Pepsin Digestion Research Articles

Effects of pH on the emulsifying, gelation and curcumin delivery properties of myofibrillar protein and carboxymethyl cellulose emulsions

This study was designed to explore the curcumin delivery properties of emulsion gel fabricated by myofibrillar protein (MP)-carboxymethyl cellulose (CMC) complexes with different pHs. Firstly, the MP-CMC complexes under pH 2, 3, 4, 5, and 6 were prepared, then the properties of these complex-stabilized emulsions were tested, and the gel properties and curcumin delivery ability of these heat-induced emulsion gels were compared. Results indicated that MP-CMC complexes showed different viscosities and combined states under different pHs. The emulsions fabricated by pH 6 complexes showed the highest emulsion activity index and emulsion stability, and the lowest droplet size value. The emulsion gel of the pH 6 complex showed the highest hardness, chewiness, gumminess, and resilience, while the pH 4 sample presented the lowest water and oil holding capacity among all samples. The pH 4 samples showed 99% curcumin-encapsulating efficiency as well as 70% curcumin retention after 4 h gastrointestinal digestion. The pH 4 samples could protect and control the release of the curcumin by protecting some proteins from pepsin digestion and controlling the release of fatty acid. As for curcumin release rate and bioavailability at the intestinal stage, the pH 6 samples showed the highest values, followed by the pH 5 samples, and the pH 4 showed the lowest. The emulsion gel prepared by pH 4 MP-CMC complexes could best protect and control the release of curcumin, whilst the pH 6 samples showed a better gel quality and a higher release rate and curcumin bioavailability.

How Modifications of Corneal Cross-Linking Protocols Influence Corneal Resistance to Enzymatic Digestion and Treatment Depth.

The purpose of this study was to determine the effects of the Photoactivated Chromophore for Keratitis Corneal Cross-Linking (PACK-CXL) protocol modifications on corneal resistance to enzymatic digestion and treatment depth. Eight hundred one ex vivo porcine eyes were randomly divided into groups of 12 to 86 corneas, treated with various epi-off PACK-CXL modifications, including acceleration (30 > 2 minutes, 5.4J/cm2), increased fluence (5.4 > 32.4J/cm2), deuterium oxide (D2O) supplementation, different carrier types (dextran versus hydroxypropyl methylcellulose [HPMC]), increased riboflavin concentration (0.1 > 0.4%), and riboflavin replenishment during irradiation (yes/no). Control group eyes did not receive PACK-CXL. A pepsin digestion assay was used to determine corneal resistance to enzymatic digestion. A phalloidin fluorescent imaging assay was used to determine the PACK-CXL treatment effect depth. Differences between groups were evaluated using a linear model and a derivative method, respectively. PACK-CXL significantly increased corneal resistance to enzymatic digestion compared to no treatment (P < 0.03). When compared to a 10 minute, 5.4J/cm2 PACK-CXL protocol, fluences of 16.2J/cm2 and higher increased corneal resistance to enzymatic digestion by 1.5- to 2-fold (P < 0.001). Other protocol modifications did not significantly change corneal resistance. A 16.2J/cm2 fluence also increased collagen compaction in the anterior stroma, whereas omitting riboflavin replenishment during irradiation increased PACK-CXL treatment depth. Increasing fluence will likely optimize PACK-CXL treatment effectiveness. Treatment acceleration reduces treatment duration without compromising effectiveness. The generated data help to optimize clinical PACK-CXL settings and direct future research efforts.

Inhibition of In Vitro Amylolysis of Wheat Starch by Gluten Peptides.

The effect of gluten peptides (GPs) isolated from a gluten proteolysate on in vitro amylolysis of gelatinized wheat starch was investigated. GPs in a pepsin hydrolysate were fractionated into fractions with molecular weights (MWs) of 500-3000, 3500-7000, 10-17, and 35-48 kDa. The fractions containing peptides with MW > 10 kDa had a strong inhibitory effect on enzyme activity and amylolysis of starch, whereas GPs with MW <10 kDa had no inhibitory effect. Binding constants estimated by surface plasmon resonance showed that peptides in the fractions with MW > 10 kDa bound more strongly to α-amylase, in contrast to peptides of MW <10 kDa. Significant correlations were observed between digestion parameters and equilibrium binding affinity. We conclude that peptides with MW >10 kDa in a pepsin digest of gluten have a strong inhibitory effect on in vitro enzymatic hydrolysis of starch due to their strong binding affinity to α-amylase.

Investigation of nutritional profile, protein solubility and in vitro digestibility of various algae species as an alternative protein source for poultry feed

Alternative protein sources, particularly seaweed and microalgae, have long been considered a potential source of protein for poultry feed. Currently, several species of algae are a special focus of animal nutrition in order to replace soybean meal (SBM) due to its high protein content and abundance of vitamins, minerals and pigments. Determining nutrient and digestibility of a new ingredient is the first step in evaluating its suitability for use in poultry feeds. This study aimed to evaluate nutritional composition and digestibility of five algae including Caulerpa lentillifera J. Agardh, Ulva rigida C. Agardh, Spirulina sp., Coelastrella sp. (KKUP1) and Scenedesmus sp. (KKUP2). We evaluated nutritive value, amino acid profiles and physical characteristics microscopically. Protein solubility and in vitro digestibility, i.e., dilute pepsin digestibility (DPD) and two-phase gastric pancreatic digestibility (GPD), were performed by incubating algae samples in porcine pepsin and porcine pancreatin with a protease, lipase and amylase enzyme solution to determine suitability of algae as a protein ingredient in poultry feed. Results showed that Spirulina sp. had the highest protein content (44.44 % of dry matter basis; P < 0.0001) and was rich in amino acids (Lys, Thr and Trp). Moreover, protein solubility, digestibility (DPD and GPDProtein) and digestible protein of Spirulina sp. were significantly higher than those of other species (52.35 %, 49–67 % and 20.70 %, respectively). On the other hand, energy digestibility (GPDEnergy) and digestible energy were highest in Ulva rigida (99.40 % and 15.29 MJ kg−1, respectively; P < 0.0001). These results imply that Spirulina sp. was more suitable for utilization as an alternative protein source for SBM substitution in poultry feed formulation. This characterization of biochemical composition and in vitro digestibility of algae represents the preliminary methodology used to initially select algae for use as a protein source in animal feed and additional in vivo tests are warranted.

Generic detection of organophosphorus nerve agent adducts to butyrylcholinesterase in plasma using liquid chromatography-tandem mass spectrometry combined with an improved procainamide-gel separation and pepsin digestion method

Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be applied to confirm exposure in humans. A sensitive method for generic detection of G- and V-series OPNA adducts to BChE in plasma was developed by combining an improved procainamide-gel separation (PGS) and pepsin digestion protocol with ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Residual matrix interferences from prior PGS purification of OPNA-BChE adducts from plasma were found to be a critical cause of significantly reduced UHPLC-MS/MS detection sensitivity. In our developed on-column PGS approach, the matrix interference was successfully removed by adding an appropriate concentration of NaCl to the washing buffer, and it could capture ≥92.5% of the BChE in plasma. The lower pH value and the longer digestion time in all previous pepsin digestion methods were found to be a key accelerated aging factor of several adducts such as tabun (GA)-, cyclohexylsarin (GF)-, and soman (GD)-BChE nonapeptide adducts, making them difficult to detect. The aging event of several OPNA-BChE nonapeptide adducts was so successfully addressed that the formic acid level in enzymatic buffer and digestion time were lowered to 0.05% (pH 2.67) and 0.5 h, respectively, and the post-digestion reaction was immediately terminated. The improved condition parameters were optimal for pepsin digestion of all types of OPNA-BChE adducts into their individual unaged nonapeptide adducts with the highest yields, expanding the applicability of the method. The method had a nearly one-fold decrease in sample preparation time through the reduction of digestion time and removal of ultrafiltration procedure after digestion. The limit of identification (LOI) were determined respectively as 0.13 ng mL−1, 0.28 ng mL−1, 0.50 ng mL−1, 0.41 ng mL−1 and 0.91 ng mL−1 for VX-, sarin (GB)-, GA-, GF-, and GD-exposed human plasma, being low exposure value compared to previously documented approaches. The approach was utilized to fully characterize the adducted (aged and unaged) BChE levels of five OPNAs in a series of their individual exposed concentration (1.00–400 nM) of plasma sample, and successfully detect OPNA exposure from all unknown plasma samples from OPCW's second and third biomedical proficiency tests. The OPNA-BChE adducts, their aged adducts, and unadducted BChE from OPNA-exposed plasma can simultaneously be measured using the method. The study provides a recommended diagnostic tool for generic verification of any OPNA exposure with high confidence by detecting its corresponding BChE adduct.

Türk Süpermarketlerindeki İthal Balıklarda Zoonotik Anisakis Larvalarının Varlığına İlişkin Devam Eden Veriler: Dondurulmuş Atlantik Uskumru (Scomber Scombrus) ve Füme Atlantik Somonu (Salmo Salar)

Anisakis pegreffii and A. simplex sensu stricto (s.s.) are the main etiological agents causing human anisakiasis. Here, we aimed to investigate based on the pepsin digestion method of the occurrence of Anisakis larvae in imported deep-frozen whole Atlantic mackerel (Scomber scombrus) from FAO 27 to Turkey and smoked Norwegian farmed Atlantic salmon fillets (Salmo salar) between 2018 and 2019. A total of 100 whole Atlantic mackerel and 180 Atlantic salmon fillets were randomly sampled from local Turkish supermarkets. No Anisakis larvae were detected in smoked Atlantic salmon fillets. In total, 827 Anisakis larvae were found in mackerel, and the prevalence was 68% (68/100). The mI and mA of Anisakis larvae in mackerel were 13.1 and 8.2, respectively. Whereas the 95.28% (788/827) of the Anisakis larvae were found in abdominal cavity/viscera, the 4.72 % (39/827) of the larvae in the muscle. The prevalence and mI of Anisakis larvae in the abdominal cavity/viscera and muscle of mackerel was 63.0% (63/100) and 42.0% (42/100), and 12.5 and 0.9, respectively. The subsample of 100 larvae was molecularly identified by ITS RFLP patterns. The 99 (99.0%) larvae were identified as A. simplex (s.s.), and 1 (1.0%) larva was A. pegreffii. Consequently, there is low or no risk of anisakiasis in smoked farmed Atlantic salmon for Turkish consumers. The 42.0% prevalence of zoonotic Anisakis species larvae in imported Atlantic mackerel fillets could have public health risk in Turkish consumers for anisakiasis or allergy.

Determination of Binding Sites on Trastuzumab and Pertuzumab to Selective Affimers Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a method to probe the solvent accessibility and conformational dynamics of a protein or a protein-ligand complex with respect to exchangeable amide hydrogens. Here, we present the application of HDX-MS to determine the binding sites of Affimer reagents to the monoclonal antibodies trastuzumab and pertuzumab, respectively. Intact and subunit level HDX-MS analysis of antibody-affimer complexes showed significant protection from HDX in the antibody Fab region upon affimer binding. Bottom-up HDX-MS experiments including online pepsin digestion revealed that the binding sites of the affimer reagents were mainly located in the complementarity-determining region (CDR) 2 of the heavy chain of the respective antibodies. Three-dimensional models of the binding interaction between the affimer reagents and the antibodies were built by homology modeling and molecular docking based on the HDX data.

UV-C light promotes the reductive cleavage of disulfide bonds in β-Lactoglobulin and improves in vitro gastric digestion

β-Lactoglobulin (β-Lg) is the main protein in whey and is known for its allergenicity and resistance to the digestion of pepsin and trypsin. The UV-C photoinduced cleavage of disulfide bonds in β-Lactoglobulin, as promoted by excitation of tryptophan residues (Trp), is shown to induce changes in the protein's secondary structure, significantly reducing the protein's resistance to pepsin digestion. The UV-C light-induced changes in the protein secondary structure are marked by an increase in the contribution of β-sheet and α-helix structures with a concomitantly smaller contribution of the β-turn structural motif. The photoinduced cleavage of disulfide bonds in β-Lg has an apparent quantum yield of ф = 0.0015 ± 0.0003 and was shown by transient absorption laser flash photolysis to arise by two different pathways: a) the reduction of the disulfide bond Cys66Cys160 occurs by direct electron transfer from the triplet-excited 3Trp to the disulfide bond due to the existence of a CysCys/Trp triad (Cys66Cys160/Trp61) and b) the reduction of the buried Cys106Cys119 disulfide bond involves a reaction with a solvated electron originated by the photoejection of electrons from the triplet-excited 3Trp decay. The in vitro gastric digestion index for UV-C-treated β-Lg is revealed to have increased significantly by 36 ± 4 % and 9 ± 2 % under simulated elderly and young adult digestive conditions, respectively. When compared to the native protein, the peptide mass fingerprint profile of digested UV-C-treated β-Lg shows a higher content and variety of peptides, including the production of some exclusive bioactive peptides such as PMHIRL and EKFDKALKALPMH.

Effect of Thermal Processing on the Conformational and Digestive Properties of Myosin

Heat treatment affects the structural properties of meat proteins, which in turn leads to changes in their sensitivity to digestive enzymes, further affecting the nutritional value of meat and meat products. The mechanism of changes in the structure and digestive properties of myosin under different heating conditions were studied. An increase in heating temperature led to the exposure of internal groups to a polar environment, but to a decrease in the sturdy α-helix structure of myosin (p < 0.05). The results of tryptophan fluorescence verified that the tertiary structure of the protein seemed to be unfolded at 70 °C. Higher protein denaturation after overheating, as proven by the sulfhydryl contents and turbidity, caused irregular aggregate generation. The excessive heating mode of treatment at 100 °C for 30 min caused myosin to exhibit a lower degree of pepsin digestion, which increased the Michaelis constant (Km value) of pepsin during the digestion, but induced the production of new peptides with longer peptide sequences. This study elucidates the effects of cooking temperature on the conformation of myosin and the change in digestibility of pepsin treatment during heating.

An in vitro protocol to characterise the resistance of food proteins to intestinal digestion.

In vitro digestion tests provide data on the form in which dietary proteins maybe presented to the gut mucosal immune system, one of many strands of evidence used in allergenicity risk assessment. A 96-well plate format in vitro intestinal digestion protocol has been developed with a high and low enzyme activity test executed at pH 6.5 and 8.0. It was applied to the systematic analysis of test proteins (including six allergens and one non-allergenic comparator) which were either completely resistant to pepsinolysis or gave rise to large persistent fragments following in vitro gastric digestion. Digestion was monitored using SDS-PAGE and densitometry. Proteins resistant to pepsin were also resistant to intestinal digestion irrespective of the protocol applied and gave rise to large persistent digestion fragments. In contrast persistent fragments from pepsin digestion were readily digested. Bile salts enhanced the digestibility of two highly resistant proteins, lysozyme ad β-lactoglobulin, changing the rank order of protein digestibility. Intestinal digestion tests that include bile salts provide a more physiologically relevant system for future investigation into how digestion products may influence the balance between tolerance and sensitisation - and hence contribute to future development of a more effective allergenicity risk assessment process.

Observation of curcumin-encapsulated Pickering emulsion stabilized by cellulose nanocrystals-whey protein isolate (CNCs-WPI) complex under in vitro lipid digestion through INFOGEST model

Curcumin-encapsulated Pickering emulsion (Cur-PE) was successfully prepared using cellulose nanocrystals (CNCs)-whey protein isolate (WPI) complex as a stabilizer to control the size and stability of the Cur-PE. Firstly, needle-like CNCs were prepared by acid hydrolysis, and the mean particle size, polydispersity index (PDI), zeta potential, and aspect ratio of the CNCs were 100.7 nm, 0.32, −43.6 mV, and 20.8, respectively. The Cur-PE-C0.5W0.1, prepared with 0.5 wt% CNCs and 0.1 wt% WPI at pH 2, had a mean droplet size of 230.0 nm, PDI of 0.275, and zeta potential of +53.5 mV. The Cur-PE-C0.5W0.1 prepared at pH 2 exhibited the highest stability during storage for 14 days. FE-SEM revealed that the droplets of the Cur-PE-C0.5W0.1 prepared at pH 2 were spherical and fully covered by CNCs. The adsorption of CNCs at the oil-water interface increases the encapsulation efficiency (89.4 %) of curcumin in the Cur-PE-C0.5W0.1 and protects curcumin from pepsin digestion in the gastric phase. However, the Cur-PE-C0.5W0.1 was sensitive to release curcumin in the intestine phase. The CNCs-WPI complex developed in this study could serve as a promising stabilizer to make Pickering emulsions stable at pH 2 for the encapsulation and delivery of curcumin to the expected target area.

In vitro antioxidant and anti-inflammatory properties of Artocarpus altilis (Parkinson) Fosberg (seedless breadfruit) fruit pulp protein hydrolysates

Protein hydrolysates from dietary sources possess many physiological and biological properties. Artocarpus altilis is an evergreen multipurpose plant with many benefits. Therefore, this study evaluates in vitro antioxidant and anti-inflammatory properties of A. altilis protein hydrolysates. Protein was isolated from A. altilis and hydrolysed with pepsin and trypsin separately using different enzyme: substrate ratios (1:8, 1:16, 1:32). Antioxidant properties investigated included Fe2+-chelating, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and hydrogen peroxide radical scavenging activities. Anti-inflammatory activities were determined using effects on hypotonic solution-induced cell lysis on red blood cell membrane stabilisation and heat-induced protein denaturation. The degree of hydrolysis of trypsin hydrolysate increased with increasing enzyme–substrate ratio, while pepsin hydrolysate decreased as the enzyme–substrate ratio increased. The dominant amino acids in A. altilis protein and hydrolysates were glutamate, aspartate and leucine. Protein hydrolysates obtained from pepsin and trypsin digestion had DPPH scavenging abilities of 43.0 ± 0.01% and 22.2 ± 0.01%, respectively. However, trypsin-hydrolysed protein had a high Fe2+-chelating ability, while pepsin-hydrolysed protein had high hydrogen peroxide scavenging ability. Trypsin-hydrolysed protein showed good membrane stability and inhibition of protein denaturation. The results indicated that A. altilis protein hydrolysates possess significant antioxidant and anti-inflammatory effects and can further lend support to food industries as functional foods.

Glycation of whey protein isolate and stachyose modulates their in vitro digestibility: Promising prebiotics as functional ingredients

Glycated conjugates have recently been designed to be targeted to probiotic in the colon. However, the underlying mechanisms of regulating the prebiotic properties and digestibility of conjugates through glycation have not been fully elucidated. In this study, WPI-stachyose conjugates were prepared through Maillard reaction and proposed as functional ingredients with prebiotic characteristics. The effect of glycation on the in vitro simulated gastrointestinal fate of WPI-stachyose conjugate was further evaluated. The results showed that the glycated conjugates of stachyose covalently bound to whey protein exhibited a characteristic cross-linked structure, which enhanced their inhibition of α-amylase activity and retardation of glucose dialysis. Besides, simulated digestion analysis demonstrated that stachyose-conjugated WPI was more resistant to pepsin and trypsin digestion than unconjugated WPI. Compared with native WPI, the protein digestibility and degree of hydrolysis of WPI-stachyose conjugates were reduced by 12.5% ± 0.66% and 5.30% ± 1.02%, respectively. In addition, rat intestinal extracts were used to detect the in vitro digestibility of stachyose. The hydrolysis rate of stachyose in the glycated conjugates was 70% lower than that of stachyose. Therefore, the resulting WPI-stachyose conjugate may be used as functional ingredients to achieve the sustained-release effect in the gastrointestinal tract. These findings demonstrate the possibility of modulating proteins and carbohydrates digestibility through glycation, and have implications for the design and manufacture of potential prebiotic molecules with controlled digestibility in food and related healthcare fields.

The degree of doneness affected molecular changes and protein digestibility of pork.

The degree of doneness has been shown to have a great impact on eating quality of meat, however, it is little known whether it affects protein digestibility of meat. In this study, we explored molecular changes and protein digestibility of pork under different degree of doneness. Pork chops were cooked in a 100°C water bath for about 26 min and a gradient decrease in doneness was obtained from outer to inner layers of samples. Compared with the raw samples, the cooked samples' active and total sulfhydryl contents, surface hydrophobicity, and turbidity increased but its solubility decreased. The inner layers with lower doneness contained higher α-helix, and fluorescence intensities of tryptophan and tyrosine residues than the outer layers with higher doneness. The pepsin and pancreatin digestibility of meat proteins in the inner layers were higher than those of the outer layers. Molecular simulation analysis showed that the most abundant protein in pork, i.e., myosin in the outer layers were more stable with an increased number of hydrogen bonds, making it difficult to be digested. These findings provided a new insight into the heterogeneity of meat nutritional quality due to the existence of doneness gradient.

Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry

Currently, fast liquid chromatographic separations at low temperatures are exclusively used for the separation of peptides generated in hydrogen deuterium exchange (HDX) workflows. However, it has been suggested that capillary electrophoresis may be a better option for use with HDX. We performed in solution HDX on peptides and bovine hemoglobin (Hb) followed by quenching, pepsin digestion, and cold capillary electrophoretic separation coupled with mass spectrometry (MS) detection for benchmarking a laboratory-built HDX-MS platform. We found that capillaries with a neutral coating to eliminate electroosmotic flow and adsorptive processes provided fast separations with upper limit peak capacities surpassing 170. In contrast, uncoated capillaries achieved 30% higher deuterium retention for an angiotensin II peptide standard owing to faster separations but with only half the peak capacity of coated capillaries. Data obtained using two different separation conditions on peptic digests of Hb showed strong agreement of the relative deuterium uptake between methods. Processed data for denatured versus native Hb after deuterium labeling for the longest timepoint in this study (50,000 s) also showed agreement with subunit interaction sites determined by crystallographic methods. All proteomic data are available under DOI: 10.6019/PXD034245.

Proximate, microbiological analysis and digestibility of pepsin in atta sexdens flour, from the region of Rioja, Perú

The consumption of insects, also known as entomophagy, is not a new eating habit; on the contrary, the insects are eaten in many countries worldwide, where insects are served as the main dish in many of them. In Peru, insect consumption is limited to regions of the central jungle where locals consume ants of the "Atta sexdens" variety in the form of toasted snacks with salt. Analysis of insects has shown a wide variation in macronutrient content relative to most wild vertebrate meats, encompassing a wide range of protein, fat and carbohydrate concentrations. In that sense, in terms of their proportional macronutrient composition, insects could serve as equivalents not only to wild meat, but also to other foods, such as seafood, nuts, legumes, vegetables or fruits. The aim of this study was to assess nutritional quality of "Atta sexdens" ant flour and determine its physical and chemical characteristics. The ants were obtained from the department of San Martín, province and district of Rioja and underwent a rigorous cleaning process to eliminate impurities such as wings, leaves and soil which are attached to the ants. The ants were dried using a Biobase oven, model BOV-T30C, and then ground in an electric grinder and finally passed through a sieve. The final product was measured for physicochemical properties such as pH, % acidity in aqueous extract and % acidity in ethanolic extract, resulting in 6.57, 0.21, and 0.29, respectively. The nutritional composition assessment was based on the AOAC standard method where the fat content of the meal was 35.40%; protein, 35.51%; carbohydrate, 16.12%; ash, 5.39%; and moisture, 7.58%. For the assessment of pepsin digestibility, the AOAC 971.09 test method was used, resulting in 99.77% activity. In regard to techno-functional properties, it was found that swelling capacity (SC) and water absorption capacity (WAC) resulted in 2.91 WAC g water/g m and 1.86 SC ml/g, respectively. On the other hand, the microbiological results were within the permissible limits for health; therefore, we can consider this ant meal a safe and innocuous product as well as an excellent food source with exceptional nutritional characteristics. Key words: Atta sexdens, flour, Entomophagy, Protein, Digestibility, Microbiology, Physicochemical properties, Proximate

Glycation with uronic acid-type reducing sugar enhances the anti-inflammatory activity of fish myofibrillar protein via the Maillard reaction

The role of carboxyl group in uronic acid in enhancing the anti-inflammatory activity of fish myofibrillar protein (Mf) was investigated, when lyophilized Mf was reacted with various reducing sugars at 60 °C and 35% relative humidity through the Maillard reaction. After pepsin and trypsin digestion, the anti-inflammatory activity was evaluated by measuring the secretions of tumor necrosis factor-α, interleukin-6, interleukin-1β, and nitric oxide in lipopolysaccharide-stimulated RAW 264.7 macrophage. The anti-inflammatory activity of Mf was not affected by glycation with glucose or galactose, whereas strongly enhanced by glycation with uronic acid-type reducing sugars: glucuronic acid, galacturonic acid, and alginate oligosaccharide. These results indicate that the presence of carboxyl group in reducing sugar is important for enhancing the anti-inflammatory activity of Mf. This study also shows that the enhanced effect could depend upon the number of carboxyl group in bound reducing sugar.

Cycloheximide promotes type I collagen maturation mainly via collagen prolyl 4-hydroxylase subunit α2.

Aberrant deposition of collagen is associated with cancer development and tissue fibrosis. Proline hydroxylation, catalyzed by collagen prolyl 4-hydroxylases (C-P4Hs), is necessary for collagen maturation and secretion. Here, we try to evaluate the mechanism of the regulation of CHX on collagen maturation. Using pepsin digestion, liquid chromatograph mass spectrometry and gene knockout, we find that treatment of mouse embryonic fibroblasts with cycloheximide (CHX) increases type I collagen proline hydroxylation partially via P4HA1 and mainly via P4HA2. Western blot analysis results show that CHX treatment reduces type I collagen but does not obviously impact the level of P4HA1/2 protein in the endoplasmic reticulum, which enhances the molar ratio of P4HA1/2 to type I collagen, and coimmunoprecipitation results confirm that more P4HA1/2 can bind to each type I collagen. Since C-P4Hs possess the capability to hydroxylate proline independent of ascorbate for a few cycles, this enhanced binding between P4HA1/2 and type I collagen can partially explain how CHX stimulates type I collagen maturation.

Species-Based Differences in Mechanical Properties, Cytocompatibility, and Printability of Methacrylated Collagen Hydrogels.

Collagen methacrylation is a promising approach to generate photo-cross-linkable cell-laden hydrogels with improved mechanical properties. However, the impact of species-based variations in amino acid composition and collagen isolation method on methacrylation degree (MD) and its subsequent effects on the physical properties of methacrylated collagen (CMA) hydrogels and cell response are unknown. Herein, we compared the effects of three collagen species (bovine, human, and rat), two collagen extraction methods (pepsin digestion and acid extraction), and two photoinitiators (lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and Irgacure-2959 (I-2959)) on the physical properties of CMA hydrogels, printability and mesenchymal stem cell (MSC) response. Human collagen showed the highest MD. LAP was more cytocompatible than I-2959. The compressive modulus and cell viability of rat CMA were significantly higher (p < 0.05) than bovine CMA. Human CMA yielded constructs with superior print fidelity. Together, these results suggest that careful selection of collagen source and cross-linking conditions is essential for biomimetic design of CMA hydrogels for tissue engineering applications.

Overcoming Incomplete Peptide Mapping of Antibody Complementarity-Determining Regions with Alternate Digestion Workflows

Peptide mapping of antibodies is an essential method to monitor peptide modifications in antibody lots that could affect the safety and efficacy of the product. Conventional protocols rely on protein digestion using proteases, such as trypsin, before mapping with mass spectrometry (MS). However, trypsin digestion may cause incomplete mapping of peptides, especially those that include highly hydrophobic peptides. Here, we show how pepsin can be used as an alternative and complementary protease for digestion that allows for improved sequence coverage, especially in proteins with highly hydrophobic regions. We also show that using guanidine hydrochloride (GuHCl) post-digestion improves peptide mapping results. Overall, these two methods—pepsin digestion and GuHCl post-digestion—can be used to provide more comprehensive antibody peptide maps, thereby enabling more thorough quality checking of biopharmaceutical products.