Ruxolitinib Improves Immune-Dysregulation Features but not Epigenetic Abnormality in a Patient with STAT1 GOF.

PURPOSE: Patients with STAT1 gain-of-function (GOF) mutations often exhibit autoimmune features. The JAK1/2 inhibitor ruxolitinib can be administered to alleviate autoimmune symptoms; however, it is unclear how immune cells are molecularly changed by ruxolitinib treatment. Then, we aimed to investigate the trnscriptional and epigenetic status of immune cells before and after ruxolitinib treatment in a patient with STAT1 GOF. METHODS: A patient with a heterozygous STAT1 GOF variant (p.Ala267Val), exhibiting autoimmune features, was treated with ruxolitinib, and peripheral blood mononuclear cells (PBMCs) were longitudinally collected. PBMCs were transcriptionally analyzed by single-cell cellular indexing of the transcriptomes and epitopes by sequencing (CITE-seq), and epigenetically analyzed by assay of transposase-accessible chromatin sequencing (ATAC-seq). RESULTS: CITE-seq analysis revealed that before treatment, the patient's PBMCs exhibited aberrantly activated inflammatory features, especially IFN-related features. In particular, monocytes showed high expression levels of a subset of IFN-stimulated genes (ISGs). Ruxolitinib treatment substantially downregulated aberrantly overexpressed ISGs, and improved autoimmune features. However, epigenetic analysis demonstrated that genetic regions of ISGs-e.g., STAT1, IRF1, MX1, and OAS1-were highly accessible even after ruxolitinib treatment. When ruxolitinib was temporarily discontinued, the patient's autoimmune features were aggravated, which is in line with sustained epigenetic abnormality. CONCLUSIONS: In a patient with STAT1 GOF, ruxolitinib treatment improved autoimmune features and downregulated aberrantly overexpressed ISGs, but did not correct epigenetic abnormality of ISGs.

Sustainable production of natural products using synthetic biology: Ginsenosides.

Synthetic biology approaches offer potential for large-scale and sustainable production of natural products with bioactive potency, including ginsenosides, providing a means to produce novel compounds with enhanced therapeutic properties. Ginseng, known for its non-toxic and potent qualities in traditional medicine, has been used for various medical needs. Ginseng has shown promise for its antioxidant and neuroprotective properties, and it has been used as a potential agent to boost immunity against various infections when used together with other drugs and vaccines. Given the increasing demand for ginsenosides and the challenges associated with traditional extraction methods, synthetic biology holds promise in the development of therapeutics. In this review, we discuss recent developments in microorganism producer engineering and ginsenoside production in microorganisms using synthetic biology approaches.

Susceptibility to Fosfomycin and Nitrofurantoin of ESBL-Positive Escherichia coli and Klebsiella pneumoniae Isolated From Urine of Pediatric Patients.

BACKGROUND: Pediatric urinary tract infection (UTI) caused by extended-spectrum ß-lactamase (ESBL)-positive gram-negative bacilli (GNB) has limited options for oral antibiotic treatment. The purpose of this study was to investigate the susceptibility of ESBL-positive Escherichia coli and Klebsiella pneumoniae isolates from pediatric urine samples to two oral antibiotics (fosfomycin and nitrofurantoin). METHODS: From November 2020 to April 2022, ESBL-positive E. coli and K. pneumoniae isolates from urine samples were collected at Samsung Medical Center, Seoul, Korea. Patients over 18 years of age or with malignancy were excluded. For repeated isolates from the same patient, only the first isolate was tested. Minimum inhibitory concentrations (MICs) were measured using agar (fosfomycin) or broth (nitrofurantoin) dilution methods. MIC50 and MIC90 were measured for fosfomycin and nitrofurantoin in both E. coli and K. pneumoniae. RESULTS: There were 117 isolates from 117 patients, with a median age of 7 months (range, 0.0-18.5 years). Among 117 isolates, 92.3% (108/117) were E. coli and 7.7% (9/117) were K. pneumoniae. Isolates from the pediatric intensive care unit (PICU) and general ward (GW) was 11.1% (13/117) and 88.9% (104/117), respectively. Among 108 E. coli isolates, MIC50 and MIC90 for fosfomycin were 0.5 µg/mL and 2 µg/mL, respectively. Fosfomycin susceptibility rate was 97.2% (105/108) with a breakpoint of 128 µg/mL. Fosfomycin susceptibility rate was significantly lower in PICU isolates than in GW isolates (81.8% vs. 99.0%, P = 0.027). For nitrofurantoin, both the MIC50 and MIC90 were 16 µg/mL. Nitrofurantoin susceptibility rate was 96.3% (104/108) with a breakpoint of 64 µg/mL based on Clinical and Laboratory Standards Institute guidelines. Among the nine K. pneumoniae isolates, the MIC50 and MIC90 for fosfomycin was 2 µg/mL and 32 µg/mL, respectively. MIC50 and MIC90 for nitrofurantoin were 64 µg/mL and 128 µg/mL, respectively. CONCLUSION: For uncomplicated UTI caused by ESBL-positive GNB in Korean children, treatment with fosfomycin and nitrofurantoin for E. coli infections can be considered as an effective oral therapy option.

Loop-mediated isothermal amplification assay for screening congenital cytomegalovirus infection in newborns.

Congenital cytomegalovirus (CMV) infection is a common cause of sensorineural hearing loss and neurodevelopmental impairment in newborns. However, congenital CMV infection cannot be diagnosed using samples collected more than 3 weeks after birth because testing after this time cannot distinguish between congenital infection and postnatal infection. Herein, we developed a robust loop-mediated isothermal amplification (LAMP) assay for the large-scale screening of newborns for congenital CMV infection. In contrast to conventional quantitative polymerase chain reaction (qPCR), which detects CMV within a dynamic range of 1.0 × 106 to 1.0 × 102 copies/µL, our quantitative LAMP assay (qLAMP) detects CMV within a dynamic range of 1.1 × 108 to 1.1 × 103 copies/µL. Moreover, the turnaround time for obtaining results following DNA extraction is 90 min in qPCR but only 15 min in qLamp. The colorimetric LAMP assay can also detect CMV down to 1.1 × 103 copies/µL within 30 min, irrespective of the type of heat source. Our LAMP assay can be utilized in central laboratories as an alternative to conventional qPCR for quantitative CMV detection, or for point-of-care testing in low-resource environments, such as developing countries, via colorimetric naked-eye detection. KEY POINTS: • LAMP assay enables large-scale screening of newborns for congenital CMV infection. • LAMP allows colorimetric or quantitative detection of congenital CMV infection. • LAMP assay can be used as a point-of-care testing tool in low-resource environments.

Online Learning-Based Hybrid Tracking Method for Unmanned Aerial Vehicles.

Tracking unmanned aerial vehicles (UAVs) in outdoor scenes poses significant challenges due to their dynamic motion, diverse sizes, and changes in appearance. This paper proposes an efficient hybrid tracking method for UAVs, comprising a detector, tracker, and integrator. The integrator combines detection and tracking, and updates the target's features online while tracking, thereby addressing the aforementioned challenges. The online update mechanism ensures robust tracking by handling object deformation, diverse types of UAVs, and changes in background. We conducted experiments on custom and public UAV datasets to train the deep learning-based detector and evaluate the tracking methods, including the commonly used UAV123 and UAVL datasets, to demonstrate generalizability. The experimental results show the effectiveness and robustness of our proposed method under challenging conditions, such as out-of-view and low-resolution scenarios, and demonstrate its performance in UAV detection tasks.

The Prevalence of Symptomatic Congenital Cytomegalovirus Disease in Korea; A 15-Year Multicenter Study and Analysis of Big Data From National Health Insurance System.

The birth prevalence of symptomatic congenital cytomegalovirus (cCMV) disease among live birth in Korea from a multicenter study was 0.06% during 2001-2015 with increasing frequency. The administrative prevalence of cCMV infection by big-data analysis from the national health insurance system was 0.01% and the average healthcare cost was US$2010 per person.

Metabolic recycling of storage lipids promotes squalene biosynthesis in yeast.

BACKGROUND: Metabolic rewiring in microbes is an economical and sustainable strategy for synthesizing valuable natural terpenes. Terpenes are the largest class of nature-derived specialized metabolites, and many have valuable pharmaceutical or biological activity. Squalene, a medicinal terpene, is used as a vaccine adjuvant to improve the efficacy of vaccines, including pandemic coronavirus disease 2019 (COVID-19) vaccines, and plays diverse biological roles as an antioxidant and anticancer agent. However, metabolic rewiring interferes with inherent metabolic pathways, often in a way that impairs the cellular growth and fitness of the microbial host. In particular, as the key starting molecule for producing various compounds including squalene, acetyl-CoA is involved in numerous biological processes with tight regulation to maintain metabolic homeostasis, which limits redirection of metabolic fluxes toward desired products. RESULTS: In this study, focusing on the recycling of surplus metabolic energy stored in lipid droplets, we show that the metabolic recycling of the surplus energy to acetyl-CoA can increase squalene production in yeast, concomitant with minimizing the metabolic interferences in inherent pathways. Moreover, by integrating multiple copies of the rate-limiting enzyme and implementing N-degron-dependent protein degradation to downregulate the competing pathway, we systematically rewired the metabolic flux toward squalene, enabling remarkable squalene production (1024.88 mg/L in a shake flask). Ultimately, further optimization of the fed-batch fermentation process enabled remarkable squalene production of 6.53 g/L. CONCLUSIONS: Our demonstration of squalene production via engineered yeast suggests that plant- or animal-based supplies of medicinal squalene can potentially be complemented or replaced by industrial fermentation. This approach will also provide a universal strategy for the more stable and sustainable production of high-value terpenes.

The Changes in Epidemiology of Imipenem-Resistant Acinetobacter baumannii Bacteremia in a Pediatric Intensive Care Unit for 17 Years.

BACKGROUND: Acinetobacter baumannii infections cause high morbidity and mortality in intensive care unit (ICU) patients. However, there are limited data on the changes of long-term epidemiology of imipenem resistance in A. baumannii bacteremia among pediatric ICU (PICU) patients. METHODS: A retrospective review was performed on patients with A. baumannii bacteremia in PICU of a tertiary teaching hospital from 2000 to 2016. Antimicrobial susceptibility tests, multilocus sequence typing (MLST), and polymerase chain reaction for antimicrobial resistance genes were performed for available isolates. RESULTS: A. baumannii bacteremia occurred in 27 patients; imipenem-sensitive A. baumannii (ISAB, n = 10, 37%) and imipenem-resistant A. baumannii (IRAB, n = 17, 63%). There was a clear shift in the antibiogram of A. baumannii during the study period. From 2000 to 2003, all isolates were ISAB (n = 6). From 2005 to 2008, both IRAB (n = 5) and ISAB (n = 4) were isolated. However, from 2009, all isolates were IRAB (n = 12). Ten isolates were available for additional test and confirmed as IRAB. MLST analysis showed that among 10 isolates, sequence type 138 was predominant (n = 7). All 10 isolates were positive for OXA-23-like and OXA-51-like carbapenemase. Of 27 bacteremia patients, 11 were male (41%), the median age at bacteremia onset was 5.2 years (range, 0-18.6 years). In 33% (9/27) of patients, A. baumannii was isolated from tracheal aspirate prior to development of bacteremia (median, 8 days; range, 5-124 days). The overall case-fatality rate was 63% (17/27) within 28 days. There was no statistical difference in the case fatality rate between ISAB and IRAB groups (50% vs. 71%; P = 0.422). CONCLUSION: IRAB bacteremia causes serious threat in patients in PICU. Proactive infection control measures and antimicrobial stewardship are crucial for managing IRAB infection in PICU.

Chain flexibility of medicinal lipids determines their selective partitioning into lipid droplets.

In guiding lipid droplets (LDs) to serve as storage vessels that insulate high-value lipophilic compounds in cells, we demonstrate that chain flexibility of lipids determines their selective migration in intracellular LDs. Focusing on commercially important medicinal lipids with biogenetic similarity but structural dissimilarity, we computationally and experimentally validate that LD remodeling should be differentiated between overproduction of structurally flexible squalene and that of rigid zeaxanthin and ß-carotene. In molecular dynamics simulations, worm-like flexible squalene is readily deformed to move through intertwined chains of triacylglycerols in the LD core, whereas rod-like rigid zeaxanthin is trapped on the LD surface due to a high free energy barrier in diffusion. By designing yeast cells with either much larger LDs or with a greater number of LDs, we observe that intracellular storage of squalene significantly increases with LD volume expansion, but that of zeaxanthin and ß-carotene is enhanced through LD surface broadening; as visually evidenced, the outcomes represent internal penetration of squalene and surface localization of zeaxanthin and ß-carotene. Our study shows the computational and experimental validation of selective lipid migration into a phase-separated organelle and reveals LD dynamics and functionalization.

Metabolite trafficking enables membrane-impermeable-terpene secretion by yeast.

Metabolites are often unable to permeate cell membranes and are thus accumulated inside cells. We investigate whether engineered microbes can exclusively secrete intracellular metabolites because sustainable metabolite secretion holds a great potential for mass-production of high-value chemicals in an efficient and continuous manner. In this study, we demonstrate a synthetic pathway for a metabolite trafficking system that enables lipophilic terpene secretion by yeast cells. When metabolite-binding proteins are tagged with signal peptides, metabolite trafficking is highly achievable; loaded metabolites can be precisely delivered to a desired location within or outside the cell. As a proof of concept, we systematically couple a terpene-binding protein with an export signal peptide and subsequently demonstrate efficient, yet selective terpene secretion by yeast (~225 mg/L for squalene and ~1.6 mg/L for ß-carotene). Other carrier proteins can also be readily fused with desired signal peptides, thereby tailoring different metabolite trafficking pathways in different microbes. To the best of our knowledge, this is the most efficient cognate pathway for metabolite secretion by microorganisms.

Pairing of orthogonal chaperones with a cytochrome P450 enhances terpene synthesis in Saccharomyces cerevisiae.

The supply of terpenes is often limited by their low extraction yield from natural resources, such as plants. Thus, microbial biosynthesis has emerged as an attractive platform for the production of terpenes. Many strategies have been applied to engineer microbes to improve terpene production capabilities; however, functional expression of heterologous proteins such as cytochrome P450 enzymes (P450s) in microbes is a major obstacle. This study reports the successful pairing of cognate chaperones and P450s for functional heterologous expression in Saccharomyces cerevisiae. This chaperone pairing was exploited to facilitate the functional assembly of the protopanaxadiol (PPD) biosynthesis pathway, which consists of a P450 oxygenase and a P450 reductase redox partner originating from Panax ginseng and Arabidopsis thaliana, respectively. We identified several chaperones required for protein folding in P. ginseng and A. thaliana and evaluated the impact of the coexpression of the corresponding chaperones on the synthesis and activity of PPD biosynthesis enzymes. Expression of a chaperone from P. ginseng (PgCPR5), a cognate of PPD biosynthesis enzymes, significantly increased PPD production by more than 2.5-fold compared with that in the corresponding control strain. Thus, pairing of chaperones with heterologous enzymes provides an effective strategy for the construction of challenging biosynthesis pathways in yeast.

Impact of maternal engrafted cytomegalovirus-specific CD8+ T cells in a patient with severe combined immunodeficiency.

OBJECTIVES: In patients with severe combined immunodeficiency (SCID), the immune system often fails to eradicate maternal cells that enter the foetus via the placenta, resulting in transplacental maternal engraftment (TME) syndrome. However, the clinical significance of TME has not been comprehensively elucidated. METHODS: Here, we describe a patient with SCID with a novel frameshift IL2RG mutation associated with maternal engrafted CD8+ T cells that had been expanded by viral infection. To evaluate the origin of the expanded T cells, we HLA-typed the myeloid and T cells of the patient and analysed the immunological characteristics of the expanded CD8+ T cells using T-cell receptor (TCR) repertoire and flow cytometry analysis. RESULTS: In our patient, the maternal engrafted CD8+ T cells expanded and exerted in vitro antiviral function against human cytomegalovirus (CMV) infection before and after haematopoietic cell transplantation (HCT). After haploidentical HCT from the maternal donor, maternal engrafted CMV-specific CD8+ T cells were maintained, successfully proliferated and activated against CMV. We found no evidence of acute graft-versus-host disease or infectious complications other than recurrent episodes of CMV viraemia, which were well controlled by ganciclovir and, possibly by, the maternal engrafted CMV-specific CD8+ T cells. CONCLUSION: Our findings elucidate a possible functional role of TME in controlling CMV infection in patient with SCID and suggest an optimal strategy for donor selection in patients with SCID with TME.

Systematic review of literature and analysis of big data from the National Health Insurance System on primary immunodeficiencies in Korea.

There are very scant data on the epidemiology of primary immunodeficiency diseases (PIDs) in Korea. Here we attempted to estimate the PID epidemiology and disease burden in Korea. A systematic review was performed of studies retrieved from the PubMed, KoreaMed, and Google Scholar databases. Studies on PIDs published in Korean or English between January 2001 and November 2018 were analyzed. The number of PID patients and the healthcare costs were estimated from Health Insurance Review and Assessment Service (HIRA) Korea data for 2017. A total of 398 PID patients were identified from 101 reports. Immunodeficiencies affecting cellular and humoral immunity were reported in 11 patients, combined immunodeficiency with associated or syndromic features in 40, predominantly antibody deficiencies in 144, diseases of immune dysregulation in 58, congenital defects of phagocytes in 104, defects in the intrinsic and innate immunity in 1, auto-inflammatory disorders in 4, complement deficiencies in 36, and phenocopies of PID in none. From the HIRA reimbursement data, a total of 1,162 outpatients and 306 inpatients were treated for 8,166 and 6,149 days, respectively. In addition, reimbursement was requested for 8,200 outpatient and 1,090 inpatient cases and $1,924,000 and $4,715,000 were reimbursed in 2017, respectively. This study systematically reviewed published studies on PID and analyzed the national open data system of the HIRA to estimate the disease burden of PID, for the first time in Korea.

Engineering Cell Wall Integrity Enables Enhanced Squalene Production in Yeast.

Microbial production of many lipophilic compounds is often limited by product toxicity to host cells. Engineering cell walls can help mitigate the damage caused by lipophilic compounds by increasing tolerance to those compounds. To determine if the cell wall engineering would be effective in enhancing lipophilic compound production, we used a previously constructed squalene-overproducing yeast strain (SQ) that produces over 600 mg/L of squalene, a model membrane-damaging lipophilic compound. This SQ strain had significantly decreased membrane rigidity, leading to increased cell lysis during fermentation. The SQ strain was engineered to restore membrane rigidity by activating the cell wall integrity (CWI) pathway, thereby further enhancing its squalene production efficiency. Maintenance of CWI was associated with improved squalene production, as shown by cell wall remodeling through regulation of Ecm33, a key regulator of the CWI pathway. Deletion of ECM33 in the SQ strain helped restore membrane rigidity and improve stress tolerance. Moreover, ECM33 deletion suppressed cell lysis and increased squalene production by approximately 12% compared to that by the parent SQ strain. Thus, this study shows that engineering of the yeast cell wall is a promising strategy for enhancing the physiological functions of industrial strains for production of lipophilic compounds.

Tailoring the Saccharomyces cerevisiae endoplasmic reticulum for functional assembly of terpene synthesis pathway.

The endoplasmic reticulum (ER) is a dynamic organelle that synthesizes and folds proteins. An imbalance between the ER protein synthesis load and its folding capacity triggers the unfolded protein response, thereby restoring normal ER functions via size adjustment. Inspired by such inherent genetic programming events, we engineered Saccharomyces cerevisiae to expand the ER by overexpressing a key ER size regulatory factor, INO2. ER space expansion enhanced ER protein synthesis and folding capacity, and relieved metabolic constraints imposed by the limited enzyme abundance. Harnessing the yeast ER for metabolic engineering, we ultimately increased the production of squalene and cytochrome P450-mediated protopanaxadiol by 71-fold and 8-fold, compared to their respective control strains without overexpression of INO2. Furthermore, genome-wide transcriptome analysis of the ER-expanded strain revealed that the significant improvement in terpene production was associated with global rewiring of the metabolic network. Therefore, the yeast ER can be engineered as a specialized compartment for enhancing terpene production, representing new possibilities for the high-level production of other value-added chemicals.

Chemical chaperone-conjugated exendin-4 as a cytoprotective agent for pancreatic ß-cells.

Endoplasmic reticulum stress has been considered a major cause of pancreatic ß-cell dysfunction and apoptosis leading to diabetes. Glucagon-like peptide-1 receptor activation and chemical chaperones have been known to reduce endoplasmic reticulum stress and improve ß-cell function and survival. The purpose of this study was to prepare and evaluate the chemical chaperone tauroursodeoxycholic acid-conjugated exendin-4 as a protective agent for pancreatic ß-cells. Mono-tauroursodeoxycholic acid-Lys27-exendin-4 conjugate (TUM1-Ex4) showed better receptor binding affinity than other conjugates with strong in vitro insulinotropic activity in rat pancreatic ß-cells and in vivo hypoglycemic activity in type 2 diabetic db/db mice. In INS-1 cells under endoplasmic reticulum stress induced by thapsigargin, TUM1-Ex4 promoted cell survival in a dose-dependent manner. In western blot analysis, TUM1-Ex4 reduced the expression of the endoplasmic reticulum stress marker GRP78 and phosphorylation of the translation initiation factor eIF2α. These results reveal that TUM1-Ex4 accelerates translational recovery and contributes to ß-cell protection and survival. The present study indicates that the chemical chaperone-coupled glucagon-like peptide-1 receptor agonist is a feasible therapeutic strategy to enhance ß-cell function and survival.

Polysaccharide-based Nanoparticles for Gene Delivery.

Nanoparticles based on nanotechnology and biotechnology have emerged as efficient carriers for various biopharmaceutical agents including proteins and genes. In particular, polysaccharides have attracted interest of many researchers in the drug delivery field due to their advantages such as biocompatibility, biodegradability, low toxicity, and ease of modification. A number of polysaccharides including chitosan, hyaluronic acid, and dextran, and their derivatives have been widely used as polymeric backbones for the formation of nanoparticles, which can be provided as valuable gene delivery carriers. In this review, we introduce the chemical and physical natures of different polysaccharides particularly used in biomedical applications, and then discuss recent progress in the development of polysaccharide-based nanoparticles for gene delivery.

Reducible Polyethylenimine Nanoparticles for Efficient siRNA Delivery in Corneal Neovascularization Therapy.

The aim of this study is to establish the safe and effective ocular delivery system of therapeutic small interfering RNA (siRNA) in corneal neovascularization therapy. The major hurdle present in siRNA-based corneal neovascularization (CNV) therapy is severe cytotoxicity caused by repetitive drug treatment. A reducible branched polyethylenimine (rBPEI)-based nanoparticle (NP) system is utilized as a new siRNA carrier as a hope for CNV therapy. The thiolated BPEI is readily self-crosslinked in mild conditions to make high molecular weight rBPEI thus allowing the creation of stable siRNA/rBPEI nanoparticles (siRNA-rBPEI-NPs). In the therapeutic region, the rBPEI polymeric matrix is effectively degraded into nontoxic LMW BPEI inside the reductive cytosol causing the rapid release of the encapsulated siRNA into the cytosol to carry out its function. The fluorescent-labeled siRNA-rBPEI-NPs can release siRNA into the entire corneal region after subconjuctival injection into the eye of Sprague Dawley rats thus confirming the proof of concept of this system.

Development of Dissociation-Enhanced Lanthanide Fluoroimmunoassay for Measuring Leptin.

Development of a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) for measuring leptin, a satiety hormone of appetite control, was conducted in sandwich assay format exploiting a microplate immobilized with an anti-leptin antibody and another antibody raised against leptin and tagged with an europium chelate. In the leptin DELFIA of this study, amounts of antibody coated to the microplate and of the bioconjugate for the second immune reaction were optimized as 0.5 µg and 200 ng per well, respectively. When plotted in double-logarithmic scale, a linear relationship of y (log10 response signal) = 0.6023× (log10 leptin concentration) + 3.4084 (r(2) = 0.9646) was obtained at the leptin concentrations of 0.01─50 ng/mL with the limit of detection of 0.01 ng/mL. Individual leptin concentrations in various samples were well convergent to the calibration curve of the current assay. When applied to the measurement of leptin in a rat serum, the present assay was found quite effective and was competitive to a commercial sandwich-type ELISA.

Enzymatic hydrolysis of anchovy fine powder at high and ambient pressure, and characterization of the hydrolyzates.

BACKGROUND: At specific conditions of high pressure, the stability and activity of some enzymes are reportedly known to increase. The aim of this study was to apply pressure-tolerant proteases to hydrolyzing anchovy fine powder (AFP) and to determine product characteristics of the resultant hydrolyzates. RESULTS: Anchovy fine powder enzyme hydrolyzates (AFPEHs) were produced at 300 MPa and ambient pressure using combinations of Flavourzyme 500MG, Alcalase 2.4L, Marugoto E and Protamex. When the same protease combination was used for hydrolysis, the contents of total soluble solids, total water-soluble nitrogen and trichloroacetic acid-soluble nitrogen in the AFPEHs produced at 300 MPa were conspicuously higher than those in the AFPEHs produced at ambient pressure. This result and electrophoretic characteristics indicated that the high-pressure process of this study accelerates protein hydrolysis compared with the ambient-pressure counterpart. Most peptides in the hydrolyzates obtained at 300 MPa had molecular masses less than 5 kDa. Functionality, sensory characteristics and the content of total free amino acids of selected hydrolyzates were also determined. CONCLUSION: The high-pressure hydrolytic process utilizing pressure-tolerant proteases was found to be an efficient method for producing protein hydrolyzates with good product characteristics.