ABSTRACTRationaleHuman hair shafts have received increased research interest owing to their easy accessibility and potential as a window for human health. Because the most abundant component in hair is protein, proteomics is a promising tool for studying the molecular composition of hair shafts. As one of the most sophisticated biomaterials, hair shafts also possess unique structures, particularly keratin intermediate filaments, posing challenges for proteomic sample processing. Previously, we discovered that incomplete trypsin proteolysis increased keratin sequence coverage but resulted in an abnormal stoichiometry between types I and II cuticular keratins (PMCID: 12130615).MethodsIn the present study, we explored the potential to re‐examine the human hair proteome through pepsin proteolysis and evaluate whether the previously observed type II to type I keratin ratio was due to enzyme biases introduced particularly by trypsin digestion.ResultsAfter optimizing the pepsin digestion conditions, we not only confirmed that previous bias was indeed contributed by trypsin but also discovered that pepsin was more effective at identifying keratin‐associated proteins, another main protein component than keratins in human hair shafts.ConclusionsSpectral counting on trypsin‐based proteomics has been widely used to study the stoichiometry of protein complexes. For the first time, we confirmed a large bias caused by the trypsin enzyme in spectral counting. We further demonstrated that the use of pepsin can effectively correct such bias. In addition, we discovered that pepsin digestion can better identify keratin‐associated proteins than trypsin proteolysis, which offers another effective tool for studying the hair proteome.

Genetics is significant contributor to acute recurrent pancreatitis. This study evaluates the prevalence of genetic variants and assesses electrophysiological characteristics in Israeli patients. Patients with ≥ two episodes of acute pancreatitis, no history of drug use, normal IgG4 levels, and normal cholangiopancreatographic findings were included. Genetic analysis targeted the PRSS1, SPINK1, CTRC, CFTR, CPA1 and TRPV6 genes, as well as the CEL-HYB1 allele. Sweat testing and nasal potential difference measurements were also performed. Seventy-nine patients (median age 13 years; range 2-61) were referred between 2015 and 2021. 45 (57.0%) carried at least one genetic variant; 18 patients carried 2 or more variants. CFTR variants were the most common (35.4%), followed by PRSS1 (11.4%), SPINK1 (10.1%), and CTRC (10.1%). The rare PRSS1 p.Lys23Arg variant was prevalent among Jewish Georgian patients (n=8). Patients with genetic variants were younger than those without (P<0.0035). Of the 71 patients who underwent nasal potential difference testing, 2 (5.6%) had abnormal results. Sweat test in 66 patients showed a mean value of 36 ± 16mmol/L, six had results above 60mmol/L; two of these carried CFTR variants TG[11]T[5] and p.Leu997Phe but had normal nasal potential. More than half of acute recurrent pancreatitis cases had a genetic basis, presenting at a younger age. CFTR variants were the most prevalent. The high prevalence of the PRSS1 p.Lys23Arg variant among Jewish Georgian individuals suggests a potential founder effect. These findings highlight the importance of genetic evaluation in the diagnosis and management of acute recurrent pancreatitis.

Effects of puerarin on growth performance, digestive ability, immune response, and resistance to ammonia stress in Pacific white shrimp (Litopenaeus vannamei) in low-salinity water.

ABSTRACTAimsMutations in the PRSS1 and SPINK1 genes are recognized as important risk factors for chronic pancreatitis (CP); however, their clinical relevance in Vietnamese populations remains unclear. This cross‐sectional study investigated the prevalence and associated factors of these mutations in Vietnamese CP patients.Methods and ResultsCP was diagnosed according to the 2020 American College of Gastroenterology Clinical Guidelines. Genetic analysis was performed via Sanger DNA sequencing. One hundred sixty CP patients were included from December 2022 to June 2024 at Cho Ray Hospital, Vietnam. Pathogenic mutations were identified in 64 patients (40.0%), with SPINK1 mutations found in 36.8% and PRSS1 mutations in 4.4%. The most frequent SPINK1 variants were c.101A>G (23.7%) and c.194+2T>C (14.3%), and their prevalence was highest in idiopathic CP cases. Multivariate logistic regression analysis revealed that younger age (OR: 0.95; 95% CI: 0.92–0.98), diabetes mellitus (OR: 2.55; 95% CI: 1.11–6.04), pancreatic duct stones (OR: 7.08; 95% CI: 2.81–20.40), and prior surgical intervention (OR: 4.14; 95% CI: 1.34–14.10) were independently associated with pathogenic mutations.ConclusionThese findings suggest a high prevalence of SPINK1 mutations, particularly c.101A>G and c.194+2T>C, among Vietnamese CP patients. The significant factors associated with genetic mutations were younger age, diabetes mellitus, pancreatic duct stones, and prior surgical intervention.

Klebsiella pneumoniae strains that are resistant to carbapenems are of great concern. Exposure to low concentrations of antibiotics may influence tolerance to antibiotics. Novel antibiotics and treatment options are thus needed, and this need is exacerbated by the rapid and global spread of antibiotic resistance. In this study, we determined the global proteome changes in a K. pneumoniae strain (CCUG 70747) carrying carbapenem resistance genes when exposed to low concentrations of ertapenem. Quantitative proteomics was achieved by the tandem mass tag labeling of peptides generated by trypsin proteolysis and mass spectrometry analysis. Bioinformatics analyses were used to observe changes in protein abundance, as well as the gene ontology (GO) terms and pathways associated with the differentially expressed proteins. The number of proteins detected with significant differential abundance were 87 at the highest concentration applied and 61 in the lowest concentration, all compared with the strain cultured without any antibiotics present. Several of these proteins, as well as the GO terms and pathways associated with the proteins, were linked to mechanisms of antibiotic resistance. However, this strain encodes a carbapenemase and other beta-lactamases, and thus, as expected, presented a reasonably modest adaptation in the global proteome upon exposure to the low concentrations of ertapenem applied. Nevertheless, our study identifies pathways that may lead to adaptation under sublethal concentrations of antibiotics leading to strains with higher tolerance.

Mechanistic insights into the interaction of α-amylase, pepsin, and trypsin with food-derived 2-Acetyl pyrrole and tetramethyl pyrazine: Integrating multispectral analysis, molecular dynamics simulation, and HS-SPME-GC-MS.

Given their frequent coexistence of plasticizers and nanoplastics, assessing their combined toxicological impacts is essential. This study investigates the interactions between di-n-octyl phthalate (DNOP) and trypsin (TRY) in the presence of polystyrene nanoplastics (PSNPs) at molecule levels. Molecular docking analysis demonstrates that DNOP could enter the active site of TRY. Spectral analyses indicate that DNOP changed TRY's conformation and residue microenvironment, which were exacerbated by the presence of PSNPs. Particle size and transmission electron microscope show that PSNPs adsorbed TRY, forming a protein corona. Enzyme activity experiments confirm that DNOP inhibited the activity of TRY, with PSNPs further enhancing this inhibitory effect. Specifically, PSNPs bind to TRY, inducing conformational changes that expose additional binding sites, while adsorbing DNOP to increase the local concentration and promote the interaction between DNOP and TRY. The findings provide novel insights when evaluating the risks associated with both PSNPs and phthalates.

Temperature is one of the most significant abiotic factors influencing vector resistance to pathogens. Research has provided substantial insights into the immunological and oxidative processes affected by temperature. However, our understanding of the complex interplay in mosquito-pathogen interactions remains limited. In this study, we investigated the impact of temperature on alternative splicing in the midgut of Aedes aegypti mosquitoes (adult females) under different thermal conditions. Few genes exhibited differential alternative splicing when comparing low (20°C) and high (36°C) temperatures to standard rearing conditions (28°C). Among these, Trypsin (TRY), Ferritin (FER), Thioredoxin (TRX) and Peptidoglycan recognition protein LC (PGRP-LC) were identified, aligning with previous findings that their expression is temperature-sensitive. Among the genes identified with alternative splicing, we focus on Thioredoxin and PGRP-LC, genes modulated by temperature variations and promising targets for future studies on their role in the competence of Ae. aegypti to transmit Zika. Experimental validation confirmed that TRX, a gene critical for pathogen defence, has a differentially spliced exon under warmer conditions, potentially altering its activity. In contrast, no differential splicing was observed for PGRP-LC across temperature treatments. These findings suggest that temperature-induced alternative splicing may play a role in shaping the mosquito's physiological responses to environmental changes, highlighting a previously underexplored layer of complexity in mosquito-pathogen dynamics.

Multiple O- and an N-glycosylation of the stalk region of the NK cell activation receptor NKp46 mediates its interaction with the Candida glabrata epithelial adhesin 1.

Each year, over 100,000 ostomy surgeries are performed in the U.S. to enhance the quality of life for those with severe digestive disorders. Peristomal Skin Complications (PSC) are a significant concern in &gt;40% of such patients, often resulting from prolonged exposure to enzymatically active effluent containing pancreatic proteases (trypsin (TRP), chymotrypsin (CTRP), and elastase (ELA)) and unconjugated primary bile acid (BA), like chenodeoxycholic acid (CDCA). Our previous work demonstrated that CDCA induces oxidative stress and cytokine production in human colonic epithelial cells. Further, altered luminal pH and BA profiles, common in inflammatory bowel disease, may worsen skin damage. Based on these findings, we hypothesize that CDCA modulates pancreatic protease activity in a pH- and dose-dependent manner, and thereby may contribute to stoma-associated epithelial damage. Protease activity was measured across a pH range (4.0–8.3) using an Invitrogen EnzCheckTM fluorescence-based assays for TRP, CTRP, ELA individually and in combination. The effect of CDCA (50–250 µM) on protease activity at different pH values were measured in triplicates and then quantified. Enzyme kinetics parameters (Km and Vmax) were analyzed using Michaelis-Menten models and reported as percentages relative to their controls. Human colonic T84 cells were treated with CDCA (25–250 µM) and protease cocktail (10% - 25%; TRP+CTRP+ELA) at varying pH to evaluate cell death and apoptosis via Annexin-V (AV) and propidium iodide (PI) staining. Examining the effect of pH on protease activity revealed that it was pH-dependent, with maximum activity at pH 8.3. Reducing pH resulted in significant activity decreases (pH 8.3: 100%, 7.4: 85%, 6.0: 65%, 5.0: 58%, 4.0: 49%). Protease cocktail consistently exhibited the highest activity at pH 8.3. Next, we identified that CDCA dose-dependently inhibited protease activity, with a pronounced ~70% reduction at pH 4.0 and 250 µM CDCA. At all pH values, the Lineweaver-Burk plot shows that CDCA reduced both Vmax and Km values (&gt;50%) values, indicative of mixed, non-competitive inhibition. CDCA induced dose-dependent apoptotic cell death in T84 cells, which was exacerbated by proteases (e.g., pH 7.4, CDCA 100 µM: 12 ± 4%; CDCA + 10% protease cocktail: 18 ± 4%). Interestingly, this apoptosis was slightly mitigated at lower pH (e.g., pH 5.0, CDCA + 10% protease cocktail: 15 ± 4%). Thus, at acidic pH and physiological dose, CDCA plays a role in reducing enzymatic activity and mitigating cell damage. Our findings suggest that mildly acidic conditions could protect peristomal skin by decreasing protease activity, potentially reducing the incidence and severity of PSC. Incorporating pH-lowering strategies for the peristomal area may serve as a novel approach to mitigate skin irritation and improve quality of life for ostomy patients. Ongoing studies in skin cell models will further elucidate these interactions and inform targeted interventions. Supported by Hollister Inc., Research and Development, University Collaborations Funds to AL, MP, NSSRP Funds, BenU to IH, AF, JS. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Effects of proteolysis pretreatment on the formation, structural changes and emulsifying properties of rice glutelin amyloid-like fibrils.

Mo1478: PRSS1 GENE MUTATION IS RARE IN INDIAN PATIENTS WITH HEREDITARY PANCREATITIS WHO HAVE DISEASE TRAJECTORY SIMILAR TO SPORADIC CP WITH SPINK1 MUTATION

Trypsin (TPN) is an important proteolytic enzyme in the digestive system and its abnormal levels are indicative of some pancreatic diseases. As an endopeptidase, TPN can cleave substrate peptide mainly by catalyzing the hydrolysis of the carboxyl side peptide bond of lysine (K) or arginine (R) residues. Based on this hydrolysis cleavage effect, two kinds of nanomaterials functionalized electrogenerated chemiluminescence (ECL) biosensors for the determination of TPN were designed as follows: A signal-ON ECL biosensor was fabricated by attaching substrate peptide (HWR*GWVC, "*" representing the cleavage site, abbreviated as HGC) labeled with ferrocene carboxylic acid (as quencher) onto the surface of NH2-MIL-53(Al) film which was incorporated with ECL emitting species (bis (2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium (N-succinimidylester)-bis (hexafluorophosphate) (Ru complex)). The presence of tryptic cleavage event can eventually lead the Fc complex to leave the electrode and results in the increase of the ECL intensity. HGC labeled with Ru-Ti3C2Tx-AuNP complex was used as capture probe and signal probe, which was attached onto NH2-MIL-53(Al) film modified glassy carbon electrode. A signal-OFF ECL biosensor was built as described above. The presence of TPN lead the Ru-Ti3C2Tx-AuNPs to leave the electrode, which resulted in the decreasing ECL intensity. As expected, the fabricated ECL biosensing methods provideexcellent sensitivity and selectivity toward the TPN activity. Thus, this strategy shows great potential application in the clinic for diagnosis of TPN-indicating diseases as well as the screening of TPN inhibitor-based anti-cancer drugs.

Background and objectives. Hereditary pancreatitis (HP) is a rare genetic condition in children, characterized by recurrent episodes of pancreatitis that often progress to chronic pancreatitis. It is primarily associated with mutations in PRSS1, SPINK1, CFTR, and CTRC genes. This review aims to assist pediatricians in recognizing early signs, implementing appropriate diagnostic protocols, and selecting optimal management strategies. Materials and methods. A comprehensive literature search was conducted using PubMed, Scopus, and Web of Science for studies published in the last 10 years. Inclusion criteria encompassed observational studies and reviews discussed genetic factors, diagnostic approaches, and treatment strategies related to hereditary pancreatitis. Results. Hereditary pancreatitis accounts for 10-20% of pediatric chronic pancreatitis cases. Genetic testing, especially next-generation sequencing, plays a crucial role in confirming the diagnosis. Conservative management strategies include pain control, pancreatic enzyme replacement therapy (PERT), nutritional support, and antioxidant supplementation. Endoscopic interventions, such as endoscopic retrograde cholangiopancreatography (ERCP) and stenting, are utilized for ductal obstructions, while surgical procedures like Puestow and Frey operations are reserved for refractory cases. Conclusions. Early diagnosis through genetic testing, combined with personalized conservative management, significantly improves patient outcomes. Further research is warranted to better understand genetic modifiers, refine therapeutic strategies, and address the psychosocial impact of the disease on children and families.

To support the sustainable development of rice and aquaculture industries, various rice-animal coculture systems have been developed. One such system, the rice-crab coculture system (RCC), has been practiced for decades in northern China. However, studies on the crab physiological status in RCC remain limited. Microorganisms play a crucial role in aquaculture by influencing animal nutrition, health, nutrient cycling, water quality, and environmental impact. Research on the gut and environmental microbiota in RCC is scarce. This study compared the growth performance, immune and digestive enzyme activities of crabs between RCC and traditional pond farming system (PF). In addition, the microbiota in crab guts, water, and sediment from both systems was investigated using 16S rRNA gene sequencing. Crabs in RCC exhibited superior growth performance and higher enzymatic activities, including acid phosphatase (ACP), alkaline phosphatase (AKP), lipase (LPS), and trypsin (TRY). Significant differences were observed in microbiota composition across crab gut, water, and sediment samples, respectively. RCC crabs had a lower abundance of Bacteroidota and a higher abundance of Firmicutes in their gut microbiota. The RCC environment was enriched with beneficial bacteria such as Rhizobiales, Methylococcales, KD4-96, C39, Xanthomonadales, and Nitrosomonadaceae. Microbial function predictions confirmed enhanced methanotrophy and nitrogen fixation in the RCC. The RCC enhances the growth rate and immune capability of crabs. Crabs from RCC consume more animal-based nutrition, which results in distinct differences in gut microbiota composition and higher levels of LPS and TRY compared to those in PF. Additionally, RCC supports environmentally beneficial bacteria that contribute to greenhouse gas reduction, carbon and nitrogen fixation, organic matter decomposition, and ammonia oxidation, benefiting both the crabs and their ecosystem. These findings enhance our understanding of crab physiology and microbial communities in RCC and PF systems.

This study explored how combining supercritical fluid extraction (SFE) and enzymatic hydrolysis influences the structure and functionality of peptides recovered from filter-pressed shrimp waste. Freeze-dried press cake (PC) was defatted via SFE and hydrolyzed using Alcalase (ALC) and trypsin (TRYP). ALC-treated PC achieved the highest protein recovery (63.49%), extraction yield (24.73%), and hydrolysis degree (18.10%) (p < 0.05). SFE-treated hydrolysates showed higher zeta potential (-47.23 to -49.93 mV) than non-SFE samples (-25.15 to -38.62 mV) but had larger droplet sizes, indicating lower emulsion stability. SC-ALC displayed reduced fluorescence intensity and a red shift in maximum wavelength. TRYP hydrolysates reduced interfacial tension (20 mN/m), similar to sodium caseinate (Na-Cas, 13 mN/m), but with lesser effects. Dilatational rheology showed TRYP hydrolysates formed stronger, solid-like structures. These results emphasize protease efficacy over SFE for extracting functional compounds, enhancing shrimp waste valorization.

Triclocarban (TCC), a widely used antimicrobial agent, may threaten ecosystems and human health via bioaccumulation, necessitating study of its protein interactions to understand molecular toxicity. In this paper, trypsin (TRY) was utilized as a model protein to explore its binding to TRY. The results revealed that the binding could result in a reduction of the enzymatic activity of TRY. Spectra analysis showed that TCC could heighten the quenching effect on the intrinsic fluorescence of TRY. The fluorescence quenching of TRY encompassed dynamic and static quenching mechanisms. The association constants (Ka) exhibited a high magnitude (∼106) at both 293 and 313 K, indicating a robust affinity between the two entities. Molecular docking studies and thermodynamic parameters (ΔH < 0, ΔS < 0) suggested hydrogen bonds and van der Waals forces are necessary for TCC’s binding to TRY. The formation of the TRY-TCC complex induced alterations in the secondary structure and local microenvironment of TRY, leading to a more relaxed skeletal structure. This paper will provide a fundamental basis for further studying the molecular toxicity of TCC in living organisms. Future in vivo studies will be essential to establish the physiological consequences of TCC-TRY binding in biological systems.

Efficient intracellular protein delivery is of great importance for the development of protein-based therapy and modern biotechnologies. However, the hydrophilic and macromolecular nature of proteins greatly hinders their ability to cross cell membranes. Herein, a calixarene modification strategy for the intracellular delivery of protein drugs is developed. The decoration of sulfonate azocalix[4]arene (SAC4A) on proteins results in a nano-multivalent effect between Protein-S and amino acids on the cell surface, leading to efficient intracellular delivery of the protein via the clathrin-mediated endocytic pathway. By using SAC4A as a novel ligand, this calixarene modification strategy efficiently delivers 7 proteins, bovine serum albumin (BSA), trypsin (TRY), horseradish peroxidase (HRP), α-chymotrypsin (α-Chyt), lysozyme (LYZ), cytochrome C (Cyt C) and ribonuclease A (RNase A), into cells and significantly enhances the cytotoxicity of Cyt C and RNase A. Moreover, SAC4A-modified Cyt C demonstrates markedly enhanced antitumor efficacy in 4T1-bearing mice without notable side effects. Considering that these proteins are varied in molecular weight and isoelectric point, this calixarene modification strategy provides a platform technology for intracellular protein delivery and the development of protein drugs targeting intracellular pathways.

Trypsin (TRY) combined with ethylenediaminetetraacetic acid (EDTA) is a widely used dissociation agent due to its efficiency and cost-effectiveness. However, its impact on preserving stem cell marker expression, such as C-X-C chemokine receptor type 4 (CXCR4) (critical for cell migration and homing) and cluster of differentiation 146 (CD146) (involved in pluripotency and angiogenesis), may be suboptimal compared to alternatives such as Accuta-se (ACC) and Accumax (ACMX), as shown previous-ly in bone marrow-derived stem cells (BM-MSCs). Limited data exist on these agents' effects on dental pulp stem cells (DPSCs). This study aims to investigate the influence of TRY, ACC, and ACMX on the expression of CXCR4 and CD146 in DPSCs. Seven characterized DPSC lines were cultured under standardized conditions and detached using TRY-EDTA, ACC, or ACMX. The expression of CXCR4 and CD146 was quantified via multicolour flow cytometry using an innovative DURAClone SC panel with supplementary anti-CXCR4 antibody. Comprehen-sive statistical analyses were performed to evaluate differences in marker preservation. No statistically significant differences in CXCR4 or CD146 expression were observed across the detachment methods (P &gt; 0.05). ACMX consistently demonstrated margi-nally higher mean expression levels for both markers (CXCR4: 84.77 %; CD146: 93.91 %) compared to ACC (CXCR4: 83.45 %; CD146: 93.41 %) and TRY (CXCR4: 83.95 %; CD146: 92.99 %). While differences were not statistically significant, ACMX consistently yielded higher mean expression of both CXCR4 and CD146, indicating a potential advantage in preserving marker integrity during the cell detachment.

Ricefield eel is an important economic fish in China. However, large-scale intensive breeding has increased the incidence of diseases in eels. In this study, we conducted an 8-week feeding trial to investigate the effects of β-glucan on the growth performance, intestinal health, and Aeromonas veronii resistance of Monopterus albus (M. albus). Three hundred healthy fish (initial body weight: 12.38 ± 0.50 g) were randomly divided into five groups: A1 (basal diet) was considered the control group, whereas A2, A3, A4, and A5 were the experimental groups. The fish in the experimental groups were fed a basal diet supplemented with 250, 500, 1000, and 2000 mg/kg β-glucan, respectively. The addition of 0.025%-0.2% β-glucan resulted in a notable enhancement of eel growth performance, with the most significant improvement observed in eels supplemented with 0.1% β-glucan (p < 0.05). Furthermore, 0.025%-0.2% β-glucan could significantly enhance the antioxidant properties of the eel intestinal tract (p < 0.05), and the addition of 0.1% β-glucan significantly improved trypsin (TPS), amylase (AMS), and lipase (LPS) activities in the intestine (p < 0.05). In terms of intestinal histology, the A3, A4, and A5 groups exhibited significantly greater villus height compared to the control group (p < 0.05). Concentrations of β-glucan at 0.1% and 0.2% enhanced the composition of the intestinal flora; specifically, the relative abundance of Proteobacteria increased, while the relative abundance of Firmicutes decreased. Moreover, the addition of 0.05%-0.2% β-glucan significantly improved the relative survival rate (SR) of A. veronii-infected eels and significantly decreased the bacterial load of the liver, spleen, and kidney (p < 0.05). In comparison to eels that did not receive β-glucan supplementation, eels supplemented with 0.2% β-glucan exhibited decreased intestinal structural damage. In summary, the addition of 0.1%-0.2% β-glucan can promote eel growth, improve intestinal digestion and antioxidant capacity, regulate intestinal flora, and enhance intestinal physical function and anti-infection ability.