Pepsin Digestion Research Articles

Anti-Obesity Effects of a Collagen with Low Digestibility and High Swelling Capacity: A Human Randomized Control Trial

Background/Objectives: Collagen is a protein formed by very long amino acid chains. When conveniently treated, it can incorporate water into the net, thus increasing its volume and mass. The present work aimed to evaluate the potential anti-obesity effects of bovine collagen that has been technologically treated to increase its water retention capacity in an acid pH medium, with the objective of inducing satiation. Methods: Collagen’s digestibility was tested with a pepsin digestion test. Its swelling capacity was tested in an acid pH medium simulating gastric conditions. Postprandial levels of ghrelin in response to collagen supplementation were tested in rats. In a randomized control trial, 64 subjects with overweight/obesity were allocated in two groups: supplemented daily with two protein bars enriched with collagen (20 g per day) for 12 weeks, or control group. Anthropometric and biochemical measurements were assessed in all the participants. Results: This collagen showed a low digestibility (<60%) and high swelling capacity (>1900%) in vitro. In humans with overweight and obesity, this collagen significantly reduced body weight, body mass index (BMI), systolic blood pressure (SBP), and fatty liver index (FLI) and increased fat-free mass when compared with the control group. A significant reduction in the sarcopenic index; total, troncular, and visceral fat (measured by DEXA); and serum leptin levels were observed in the collagen group at the end of the intervention, with no differences with respect to controls. Collagen reduced the sensation of hunger and increased fullness and satisfaction. In male Wistar rats, collagen decreased postprandial blood ghrelin levels. Conclusions: Collagen supplementation (20 g per day for 12 weeks) reduced body weight, BMI, waist circumference, fat mass, FLI, and SBP in humans with overweight and obesity, which might be related to the increased sensation of fullness and satisfaction reported by the volunteers after the intake.

Enhanced anti-inflammatory effect of fish myofibrillar protein by introducing pectin oligosaccharide and its molecular mechanisms

This study investigated the efficacy of glycation with edible uronic acid-containing oligosaccharides via the Maillard reaction to enhance the anti-inflammatory effect of fish myofibrillar protein (Mf). Lyophilized Mf was reacted with pectin oligosaccharide (PO, half of the total protein weight) at 60 °C and 35 % relative humidity for up to 12 h to produce glycated Mf (Mf-PO). After pepsin and trypsin digestion, the anti-inflammatory effect was assessed by measuring the secretions of proinflammatory cytokines in LPS-stimulated RAW 264.7 macrophages, and the anti-inflammatory effect of Mf was enhanced by PO-glycation without marked lysine loss and browning. The effects on the expressions of genes related to the LPS-stimulated signaling pathway in macrophages were also examined. PO-glycation suppressed LPS-stimulated inflammation by suppressing expression of cd14 and enhancing suppressive effect of Mf on the TLR4-MyD88-dependent inflammatory signaling pathway. Therefore, as an edible reducing sugar, PO could be an effective bioindustrial material for developing anti-inflammatory Mf.

Insights into the Structural and Thermal Properties of Pepsin-Soluble Collagen from Daggertooth pike conger (Muraenesox cinereus)

Pepsin soluble collagen (PSC) is a type-1 collagen which is abundant in skin. The pepsin soluble collagen was successfully extracted from the skin of Daggertooth pike conger (Muraenesox cinereus) by using the conventional method of salt precipitation followed by dialysis. Pepsin-soluble collagen showed a yield of 10.12% on wet matter basis and 25.5% on dry matter basis. The triple helical structure of collagen was confirmed by Fourier Transform Infrared Spectroscopy (FTIR) and UV-Visible spectrophotometry. Isolated PSC was confirmed as type 1 collagen by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). FTIR spectra of PSC suggested that the secondary structure of the triple helical collagen was intact even after pepsin digestion. Pepsin-soluble collagen was soluble in acidic pH with maximum solubility at pH 4. The solubility was found to be decreased with an increase in the concentration of sodium chloride with a minimum solubility at 4%. The present PSC isolate from eel fish exhibited comparatively high thermal stability of 36 ͦ C. Morphological analysis by Scanning electron microscopy (SEM) revealed a porous structure and comparatively high thermal stability makes it a promising biomaterial.

A novel angiotensin I-converting enzyme inhibitory peptide APPLRP from Grifola frondosa ameliorated the Ang II-induced vascular modeling in zebrafish model by mediating smooth muscle cells

Grifola frondosa has garnered significant popularity as an edible mushroom attributable to its exceptional taste and nutritional benefits. This study isolated APPLRP, a potent ACE-inhibitory peptide, from the alcohol-soluble fraction of Grifola frondosa. The underlying mechanisms of APPLRP in antihypertension were explored through computational chemistry, cell experiments, and zebrafish model. Results demonstrated that APPLRP was an active competitive ACE inhibitor (IC50 = 29.93 μM) that could bind to the active pocket S2 and S1′ of ACE. APPLRP exhibited resistance to pepsin and pancreatin digestion. In vitro experiments revealed that APPLRP significantly attenuated Ang II-induced VSMCs proliferation and migration by down-regulating AT1R expression and inhibiting ERK1/2 and STAT3 phosphorylation. APPLRP intervention significantly ameliorated myocardial fibrosis, as evidenced by reductions in cardiac output, blood flow velocity, and cardiac collagen deposition levels in Ang II-induced hypertensive zebrafish model. Furthermore, APPLRP improved vascular remodeling in hypertensive zebrafish, indicated by increased vessel diameter and decreased vessel wall thickness. Notably, APPLRP treatment resulted in down-regulation of ACE and up-regulation of ACE2 expression in the vessels of hypertensive zebrafish. These findings indicated that APPLRP was a representative component of Grifola frondosa peptides, and its antihypertensive effects were associated with ACE inhibition and the improvement of VSMCs-mediated vascular remodeling.

TRICHINELLA AND AT LEAST THREE SPECIES OF SARCOCYSTIS PARASITIZE THE MUSCLES OF BOBCATS (LYNX RUFUS) FROM MISSISSIPPI.

Muscles of 25 bobcats (Lynx rufus) from remote areas of Mississippi in 2017 were tested for parasites. Testing for Sarcocystis infections included microscopic examination of fresh unstained muscle squashes, pepsin digestion of hearts and tongues, and histological sections of paraffin-embedded tissues. Sarcocystis spp. infections were detected in the muscles of 21 (84%) by a combination of methods. Sarcocysts were detected in the unstained tongue squashes of 2 bobcats. Sarcocystis sp. bradyzoites were detected in the pepsin digests of 3 of 19 hearts, and 12 of 19 tongues. In paraffin-embedded histological sections, sarcocysts were detected in 7 of 25 hearts, 17 of 25 tongues, and 5 of 23 limb muscles. Based on the character of the cyst wall, at least 3 morphologic types of sarcocysts were detected: those with small spikes on the cyst wall, corresponding to Sarcocystis felis, those with long villar protrusions, corresponding to Sarcocystis neurona, and those lacking visible cyst wall protrusions, representing an unidentified type of sarcocyst. Myositis associated with sarcocysts was seen in the tongues of 3, and in the limb muscles of 1 bobcat. Multilocus genotyping of the DNA extracted from paraffin-embedded sections from 2 bobcats, employing 18S, 28S, COI, ITS-1, and 5.8S and rpoB genes, diagnosed Sarcocystis caninum, S. felis, Sarcocystis lutrae, and S. neurona. An encapsulated species of Trichinella was identified in the tongue of 1; it represents the first documented occurrences in bobcats from Mississippi. Taken together, these observations suggest intensive exposure of these wild carnivores to Trichinella tissue cysts, implies predation or scavenging on these tissues promotes parasite transmission, and raises caution concerning zoonotic risk when such meat is rendered for human consumption.

Optimization of Hydrolytic Enzyme Production from Tempeh Starter to Increase In Vitro Pepsin Digestibility of Black Soldier Fly Maggot

This research aims to evaluate the optimal level and incubation time for the production of hydrolytic enzymes from tempeh starter (TS), in order to increase the in vitro pepsin digestibility of Black Soldier Fly (BSF), Hermetia illucens, maggot flour. The materials used were commercial tempeh starter, pepsin, and 15 days of age dried BSF maggot. In this study, two experiments were conducted. In a factorial completely randomized design, the analysis of variance (ANOVA) was performed to assess the significance of treatment effects, and orthogonal polynomial contrasts were employed to identify specific linear and quadratic trends among the group means. In experiment 1, research was carried out to determine the optimal starter level and incubation time to produce hydrolytic enzymes from tempeh starter. In experiment 2, crude enzyme from the best treatment in the first stage was used to increase the pepsin digestibility of BSF maggot flour. The optimal protein content and enzyme activities of tempeh starter was 0.10-0.13% starter with an incubation time of 2.00-3.47 days. Meanwhile, the optimal pepsin digestibility of BSF maggot flour was 1.51% enzymes with a hydrolysis time of 23.87 hours. This analysis measurement of protein content and enzymatic activity of tempeh starter and in vitro digestibility of BSF flour represent the preliminary methodology used to initially select hydrolyzed BSF flour to be a source of protein in animal feed.

Evaluation of the potential food allergy risks of human lactoferrin expressed in Komagataella phaffii.

Prior to the introduction of novel food ingredients into the food supply, safety risk assessments are required, and numerous prediction models have been developed and validated to evaluate safety. The allergenic risk potential of Helaina recombinant human lactoferrin (rhLF, Effera™), produced in Komagataella phaffii (K. phaffii) was assessed by literature search, bioinformatics sequence comparisons to known allergens, glycan allergenicity assessment, and a simulated pepsin digestion model. The literature search identified no allergenic risk for Helaina rhLF, K. phaffii, or its glycans. Bioinformatics search strategies showed no significant risk for cross-reactivity or allergenicity between rhLF or the 36 residual host proteins and known human allergens. Helaina rhLF was also rapidly digested in simulated gastric fluid and its digestibility profile was comparable to human milk lactoferrin (hmLF), further demonstrating a low allergenic risk and similarity to the hmLF protein. Collectively, these results demonstrate a low allergenic risk potential of Helaina rhLF and do not indicate the need for further clinical testing or serum IgE binding to evaluate Helaina rhLF for risk of food allergy prior to introduction into the food supply.

Investigation of bioactive peptides from Scolymus hispanicus by using in silico methods

Due to increases in the soil pollution related to agricultural areas, the interests on the wild edible greens have been increasing nowadays. Scolymus hispanicus (=Golden thistle) is a wild edible plant which is widely and naturally spread in Türkiye. An in silico-based bioinformatics approach has been proposed for the evaluation of bioactive peptides from this species. In silico digestion and also bioactive peptides of RubisCO from S.hispanicus were studied by using BIOPEP-UWM. Protparam and Clustal Omega were also used to determine physicochemical parameters and sequence similarity, respectively. The AE values related to angiotensin converting enzyme and dipeptidyl peptidase-IV were 0.0847 and 0.1059 after in silico pepsin digestion (pH>2), respectively. While the antioxidant property obtained after pepsin (pH>2) digestion was found to be 0.0127, the value of 0.042 was obtained for ficin on this parameter. BIOPEP-UWM also exhibit important properties related to the bioactivities of the peptides such as antioxidant, dipeptidyl peptidase-IV and angiotensin converting enzyme inhibitions. From the results, it could be said that S. hispanicus has very important bioactive peptides which could be evaluated in the production of functional foods. Moreover, isolated bioactive peptides and also secondary metabolites can also be utilized in pharmaceutical industry. Further in vitro and in vivo studies are strongly recommended on S. hispanicus.

Valorization of broiler edible byproducts: a chicken-liver hydrolysate with hepatoprotection against binge drinking

Over 10,000 metric-ton broiler livers are produced annually in Taiwan. Concerning unpleasant odor and healthy issue, broiler livers are not attractive to consumers. Although the patented chicken-liver hydrolysates (CLHs) through pepsin digestion possess several biofunctionalities, there is no study on hepatoprotection of CLH-based formula capsule (GBHP01) against binge drinking (Whiskey, 50% Alc./Vol.). GBHP01 led to an accelerated blood-alcohol clearance in rats, as evidenced by lowering blood-alcohol increment within 0 to 4 h, increasing blood-alcohol decrement within 4 to 8 h, and smaller blood alcohol concentration areas under the curve (BAC AUC) in the 8-h period (p < 0.05). The ameliorative effects of GBHP01 against binge drinking in rats over 6 wk were attributed to accelerated alcohol metabolism by further increasing alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities while downregulating cytochrome P450 2E1 (CYP2E1) protein expression, elevating antioxidant capacity, decreasing zonula occludens-1 (ZO-1) protein decrement and serum endotoxin, and reducing inflammation related protein levels, that is, toll-like receptor 4 (TLR4) and mitogen-activated protein kinase (MAPK), and proinflammatory cytokines. The development of CLH supplements could not only enhance the added value of broiler livers through nutraceutical development but also offer a strategy to maximize the utilization of poultry processing residues, as shown in this study.

Identification and Characterization of a Pepsin- and Chymotrypsin-Resistant Peptide in the α Subunit of the 11S Globulin Legumin from Common Bean (Phaseolus vulgaris L.).

The 11S globulin legumin typically accounts for approximately 3% of the total protein in common beans (Phaseolus vulgaris). It was previously reported that a legumin peptide of approximately 20 kDa is resistant to pepsin digestion. Sequence prediction suggested that the pepsin-resistant peptide is located at the C-terminal end of the α-subunit, within a glutamic acid-rich domain, overlapping with a chymotrypsin-resistant peptide. Using purified legumin, the peptide of approximately 20 kDa was found to be resistant to pepsin digestion in a pH-dependent manner, and its location was determined by two-dimensional gel electrophoresis and LC-MS-MS. The location of the chymotrypsin-resistant peptide was confirmed by immunoblotting with peptide-specific polyclonal antibodies. The presence of a consensus site for proline hydroxylation and arabinosylation, the detection of hydroxyproline residues, purification by lectin affinity chromatography, and a difference in electrophoretic migration between the chymotrypsin- and pepsin-resistant peptides suggest the presence of a large O-glycan within these peptides.

Identification of DPP-IV inhibitory peptides derived from buffalo colostrum: Mining through bioinformatics, in silico and in vitro approaches.

Bioactive peptides derived from foods provide physiological health benefits beyond nutrition. This study focused on profiling small peptide inhibitors against two key serine proteases, dipeptidyl peptidase-IV (DPP-IV) and prolyl oligopeptidase (POP). DPP-IV is a well-known protein involved in diverse pathways regulating inflammation, renal, cardiovascular physiology, and glucose homeostasis. POP is yet another key target protein for neurodegenerative disorders. The study evaluated peptide libraries of buffalo colostrum whey and fat globule membrane proteins derived from pepsin and pepsin-pancreatin digestion through in silico web tools and structure-based analysis by molecular docking and binding free-energy estimation, followed by in vitro assay for DPP-IV inhibition for the lead peptides. The bioinformatic study indicated 49 peptides presented motifs with DPP-IV inhibition while 5 peptides with sequences for POP inhibition. In the molecular docking interactions study, 22 peptides interacted with active site residues of DPP-IV and 3 peptides with that of POP. The synthesized peptides, SFVSEVPEL and LTFQHNF inhibited DPP-IV in vitro with an IC50 of 193.5 μM and 1.782 mM, respectively. The study revealed the key residues for inhibition of DPP-IV and POP thus affirming the DPP-IV inhibitory potential of milk-derived peptides.

Evaluation of lauric acid enhancement of black soldier fly larvae from coconut.

The current study evaluated the potential enhancement of lauric acid (LA) in black soldier fly, Hermetia illucens, (L.) (Diptera: Stratiomyidae) larvae (BSFL), a source of this short-chain fatty acid which has antimicrobial and immunostimulatory properties. Replicate groups of BSFL were reared on either the coconut or Gainesville diet for 7 days. After the rearing period, BSFL were harvested, purged, dried, and subjected to proximate, fatty acid and amino acid compositions, and pepsin digestibility analyses. Results demonstrate changes in proximate composition. BSFL reared on the coconut had significantly (P = 0.002) higher lipid content (47.3% vs. 25.2%) on a dry-matter basis. The LA concentration in BSFL produced on the coconut was 31% greater than those reared on Gainesville, resulting in almost 150% more LA. Furthermore, BSFL-fed coconut had reduced crude protein (29.7% of dry weight) and ash (3.7% of dry weight) relative to those fed Gainesville (43.4% and 7.5% for crude protein and ash, respectively) but higher pepsin digestibility (91.0% vs. 87.0%). The relative amounts of various amino acids in the 2 BSFL meals did not differ extensively, with statistically lower concentrations of only phenylalanine and tryptophan and higher concentrations of alanine, arginine, isoleucine, leucine, and serine in BSFL reared on coconut. Results demonstrate that the nutritional composition of BSFL can be manipulated, and an enhancement of LA concentrations of 150% was achieved with coconut, which has value for BSFL as a feed for various livestock, including aquaculture. Lower protein content is a tradeoff in terms of BSFL value as a feed additive.

An injectable dental pulp-derived decellularized matrix hydrogel promotes dentin repair through modulation of macrophage response

Maintaining the viability of damaged pulp is critical in clinical dentistry. Pulp capping, by placing dental material over the exposed pulp, is a main approach to promote pulp-dentin healing and mineralized tissue formation. The dental materials are desired to impact on intricate physiological mechanisms in the healing process, including early regulation of inflammation, immunity, and cellular events. In this study, we developed an injectable dental pulp-derived decellularized matrix (DPM) hydrogel to modulate macrophage responses and promote dentin repair. The DPM derived from porcine dental pulp has high collagen retention and low DNA content. The DPM was solubilized by pepsin digestion (named p-DPM) and subsequently injected through a 25G needle to form hydrogel facilely at 37 °C. In vitro results demonstrated that the p-DPM induced the M2-polarization of macrophages and the migration, proliferation, and dentin differentiation of human dental pulp stem cells from deciduous teeth (SHEDs). In a mouse subcutaneous injection test, the p-DPM hydrogel was found to facilitate cell recruitment and M2 polarization during the early phase of implantation. Additionally, the acute pulp restoration in rat models proved that injectable p-DPM hydrogel as a pulp-capping agent had excellent efficacy in dentin regeneration. This study demonstrates that the DPM promotes dentin repair by modulating macrophage responses, and has a potential for pulp-capping applications in dental practice.

Determination of Phytase-hydrolyzable Phosphorus in Selected Animal Feed Ingredients by In Vitro Digestion Method

Feed phytase is one of the most successful biotechnological applications that led to better farm performance and effective phosphorus (P) reduction in animal excrement. Better estimation of phytase-hydrolyzable P (PHP) in feeds can further improve the estimation of animal rations for higher productivity. PHP in yellow corn, feed wheat, wheat pollard, rice bran, soybean meal, copra meal, and monodicalcium phosphate (MDCP) was determined by in vitro digestion. To determine if PHP values of feed ingredients are additive, three formulated diets were also subjected to the same method. The procedure consisted of phytase, pepsin, and pancreatin digestion periods – each of which simulated the different sections of poultry’s gastrointestinal tract. After these three incubations, the P contents in the dialysate and residual fluids – with and without enzyme – were analyzed. Results showed that yellow corn contained the highest PHP value with 52.54% of its total soluble P – followed by soybean meal, rice bran, copra meal, and feed wheat with 52.32, 39.86, 26.77, and 21.9%, respectively. Wheat pollard and MDCP had –58.47 and –2.63%, respectively. On the other hand, formulated diets 1, 2, and 3 contained actual PHPs of 32.3, 14.02, and 38.68% – as well as calculated values of 33.22, 29.13, and 40.1%, respectively. Statistical analyses showed no significant difference between the actual and calculated values. Therefore, PHP is additive when used in diet formulation. Lastly, comparing the P in the dialysate from the experiment and the available P present in the seven feed samples based on literature indicated that the experimental values were lower than those of the literature data for all samples. This discrepancy can be attributed to the type of samples analyzed; the experimental data were obtained upon in vitro assay of individual feeds, whereas the literature values were based on an as-fed basis, which may contain other components normally fed to animals.

Identification of Sarcocystis spp. in Slaughtered Sheep from Spain and Evaluation of Bradyzoite Viability after Freezing.

Sarcocystis spp. are complex apicomplexan parasites that cause a substantial economic impact on livestock used for meat production. These parasites are present worldwide. Our study aimed to identify Sarcocystis species affecting sheep meat in southern-central Spain and to evaluate the effectiveness of freezing for parasite inactivation. A total of 210 condemned samples of sheep meat were thoroughly assessed grossly and microscopically; the presence of macro- and microcysts was confirmed. The samples were then frozen at -20 °C for various time intervals (24, 48, 72, 96, 120, and 144 h) and compared with untreated samples. Bradyzoites were isolated through pepsin digestion for subsequent molecular analysis and viability assessment, employing trypan blue and double fluorescence staining techniques. Our measurements confirmed the presence of S. tenella, S. gigantea, and S. medusiformis in Spanish domestic sheep. Freezing for 96 to 144 h resulted in a significant reduction in parasite viability, with a robust correlation observed between the two staining methods. Both stains effectively measured the viability of Sarcocystis, thereby promising future advances in meat safety.

Proteolysis of dry-cured ham according to the type and temperature of starter mixed with Debaryomyces hansenii and Penicillium nalgiovense

This study analyzed the impact of yeast and mold on the proteolysis of dry-cured ham protein.Debaryomyces hansenii, isolated from Doenjang (DJ) and fermented sausage (FS), was used as a starter in the preparation. The experiment involved three repetitions, using 10 gluteus medius muscles each in the 2, 4, and 6-week aging periods. It assessed the following parameters: aging yield, moisture and crude protein content, pH, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profile, non-protein nitrogen (NPN) content, shear force, and in-vitro digestibility. SDS-PAGE analysis reve protein aled that proteolysis occurred as the aging period increased, and the treatment groups DJ25, FS25, and DF25 displayed thicker bands at 25 kDa than the treatment groups DJ20, FS20, and DF20. The NPN tended to increase as the aging period increased, and treatment groups consistently exhibited higher NPN levels than the control at the same aging temperature. The shear force DJ25, FS25, and DF25 tended to have lower shear force values than the other treatments in the final week. The in-vitro digestion profile showed an increased digestion rate of pepsin and trypsin as the aging period increased. DJ25, FS25, and DF25 exhibited significantly higher pepsin and trypsin digestion rates than DJ20, FS20, and DF20 (P < 0.05). In conclusion, the findings indicate that aging dry-cured ham at 25 °C yields higher proteolysis, lower shear force, and higher digestion rates, suggesting that such a process would result in enhanced consumer preference and superior product quality.

Evaluation of 177Lu-Labeled Pertuzumab F(ab')2 Fragments for HER2-Positive Cancer Targeting: A Comparative In Vitro and In Vivo Study.

Background: Radiolabeled antibody fragments present a promising opportunity as theranostic agents, offering distinct advantages over whole antibodies. In this study, the authors investigate the potential of [177Lu]Lu-DTPA-F(ab')2-pertuzumab as a theranostic agent for precise targeting of human epidermal growth factor receptor 2 (HER2)-positive cancers. Additionally, the authors aim to quantitatively assess the binding synergism in the presence of cold trastuzumab. Materials and Methods: F(ab')2-pertuzumab was prepared by pepsin digestion and conjugated with a bifunctional chelator. The immunoconjugate was radiolabeled with 177Lu and characterized by chromatography techniques. Binding parameters (affinity, specificity, and immunoreactivity) and cellular binding enhancement studies were evaluated in HER2-overexpressing and triple-negative cell lines. The in vivo enhancement in tumor uptake of the radiolabeled immunoformulation was assessed in severe combined immunodeficient (SCID) mice bearing tumors, both in the presence and absence of unlabeled trastuzumab. Results: The formulation of [177Lu]Lu-DTPA-F(ab')2-pertuzumab could be prepared in high yields and with consistent radiochemical purity, ensuring reproducibility. Comprehensive in vitro and in vivo evaluation studies confirmed high specificity and immunoreactivity of the formulation toward HER2 receptors. Binding synergism of radiolabeled pertuzumab fragments in the presence of trastuzumab to HER2 receptors was observed. Conclusions: The radioformulation of [177Lu]Lu-DTPA-F(ab')2-pertuzumab holds great promise as a targeted approach for addressing HER2-positive cancers. A potentially effective strategy to amplify therapeutic efficacy involves dual epitope targeting by combining radiolabeled pertuzumab with cold trastuzumab.

A millimeter water-in-oil droplet as an alternative back exchange prevention strategy for hydrogen/deuterium exchange mass spectrometry of peptides/proteins.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a versatile bioanalytical technique for protein analysis. Since the reliability of HDX-MS analysis considerably depends on the retention of deuterium labels in the post-labeling workflow, deuterium/hydrogen (D/H) back exchange prevention strategies, including decreasing the pH, temperature, and exposure time to protic sources of the deuterated samples, are widely adopted in the conventional HDX-MS protocol. Herein, an alternative and effective back exchange prevention strategy based on the encapsulation of a millimeter droplet of a labeled peptide solution in a water-immiscible organic solvent (cyclohexane) is proposed. Cyclohexane was used to prevent the undesirable uptake of water by the droplet from the atmospheric vapor through the air-water interface. Using the pepsin digest of deuterated myoglobin, our results show that back exchange kinetics of deuterated peptides is retarded in a millimeter droplet as compared to that in the bulk solution. Performing pepsin digestion directly in a water-in-oil droplet at room temperature (18-21 °C) was found to preserve more deuterium labels than that in the bulk digestion with an ice-water bath. Based on the present findings, it is proposed that keeping deuterated peptides in the form of water-in-oil droplets during the post-labelling workflow will facilitate the preservation of deuterium labels on the peptide backbone and thereby enhance the reliability of the H/D exchange data.

Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels.

Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.

Critical add-on SEM observations on oral armature and life cycle of raphidascaridoid worm, Indospinezia multispinatum Jaiswal and Malhotra (2017)

Additional observations of taxonomic significance with unique life cycle details were recorded after the publication of specialized liver-inhabiting roundworms, Indospinezia multispinatum Jaiswal and Malhotra (2017) in the Gangetic gar-fish, Xenentodon cancila from Prayagraj riverine ecosystems. Typically strong musculature supporting triangularly placed one dorsal and two ventro-lateral lips has been observed and their characteristics recorded. The muscular complement that comprised each of the 3 lips, was 3-layered, each of which was stacked one over the other, around a medial furrow. The 2nd stage larva armed with cephalic tooth was the specific discovery which was extracted by Pepsin Digestion method, during compilation of various larval stages that made up complete life-cycle of the roundworm under study. Presumption based conclusions derived on larval stages in the life cycle of Goezia bangladeshi by Akther,1 et al led to erroneous interpretations and hence made the taxonomic position of Goezia bangladeshi circumspect unless appropriate larvae are studied. Therefore, Goezia bangladeshi is termed as species inquirendae pending complete description of its larvae.