Infant milk formula, produced by membrane filtration, promotes mucus production in the upper small intestine of young pigs.

Human breast milk promotes maturation of the infant gastrointestinal barrier, including the promotion of mucus production. In the quest to produce next generation infant milk formula (IMF), we have produced IMF by membrane filtration (MEM-IMF). With a higher quantity of native whey protein, MEM-IMF more closely mimics human breast milk than IMF produced using conventional heat treatment (HT-IMF). After a 4-week dietary intervention in young pigs, animals fed a MEM-IMF diet had a higher number of goblet cells, acidic mucus and mucin-2 in the jejunum compared to pigs fed HT-IMF (P < 0.05). In the duodenum, MEM-IMF fed pigs had increased trypsin activity in the gut lumen, increased mRNA transcript levels of claudin 1 in the mucosal scrapings and increased lactase activity in brush border membrane vesicles than those pigs fed HT-IMF (P < 0.05). In conclusion, MEM-IMF is superior to HT-IMF in the promotion of mucus production in the young gut.

Trypsin Encapsulation in the Zeolitic Imidazolate Framework for Low-Molecular Weight Protein Analysis with High Selectivity and Efficiency.

Low-molecular weight proteins (LWPs) are important sources of biological information in biomarkers, signaling molecules, and pathology. However, the separation and analysis of LWPs in complex biological samples are challenging, mainly due to their low abundance and the complex sample pretreatment procedure. Herein, trypsin modified by poly(acrylic acid) (PAA) was encapsulated by a zeolitic imidazolate framework (ZIF-L). Mesopores were formed on the ZIF-L with the introduction of PAA. An alternative strategy for separation and pretreatment of LWPs was developed based on the prepared ZIF-L-encapsulated trypsin with adjustable pore size. The mesoporous structure of the prepared materials selectively excluded high-molecular weight proteins from the reaction system, allowing LWPs to enter the pores and react with the internal trypsin, resulting in an improved separation efficiency. The hydrophobicity of the ZIF-L simplified the digestion process by inducing significant structural changes in substrate proteins. In addition, the enzymatic activity was significantly enhanced by the developed encapsulation method that maintained the enzyme conformation, allowed low mass transfer resistance, and possessed a high enzyme-to-substrate ratio. As a result, the ZIF-L-encapsulated trypsin can achieve highly selective separation, valid denaturation, and efficient digestion of LWPs in a short time by simply mixing with substrate proteins, greatly simplifying the separation and pretreatment process of the traditional hydrolysis method. The prepared materials and the developed strategy demonstrated an excellent size-selective assay performance in model protein mixtures, showing great potential in the application of proteomics analysis.

Enzymatic and Algebraic Methodology to Determine the Contents of Kunitz and Bowman-Birk Inhibitors and Their Contributions to Total Trypsin or Chymotrypsin Inhibition in Soybeans.

Soybeans are the number one source of plant proteins for food and feed, but the natural presence of protein protease inhibitors (PIs), namely, the Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI), exerts antinutritional effects. This communication describes a new methodology for simultaneously quantitating all parameters of PIs in soybeans. It consists of seven steps and featured enzymatically measuring trypsin and chymotrypsin inhibitory activities, respectively, and subsequently determining the contents of reactive KTI and BBI and the contributions of each toward total PI mass and total trypsin or chymotrypsin inhibition by solving a proposed system of linear equations with two variables (C = dB + eK and T = xB + yK). This enzymatic and algebraic (EA) methodology was based on differential inhibitions of KTI and BBI toward trypsin and chymotrypsin and validated by applications to a series of mixtures of purified KTI and BBI, two KTI-null and two conventional soybeans, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The EA methodology allowed calculations of PI composition and the contributions of individual inhibitors toward total inhibition with ease. It was first found that although BBI constituted only about 30% of the total PI mass in conventional raw soybeans, it contributed about 80% toward total chymotrypsin inhibitor activity and about 45% toward trypsin inhibitor activity. Therefore, BBI caused more total protease inhibitions than those of KTI. Furthermore, the so-called KTI-null soybean mutants still contained measurable KTI content and thus should be named KTI-low soybeans.

A lava-inspired proteolytic enzyme therapy on cancer with a PEG-based hydrogel enhances tumor distribution and penetration of liposomes.

The enhanced permeability and retention (EPR) effect has become the guiding principle for nanomedicine against cancer for a long time. However, several biological barriers severely resist therapeutic agents' penetration and retention into the deep tumor tissues, resulting in poor EPR effect and high tumor mortality. Inspired by lava, we proposed a proteolytic enzyme therapy to improve the tumor distribution and penetration of nanomedicine. A trypsin-crosslinked hydrogel (Trypsin@PSA Gel) was developed to maintain trypsin's activity. The hydrogel postponed trypsin's self-degradation and sustained the release. Trypsin promoted the cellular uptake of nanoformulations in breast cancer cells, enhanced the penetration through endothelial cells, and degraded total and membrane proteins. Proteomic analysis reveals that trypsin affected ECM components and down-regulated multiple pathways associated with cancer progression. Intratumoral injection of Trypsin@PSA Gel significantly increased the distribution of liposomes in tumors and reduced tumor vasculature. Combination treatment with intravenous injection of gambogic acid-loaded liposomes and intratumoral injection of Trypsin@PSA Gel inhibited tumor growth. The current study provides one of the first investigations into the enhanced tumor distribution of liposomes induced by a novel proteolytic enzyme therapy.

Integration of caveolin-mediated cytosolic delivery and enzyme-responsive releasing of squalenoyl nanoparticles enhance the anti-cancer efficacy of chidamide in pancreatic cancer.

We explored the potential of overcoming the dense interstitial barrier in pancreatic cancer treatment by enhancing the uptake of hydrophilic chemotherapeutic drugs. In this study, we synthesized the squalenoyl-chidamide prodrug (SQ-CHI), linking lipophilic squalene (SQ) with the hydrophilic antitumor drug chidamide (CHI) through a trypsin-responsive bond. Self-assembled nanoparticles with sigma receptor-bound aminoethyl anisamide (AEAA) modification, forming AEAA-PEG-SQ-CHI NPs (A-C NPs, size 116.6 ± 0.4 nm), and reference nanoparticles without AEAA modification, forming mPEG-SQ-CHI NPs (M-C NPs, size 88.3 ± 0.3 nm), were prepared. A-C NPs exhibited significantly higher in vitro CHI release (74.7 %) in 0.5 % trypsin medium compared to release (20.2 %) in medium without trypsin. In vitro cell uptake assays revealed 3.6 and 2.3times higher permeation of A-C NPs into tumorspheres of PSN-1/HPSC or CFPAC-1/HPSC, respectively, compared to M-C NPs. Following intraperitoneal administration to subcutaneous tumor-bearing nude mice, the A-C NPs group demonstrated significant anti-pancreatic cancer efficacy, inducing cancer cell apoptosis and inhibiting proliferation in vivo. Mechanistic studies revealed that AEAA surface modification on nanoparticles promoted intracellular uptake through caveolin-mediated endocytosis. This nanoparticle system presents a novel therapeutic approach for pancreatic cancer treatment, offering a delivery strategy to enhance efficacy through improved tumor permeation, trypsin-responsive drug release, and specific cell surface receptor-mediated intracellular uptake.

Mechanism for improving the in vitro digestive properties of coconut milk by modifying the structure and properties of coconut proteins with monosodium glutamate.

In this paper, the effect of monosodium glutamate (MSG) on coconut protein (CP) solubility, surface hydrophobicity, emulsification activity, ultraviolet spectroscopy and fluorescence spectroscopy was investigated. Meanwhile, the changes in the in vitro digestive properties of coconut milk were also further analyzed. MSG treatment altered the solubility and surface hydrophobicity of CP, thereby improving protein digestibility. Molecular docking showed that CP bound to pepsin and trypsin mainly through hydrogen bonds and salt bridges. And MSG increased the cleavable sites of pepsin and trypsin on CP, thus further improving the protein digestibility. In addition, MSG increased the Na+ concentration in coconut milk, promoted flocculation and aggregation between coconut milk droplets, which prevented the binding of lipase and oil droplets and inhibited lipid digestion. These findings may provide new ideas and insights to improve the digestive properties of plant-based milk.

Detection of Sensitization Profiles with Cellular In Vitro Tests in Wheat Allergy Dependent on Augmentation Factors (WALDA).

Wheat allergy dependent on augmentation factors (WALDA) is the most common gluten allergy in adults. IgE-mediated sensitizations are directed towards ω5-gliadin but also to other wheat allergens. The value of the different in vitro cellular tests, namely the basophil activation test (BAT) and the active (aBHRA) and passive basophil histamine-release assays (pBHRA), in the detection of sensitization profiles beyond ω5-gliadin has not been compared. Therefore, 13 patients with challenge-confirmed, ω5-gliadin-positive WALDA and 11 healthy controls were enrolled. Specific IgE (sIgE), skin prick tests, BATs, aBHRA, and pBHRA were performed with allergen test solutions derived from wheat and other cereals, and results were analyzed and compared. This study reveals a distinct and highly individual reactivity of ω5-gliadin-positive WALDA patients to a range of wheat allergens beyond ω5-gliadin in cellular in vitro tests and SPT. In the BAT, for all tested allergens (gluten, high-molecular-weight glutenin subunits, α-amylase/trypsin inhibitors (ATIs), alcohol-free wheat beer, hydrolyzed wheat proteins (HWPs), rye gluten and secalins), basophil activation in patients was significantly higher than in controls (p = 0.004-p < 0.001). Similarly, significant histamine release was detected in the aBHRA for all test substances, exceeding the cut-off of 10 ng/mL in all tested allergens in 50% of patients. The dependency of tests on sIgE levels against ω5-gliadin differed; in the pBHRA, histamine release to any test substances could only be detected in patients with sIgE against ω5-gliadin ≥ 7.7 kU/L, whereas aBHRA also showed high reactivity in less sensitized patients. In most patients, reactivity to HWPs, ATIs, and rye allergens was observed. Additionally, alcohol-free wheat beer was first described as a promising test substance in ω5-gliadin-positive WALDA. Thus, BAT and aBHRA are valuable tools for the identification of sensitization profiles in WALDA.

A photoelectrochemical biosensor based on self-calibration platform of carbon-rich plasmonic probe with near-infrared driving signal amplification.

Exploring the photochemical (PEC) method induced by low-energy light source makes great significance to achieve high stability and accurate analysis. A sensing platform driven by near-infrared (NIR) light was designed by making the biochemically encoded carbon rich plasmonic hybrid (CPH) probe, the peptide@C-Mo2C. The inherent plasmonic effect of C-Mo2C CPH can directly absorb NIR light, thus starting effective electronic-hole pairs separation. Moreover, the photothermal effect of C-Mo2C CPH also promoted the reaction yield of photothermal catalyst reaction on sensing interface to assist the PEC signal amplification. In the presence of target trypsin, it cleaves the peptides, resulting in the release of peptide@C-Mo2C probe from interface, which leads to a relative decrease in PEC signal. More importantly, a self-calibration system consisting of two independent PEC test channels attempted to eliminate the influence of background signal and baseline drift. The test channel was used to specify the recognition target, while the blank channel was used as a reference. Therefore, the signal difference between two channels was recorded, so as to obtain results with less error and higher stability. In this NIR driven PEC sensor, the carbon rich probe with direct and efficient NIR light conversion promoted the sensitivity and a self-calibration system guaranteed the stability which provided innovative thoughts for developing ingenious PEC sensor.

MDR49 coding for both P-glycoprotein and TMOF transporter functions in ivermectin resistance, trypsin activity inhibition, and fertility in the yellow fever mosquito, Aedes aegypti.

This study investigated the function of the MDR49 gene in Aedes aegypti. MDR49 mutants were constructed using CRISPR/Cas9 technology; the mutation led to increased sensitivity to ivermectin (LC50: from 1.3090 mg L-1 to 0.5904 mg L-1), and a reduction in midgut trypsin activity. These findings suggest that the P-gp encoded by MDR49 confers resistance to ivermectin and impacts the reproductive function in Ae. aegypti. RNA interference technology showed that knockdown of MDR49 gene resulted in a significant decrease in the expression of VGA1 after a blood meal, as well as a decrease in the number of eggs laid and their hatching rate. LC-MS revealed that following ivermectin treatment, the MDR493d+2s/3d+2s strain larvae exhibited significantly higher drug concentrations in the head and fat body compared to the wild type. Modeling of inward-facing P-gp and molecular docking found almost no difference in the affinity of P-gp for ivermectin before and after the mutation. However, modeling of the outward-facing conformation demonstrated that the flexible linker loop between TM5 and TM6 of P-gp undergoes changes after the mutation, resulting in a decrease in trypsin activity and an increase in sensitivity to ivermectin. These results provide useful insights into ivermectin resistance and the other roles played by the MDR49 gene.

Characteristics of Trypsin genes and their roles in insecticide resistance based on omics and functional analyses in the malaria vector Anopheles sinensis.

Trypsin is one of the most diverse and widely studied protease hydrolases. However, the diversity and characteristics of the Trypsin superfamily of genes have not been well understood, and their role in insecticide resistance is yet to be investigated. In this study, a total of 342 Trypsin genes were identified and classified into seven families based on homology, characteristic domains and phylogenetics in Anopheles sinensis, and the LY-Domain and CLECT-Domain families are specific to the species. Four Trypsin genes, (Astry2b, Astry43a, Astry90, Astry113c) were identified to be associated with pyrethroid resistance based on transcriptome analyses of three field resistant populations and qRT-PCR validation, and the knock-down of these genes significantly decrease the pyrethroid resistance of Anopheles sinensis based on RNAi. The activity of Astry43a can be reduced by five selected insecticides (indoxacarb, DDT, temephos, imidacloprid and deltamethrin); and however, the Astry43a could not directly metabolize these five insecticides, like the trypsin NYD-Tr did in earlier reports. This study provides the overall information frame of Trypsin genes, and proposes the role of Trypsin genes to insecticide resistance. Further researches are necessary to investigate the metabolism function of these trypsins to insecticides.

Antioxidant and Antimicrobial Potential of Peptide from Rabbit Meat Hydrolysate Prepared by Trypsin and Zingibain.

<b>Background and Objective:</b> Rabbit meat is a livestock product potentially viable as a protein source to obtain peptides. Antioxidant and antimicrobial peptides are ingredients extracted from various foods through enzymatic hydrolysis, chemical hydrolysis and fermentation to produce health-promoting foods. This research aims to investigate the potential of rabbit meat as a source of antioxidant and antimicrobial peptides through hydrolysis using trypsin and zingibain enzymes. <b>Materials and Methods:</b> This research conducted an explorative-descriptive approach, focusing on antioxidant and antimicrobial activity. Rabbit meat was extracted using trypsin, zingibain and a combination of trypsin and crude extract zingibain. The hydrolyzed rabbit meat extract was tested at intervals of 0, 2, 6, 16, 24, 40 and 48 hrs to determine the degree of hydrolysis and the profile of hydrolyzed proteins with electrophoresis SDS PAGE. The antioxidant activity was tested using the DPPH method and the antimicrobial activity using agar well diffusion method. <b>Results:</b> The degree of hydrolysis increased with the hydrolysis time. The highest protein content of rabbit meat extract hydrolyzed with trypsin was 287.65 mg/mL, observed during 12 hrs hydrolysis. The optimum conditions for the hydrolysis of rabbit meat protein were obtained at 24 hrs, with an IC₅₀ value of 52.45% hydrolyzed by trypsin. As per antimicrobial activities, <i>Escherichia coli</i> and <i>Salmonella</i> sp. were more effective in inhibiting rabbit meat hydrolysates compared to <i>Pseudomonas aeruginosa</i> and <i>Staphylococcus aureus</i>. The inhibition of all pathogen increased until 12 hrs hydrolysis but decreased in 24 hrs hydrolysis. <b>Conclusion:</b> The combination zingibain enzyme and trypsin is feasible for hydrolyzing rabbit meat and the optimum hydrolysis time was 24 hrs with IC₅₀ 52.45 ppm, although accompanied by reduction in antibacterial activities.

Refinement of paramagnetic bead-based digestion protocol for automatic sample preparation using an artificial neural network.

Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead-based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic sample preparation for MS analysis. For this purpose, an artificial neural network (ANN) was applied to maximize the number of peptides without missed cleavages identified in HeLa extract by combining factors such as the quantity (µg) of trypsin/Lys-C and beads (MagReSyn® Amine), % (w/v) SDS, % (v/v) acetonitrile, and time of digestion (h). ANN model predicted the optimal conditions for the digestion of 50 µg of HeLa extract, pointing to the use of 2.5% (w/v) SDS and 300 µg of beads for sample preparation and long-term digestion (16h) with 0.15 µg Lys-C and 2.5 µg trypsin (≈1:17 ratio). Based on the results of the ANN model, the manual protocol was automated in OT-2. The performance of the automatic protocol was evaluated with different sample types, including human plasma, Arabidopsis thaliana leaves, Escherichia coli cells, and mouse tissue cortex, showing great reproducibility and low sample-to-sample variability in all cases. In addition, we tested the performance of this method in the preparation of a challenging biological fluid such as rat bile, a proximal fluid that is rich in bile salts, bilirubin, cholesterol, and fatty acids, among other MS interferents. Compared to other protocols described in the literature for the extraction and digestion of bile proteins, the method described here allowed identify 385 unique proteins, thus contributing to improving the coverage of the bile proteome.

Characterization and Hydrolysis Mechanism Analysis of a Cold-Adapted Trypsin-Like Protease from Antarctic Krill.

Cold-adapted proteases are capable of efficient protein hydrolysis at reduced temperatures, which offer significant potential applications in the area of low temperature food processing. In this paper, we attempted to characterize cold-adapted proteases from Antarctic krill. Antarctic krill possesses an extremely active autolytic enzyme system in their bodies, and the production of peptides and free amino acids accompanies the rapid breakdown of muscle proteins following the death. The crucial role of trypsin in this process is recognized. A cold-adapted trypsin named OUC-Pp-20 from Antarctic krill genome was cloned and expressed in Pichia pastoris. Recombinant trypsin is a monomeric protein of 26.8 ± 1.0 kDa with optimum reaction temperature at 25 °C. In addition, the catalytic specificity of OUC-Pp-20 was assessed by identifying its hydrolysis sites through LC-MS/MS. OUC-Pp-20 appeared to prefer Gln and Asn at the P1 position, which is an amino acid with an amide group in its side chain. Hydrolysis reactions on milk and shrimp meat revealed that it can effectively degrade allergenic components in milk and arginine kinase in shrimp meat. These findings update the current knowledge of cold-adapted trypsin and demonstrate the potential application of OUC-Pp-20 in low temperature food processing.

Improved Detection of Tryptic Peptides from Tissue Sections Using Desorption Electrospray Ionization Mass Spectrometry Imaging.

DESI-MSI is an ambient ionization technique used frequently for the detection of lipids, small molecules, and drug targets. Until recently, DESI had only limited use for the detection of proteins and peptides due to the setup and needs around deconvolution of data resulting in a small number of species being detected at lower spatial resolution. There are known differences in the ion species detected using DESI and MALDI for nonpeptide molecules, and here, we identify that this extends to proteomic species. DESI MS images were obtained for tissue sections of mouse and rat brain using a precommercial heated inlet (approximately 450 °C) to the mass spectrometer. Ion mobility separation resolved spectral overlap of peptide ions and significantly improved the detection of multiply charged species. The images acquired were of pixel size 100 µm (rat brain) and 50 µm (mouse brain), respectively. Observed tryptic peptides were filtered against proteomic target lists, generated by LC-MS, enabling tentative protein assignment for each peptide ion image. Precise localizations of peptide ions identified by DESI and MALDI were found to be comparable. Some spatially localized peptides ions were observed in DESI that were not found in the MALDI replicates, typically, multiply charged species with a low mass to charge ratio. This method demonstrates the potential of DESI-MSI to detect large numbers of tryptic peptides from tissue sections with enhanced spatial resolution when compared to previous DESI-MSI studies.

The effect of feeding on serum concentrations of cobalamin, folate, trypsin-like immunoreactivity, and pancreatic lipase immunoreactivity in dogs with signs of chronic gastrointestinal disease.

BACKGROUND: It is unknown if serum concentrations of cobalamin, folate, canine pancreatic lipase immunoreactivity (cPLI), and canine trypsin-like immunoreactivity (cTLI) obtained postprandially are equivalent to measurements obtained after withholding food in dogs with suspected gastrointestinal disease. HYPOTHESIS/OBJECTIVES: Measurements of serum concentrations of cobalamin, folate, cPLI, and cTLI postprandially will be equivalent to measurements after 12 hours of withholding food in dogs with signs of chronic gastrointestinal disease. Changes observed will not alter clinical interpretation. ANIMALS: 51 client-owned dogs with signs of gastrointestinal disease. METHODS: Prospective single arm clinical trial. Serum concentrations of cobalamin, folate, cPLI and cTLI 2, 4, and 8 hours postprandially were compared by equivalence testing to values after withholding food for 12 hours (baseline). RESULTS: Mean serum cobalamin concentrations 2 hours (498.1 ± 213.1 ng/L; P = 0.024) and 4 hours (501.9 ± 207.4 ng/L; P = 0.008) postprandial were equivalent to baseline (517.3 ± 211.5 ng/L). Mean serum cTLI 2 hours (31.3 ± 14 µg/L; P < 0.001) and 4 hours (29.6 ± 13.1 µg/L; P = 0.027) postprandial were equivalent to baseline (31.1 ± 15 µg/L). Mean serum folate concentration 2 hours postprandial (15 ± 7.7 µg/L) was equivalent to baseline (13.7 ± 8.3 µg/L; P < 0.001). Equivalence could not be assessed for cPLI due to results below the lower limit of quantification. Feeding altered the clinical interpretation in 27% (cobalamin), 35% (folate), 20% (cTLI), and 12% (cPLI) of dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: The clinical interpretation for a substantial number of samples changed after feeding, therefore withholding food before sample collection is prudent.

An in silico scheme for optimizing the enzymatic acquisition of natural biologically active peptides based on machine learning and virtual digestion.

BACKGROUND: As a potential natural active substance, natural biologically active peptides (NBAPs) are recently attracting increasing attention. The traditional proteolysis methods of obtaining effective NBAPs are considerably vexing, especially since multiple proteases can be used, which blocks the exploration of available NBAPs. Although the development of virtual digesting brings some degree of convenience, the activity of the obtained peptides remains unclear, which would still not allow efficient access to the NBAPs. It is necessary to develop an efficient and accurate strategy for acquiring NBAPs. RESULTS: A new in silico scheme named SSA-LSTM-VD, which combines a sparrow search algorithm-long short-term memory (SSA-LSTM) deep learning and virtually digested, was presented to optimize the proteolysis acquisition of NBAPs. Therein, SSA-LSTM reached the highest Efficiency value reached 98.00 % compared to traditional machine learning algorithms, and basic LSTM algorithm. SSA-LSTM was trained to predict the activity of peptides in the proteins virtually digested results, obtain the percentage of target active peptide, and select the appropriate protease for the actual experiment. As an application, SSA-LSTM was employed to predict the percentage of neuroprotective peptides in the virtual digested result of walnut protein, and trypsin was ultimately found to possess the highest value (85.29 %). The walnut protein was digested by trypsin (WPTrH) and the peptide sequence obtained was analyzed closely matches the theoretical neuroprotective peptide. More importantly, the neuroprotective effects of WPTrH had been demonstrated in nerve damage mouse models. SIGNIFICANCE: The proposed SSA-LSTM-VD in this paper makes the acquisition of NBAPs efficient and accurate. The approach combines deep learning and virtually digested skillfully. Utilizing the SSA-LSTM-VD based strategy holds promise for discovering and developing peptides with neuroprotective properties or other desired biological activities.

Photoelectrochemical quenching-recovery biosensor based on NSCQDs/Fe2O3@Bi2S3 for the detection of trypsin.

BACKGROUND: The content of trypsin will change when pancreatic diseases occur, therefore developing a high-performance method for trypsin detection is of great significance for guiding patients on medication plans and improving their prognosis. Photoelectrochemical (PEC) analysis techniques have emerged as a solution to apply for bioassays. RESULTS: Herein, the Fe2O3@Bi2S3 and Nitrogen and sulfur co-doped carbon quantum dots (NSCQDs) were successfully synthesized by a hydrothermal method. Subsequently, NSCQDs/Fe2O3@Bi2S3 with a photocurrent amplification effect covered on fluorine-doped tin oxide (FTO) electrode as the substrate material and apoferritin (APO) as a bio-recognition element to quench the photocurrent of the substrate material which can be excited with light. Due to the decomposition specifically between APO and trypsin, the photocurrent response increased. The linear range for trypsin detection showed satisfied results from 2 to 1000 ng mL-1 under optimal conditions, with a detection limit of 0.42 ng mL-1 and a recovery rate of 97.41 %-103.02 %, enabling efficient quantitative analysis of trypsin. SIGNIFICANCE: In this experiment, a PEC biosensor with simple operation, low detection limit, excellent selectivity and strong stability was successfully prepared, enabling quantitative analysis of trypsin in human serum samples through the quenching-recovery mechanism. It holds great significance for diagnosis and serves as a practical method for the detection of trypsin in the future.

Effect of moxibustion on colonic low-grade inflammatory response in rats with diarrhea-predominant irritable bowel syndrome based on mast cell degranulation. / åŸºäºŽè‚¥å¤§ç»†èƒžè„±é¢—ç²’æŽ¢è®¨è‰¾ç¸å¯¹è ¹æ³»åž‹è‚ æ˜“æ¿€ç»¼åˆå¾å¤§é¼ ç»“è‚ ä½Žåº¦ç‚Žæ€§ååº”çš„å½±å“.

OBJECTIVES: To observe the effects of moxibustion on colonic mast cell degranulation and inflammatory factor expression in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), and explore the potential mechanism of moxibustion in treating IBS-D. METHODS: Forty-five rat pups born from 5 healthy SPF-grade pregnant SD rats, with 8 rats were randomly selected as the normal group. The remaining 37 rats were intervened with maternal separation, acetic acid enema, and chronic restraint stress to establish the IBS-D model. The successfully modeled 32 rats were then randomly assigned to a model group, a ketotifen group, a moxibustion group, and a moxibustion-medication group, with 8 rats in each group. The rats in the ketotifen group were intervened with intragastric administration of ketotifen solution (10 mL/kg); the rats in the moxibustion group were intervened with suspended moxibustion on bilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37); the rats in the moxibustion-medication group were intervened with suspended moxibustion combined with intragastric administration of ketotifen solution. All interventions were administered once daily for 7 consecutive days. The diarrhea rate and minimum volume threshold of abdominal withdrawal reflex (AWR) were calculated before and after modeling, as well as after intervention. After intervention, colonic tissue morphology was observed using HE staining; colonic mucosal ultrastructure was examined by scanning electron microscopy; colonic mast cell ultrastructure was observed using transmission electron microscopy; mast cell degranulation was assessed by toluidine blue staining; serum and colonic levels of histamine, interleukin (IL)-1ß, IL-6, IL-1α, trypsin-like enzyme, and protease-activated receptor 2 (PAR-2) were measured by ELISA; the Western blot and real-time quantitative PCR were employed to evaluate the protein and mRNA expression of colonic IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2; the immunofluorescence was used to detect the positive expression of histamine, IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colonic tissue. RESULTS: Compared to the normal group, the rats in the model group exhibited extensive infiltration of inflammatory cells in colonic tissue, severe damage to the colonic mucosa, disordered arrangement of villi, reduced electron density, and a significant decrease in granule quantity within mast cells. The diarrhea rate and mast cell degranulation rate were increased (P<0.01), AWR minimum volume threshold was decreased (P<0.01); the serum and colonic levels of histamine, IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 were elevated (P<0.01); the positive expression of histamine, as well as protein, mRNA and positive expression of IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colon were all elevated (P<0.01). Compared to the model group, the rats in the ketotifen group, the moxibustion group, and the moxibustion-medication group exhibited significantly reduced infiltration of inflammatory cells in colonic tissue, relatively intact colonic mucosa, orderly arranged villi, increased electron density, and an augmented number of mast cell granules; the diarrhea rate and mast cell degranulation rate were decreased (P<0.01), and AWR minimum volume threshold was increased (P<0.01); the serum and colonic levels of histamine, IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 were reduced (P<0.01); the positive expression of histamine, as well as protein, mRNA and positive expression of IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colon were all decreased (P<0.01). Compared to the ketotifen group, the moxibustion group showed decreased serum levels of histamine, IL-6, and trypsin-like enzyme (P<0.01, P<0.05), as well as reduced colonic levels of IL-1ß and IL-6 (P<0.01, P<0.05); the protein expression of colonic IL-1ß, IL-1α, and PAR-2 was reduced (P<0.05), and the positive expression of colonic IL-1ß and trypsin-like enzyme was reduced (P<0.01, P<0.05). Compared to both the ketotifen group and the moxibustion group, the moxibustion-medication group exhibited decreased diarrhea rate and mast cell degranulation rate (P<0.01), an increased AWR minimum volume threshold (P<0.01), reduced serum and colonic levels of histamine, IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 (P<0.01), decreased protein expression of colonic IL-1ß, trypsin-like enzyme, and PAR-2 (P<0.01, P<0.05), reduced mRNA and positive expression of colonic IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 (P<0.01, P<0.05), and decreased positive expression of colonic histamine (P<0.01). CONCLUSIONS: Moxibustion on "Tianshu" (ST 25) and "Shangjuxu" (ST 37) might inhibit low-grade inflammatory reactions in the colon of IBS-D model rats. The mechanism may be related to the inhibition of histamine and trypsin-like enzyme secreted by mast cell, thereby reducing the expression of related inflammatory factors.

High-throughput method for Peptide mapping and Amino acid sequencing for Calcitonin Salmon in Calcitonin Salmon injection using Ultra High Performance Liquid Chromatography - High Resolution Mass Spectrometry (UHPLC-HRMS) with the application of Bioinformatic tools.

BACKGROUND: Tandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic peptide drugs, and peptide drug products including side chain modifications in the Peptide drugs. This article explains a step-by-step guide to developing a high-throughput method using high resolution mass spectrometry for characterization of Calcitonin Salmon injection containing high proportion of UV-active excipients. METHODS: The major challenge in the method development of Amino acid sequencing and Peptide mapping was presence of phenol in drug product. Phenol is a UV-active excipient and reacts with both Dithiothreitol (DTT) and Trypsin. Hence Calcitonin Salmon was extracted from the Calcitonin Salmon injection using solid phase extraction after the extraction, Amino acid sequencing and peptide mapping study was performed. Upon incubation of Calcitonin Salmon with Trypsin and DTT, digested fragments were generated which were separated by mass compatible reverse phase chromatography and the molecular mass of each fragment was determined using HRMS. RESULTS: A reverse phase chromatographic method was developed using UHPLC-HRMS for the determination of direct mass, peptide mapping and to determine the amino acid sequencing in the Calcitonin Salmon injection. The method was found Specific and fragments after trypsin digest are well resolved from each other and the molecular mass of each fragment was determined using HRMS. Sequencing was performed using automated identification of b and y ions annotation and identifications based on MS/MS spectra using Biopharma finder and Proteome discoverer software. CONCLUSION: Using this approach 100% protein coverage was obtained and protein was identified as Calcitonin Salmon and the observed masses of tryptic digest of peptide was found similar with theoretical masses. The method can be used for both UV and MS based Peptide mapping and whereas the UV based peptide mapping method can be used as identification test for Calcitonin Salmon drug substance and drug product in quality control.

Rational design of soluble expressed human aldehyde dehydrogenase 2 with high stability and activity in pepsin and trypsin.

Acetaldehyde dehydrogenase 2 (ALDH2) is a crucial enzyme in alcohol metabolism, and oral administration of ALDH2 is a promising method for alcohol detoxification. However, recombinant ALDH2 is susceptible to hydrolysis by digestive enzymes in the gastrointestinal tract and is expressed as inactive inclusion bodies in E. coli. In this study, we performed three rounds of rational design to address these issues. Specifically, the surface digestive sites of pepsin and trypsin were replaced with other polar amino acids, while hydrophobic amino acids were incorporated to reshape the catalytic cavity of ALDH2. The resulting mutant DE2-852 exhibited a 45-fold increase in soluble expression levels, while its stability against trypsin and pepsin increased by eightfold and twofold, respectively. Its catalytic efficiency (kcat/Km) at pH 7.2 and 3.2 improved by more than four and five times, respectively, with increased Vmax and decreased Km values. The enhanced properties of DE2-852 were attributed to the D457Y mutation, which created a more compact protein structure and facilitated a faster collision between the substrate and catalytic residues. These results laid the foundation for the oral administration and mass preparation of highly active ALDH2 and offered insights into the oral application of other proteins.