedling. In this study, the rate of embryonic development, embryo antioxidant ability and hatching rate of swimming crab in different water temperatures in an off-season breeding mode (breeding in early autumn) and digestive enzyme activity of newly hatched larvae were compared. The results showed that the duration of each development stage of swimming crab embryos was gradually reduced with increasing water temperature. The total development time was 9.43 d at 27°C and only 6.88 d at 33°C. These effects were accompanied by an increase in the development rate from 0.11 d-1 to 0.15 d-1. The total effective accumulated temperature under the 4 temperature conditions was basically maintained at about 150°C·d (147.08 ∼ 153.62°C·d), and there was no significant difference between different groups. The development of embryos at 27°C and 29°C was very synchronous, and no abnormal embryos were observed. Conversely, at 31°C, the later stage of development exhibited asynchrony, and diapause and death were noted in some embryos. At 33°C, more embryos died. The embryo hatching rate was approximately 70% at 27°C and 29°C, and the rate was significantly reduced at 31°C and 33°C. Specifically, the rate was only 13.89% at 33°C. As the water temperature increased, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) as well as the total antioxidant capacity (T-AOC) and malondialdehyde (MDA) levels of embryos increased first and then decreased, reaching the highest value at 31°C. Catalase (CAT) activity exhibited the opposite trend which was the lowest at 29 °C with a value of only 0.17 U/mg prot and the highest at 33°C with a value of up to 0.51 U/mg prot. At temperatures of 27°C, 29°C and 31°C, the differences in various digestive enzymes of newly hatched larvae primarily manifested as the high activities of pepsin (PEP) and α-amylase (AMS) at 31°C, and other differences were not obvious. At 33°C during embryonic development, the activities of various digestive enzymes were relatively low, especially the activities of trypsin (TPS) and cellulase (CL), which were significantly lower than those of the other three temperature groups. Therefore, judging from the antioxidant index of embryos and the digestive enzyme activity of newly hatched larvae, the embryonic development was still good when the water temperature was 31°C, accompanied by water temperatures above 31°C that seriously affected enzyme activity. Overall, a temperature below 31°C represents the appropriate temperature for embryonic development in autumn in swimming crabs.

Influence of Asp Isomerization on Trypsin and Trypsin-like Proteolysis.

Long-lived proteins (LLPs), although less common than their short-lived counterparts, are increasingly recognized to play important roles in age-related diseases such as Alzheimer's. In particular, spontaneous chemical modifications can accrue over time that serve as both indicators of and contributors to disrupted autophagy. For example, isomerization in LLPs is common and occurs in the absence of protein turnover while simultaneously interfering with the protein turnover by impeding proteolysis. In addition to the biological implications this creates, isomerization may also interfere with its own analysis. To clarify, bottom-up proteomics experiments rely on protein digestion by proteases, most commonly trypsin, but the extent to which isomerization might interfere with trypsin digestion is unknown. Here, we use a combination of liquid chromatography and mass spectrometry to examine the effect of isomerization on proteolysis by trypsin and chymotrypsin. Isomerized aspartic acid and serine residues (which represent the most common sites of isomerization in LLPs) were placed at various locations relative to the preferred protease cleavage point to evaluate the influence on digestion efficiency. Trypsin was found to be relatively tolerant of isomerization, except when present at the residue immediately C-terminal to Arg/Lys. For chymotrypsin, the influence of isomerization on digestion was less predictable, resulting in long-range interference for some isomer/peptide combinations. Given the trypsin- and chymotrypsin-like behaviors of the 20S proteasome, and to further establish the biological relevance of isomerization in LLPs, substrates with isomerized sites were also tested against proteasomal degradation. Significant disruption of 20S proteolysis was observed, suggesting that if LLPs persist long enough to isomerize, it will be difficult for the cells to digest them.