The manufacturing of lactobacillus microcapsules by freezing with egg yolk: The analysis of microstructure and the preservation effect against freezing and acid treatments

Encapsulation is an effective strategy to prevent probiotics from environmental damages. The survivability of Lactobacillus is generally reduced under frozen or acid environments. In this study, an attempt was made to encapsulate Lactobacillus into the egg yolk aggregates formed upon freezing. Lactobacillus broth was mixed with liquid egg yolk and frozen at selected temperatures and time periods. After thawing, the number of surviving bacteria was determined. The results showed that freezing with the addition of egg yolk improved Lactobacillus survivability. It was confirmed that freezing increased the number of egg yolk aggregates, and those aggregates coated Lactobacillus after freezing. The encapsulated Lactobacillus was treated at pH 2.5 for 10 min, and the results showed that the encapsulation process increased the acid resistance of Lactobacillus. These results indicate that freezing-induced encapsulation with egg yolk could effectively protect Lactobacillus against freezing and acid treatment. This finding could be useful for the design and preservation of probiotics-based food products

Arginine prevents thermal aggregation of hen egg white proteins

The control of aggregation and solubilization of hen egg white protein (HEWP) is an important issue for industrial applications of one of the most familiar food protein sources. Here, we investigated the effects of edible amino acids on heat-induced aggregation of HEWP. The addition of 0.6 M arginine (Arg) completely suppressed the formation of insoluble aggregates of 1 mg mL− 1 HEWP following heat treatment, even at 90 °C for 20 min. In contrast, lysine (Lys), glycine (Gly), and sodium chloride (NaCl) did little to suppress the aggregation of HEWP under the same conditions. SDS-PAGE indicated that Arg suppresses the thermal aggregation of almost all types of HEWP at 1 mg mL− 1. However, Arg did not suppress the thermal aggregation of HEWP at concentrations ≥ 10 mg mL− 1 and prompted the formation of aggregates. Transmission electron micrographs revealed a high-density structure of unfolded proteins in the presence of Arg. These results indicate that Arg exerts a greater suppressive effect on a protein mixture, such as HEWP, than on a single model protein. These observations may propose Arg as a safe and reasonable additive to HEWP for the elimination of microorganisms by allowing an increase in sterilization temperature

Co-aggregation of ovalbumin and lysozyme

Hen egg white has excellent heat-induced gelation properties. However, the molecular mechanisms underlying the aggregation of egg white proteins have not been elucidated due to their complex composition. Here, we focused on the thermal co-aggregation process of the main component, ovalbumin (OVA), with well-studied lysozyme (LYZ) in terms of protein composition, aggregation rate, intermolecular forces, and morphology. Size exclusion chromatographic analysis of OVA–LYZ mixture by heat treatment at 70 °C indicated that the aggregation-rate constant of LYZ increased 64-fold in the presence of equimolar OVA. In contrast, the aggregation rate of OVA was not dependent on the presence of LYZ. Enzyme assay and SDS-PAGE analysis showed that LYZ forms precipitates with unfolded OVA via reversible non-covalent interactions and irreversible disulfide bonds. The unfolding of OVA triggers co-aggregation by exposure of the aggregation-prone region, followed by disulfide bond exchange between OVA and LYZ. LYZ links covalently to small OVA aggregates through disulfide bonds, leading to the hierarchical growth of OVA–LYZ aggregates with larger networks. These results provide information regarding the thermal co-aggregation of proteins in hen egg white

Osteocrin ameliorates adriamycin nephropathy via p38 mitogen-activated protein kinase inhibition

Natriuretic peptides exert multiple effects by binding to natriuretic peptide receptors (NPRs). Osteocrin (OSTN) binds with high affinity to NPR-C, a clearance receptor for natriuretic peptides, and inhibits degradation of natriuretic peptides and consequently enhances guanylyl cyclase-A (GC-A/NPR1) signaling. However, the roles of OSTN in the kidney have not been well clarified. Adriamycin (ADR) nephropathy in wild-type mice showed albuminuria, glomerular basement membrane changes, increased podocyte injuries, infiltration of macrophages, and p38 mitogen-activated protein kinase (MAPK) activation. All these phenotypes were improved in OSTN- transgenic (Tg) mice and NPR3 knockout (KO) mice, with no further improvement in OSTN-Tg/NPR3 KO double mutant mice, indicating that OSTN works through NPR3. On the contrary, OSTN KO mice increased urinary albumin levels, and pharmacological blockade of p38 MAPK in OSTN KO mice ameliorated ADR nephropathy. In vitro, combination treatment with ANP and OSTN, or FR167653, p38 MAPK inhibitor, reduced Ccl2 and Des mRNA expression in murine podocytes (MPC5). OSTN increased intracellular cyclic guanosine monophosphate (cGMP) in MPC5 through GC-A. We have elucidated that circulating OSTN improves ADR nephropathy by enhancing GC-A signaling and consequently suppressing p38 MAPK activation. These results suggest that OSTN could be a promising therapeutic agent for podocyte injury

Production and nonclinical evaluation of an autologous iPSC-derived platelet product for the iPLAT1 clinical trial

血小板減少症に対するiPS細胞由来血小板の自己輸血に関する臨床研究」の成果公表（論文発表）について. 京都大学プレスリリース. 2022-09-30.Donor-derived platelets are used to treat or prevent hemorrhage in patients with thrombocytopenia. However, ∼5% or more of these patients are complicated with alloimmune platelet transfusion refractoriness (allo-PTR) due to alloantibodies against HLA-I or human platelet antigens (HPA). In these cases, platelets from compatible donors are necessary, but it is difficult to find such donors for patients with rare HLA-I or HPA. To produce platelet products for patients with aplastic anemia with allo-PTR due to rare HPA-1 mismatch in Japan, we developed an ex vivo good manufacturing process (GMP)–based production system for an induced pluripotent stem cell–derived platelet product (iPSC-PLTs). Immortalized megakaryocyte progenitor cell lines (imMKCLs) were established from patient iPSCs, and a competent imMKCL clone was selected for the master cell bank (MCB) and confirmed for safety, including negativity of pathogens. From this MCB, iPSC-PLTs were produced using turbulent flow bioreactors and new drugs. In extensive nonclinical studies, iPSC-PLTs were confirmed for quality, safety, and efficacy, including hemostasis in a rabbit model. This report presents a complete system for the GMP-based production of iPSC-PLTs and the required nonclinical studies and thus supports the iPLAT1 study, the first-in-human clinical trial of iPSC-PLTs in a patient with allo-PTR and no compatible donor using the autologous product. It also serves as a comprehensive reference for the development of widely applicable allogeneic iPSC-PLTs and other cell products that use iPSC-derived progenitor cells as MCB

Incorporation of 8-hydroxyguanosine 5'-triphosphate (8-oxo-7,8-dihydroguanosine 5'-triphosphate) by bacterial and human RNA polymerases

Oxidized RNA precursors formed in the nucleotide pool may be incorporated into RNA. In this study, the incorporation of 8-hydroxyguanosine 5'-triphosphate (8-OH-GTP; 8-oxo-7,8-dihydroguanosine 5'-triphosphate) into RNA by Escherichia coli RNA polymerase was examined in vitro, using a primer RNA and a template DNA with defined sequences. 8-OH-GTP was incorporated opposite C and A in the template DNA. Surprisingly, 8-OH-GTP was quite efficiently incorporated by the bacterial RNA polymerase, in contrast to the incorporation of the 2'-deoxyribo-counterpart by DNA polymerases, as indicated by the kinetic parameters. The primer was further extended by the addition of a ribonucleotide complementary to the nucleobase adjacent to C or A (the nucleobase opposite which 8-OH-GTP was inserted). Thus, the incorporation of 8-OH-GTP did not completely inhibit further RNA chain elongation. 8-OH-GTP was also incorporated opposite C and A by human RNA polymerase II. These results suggest that 8-OH-GTP in the nucleotide pool call cause the formation of oxidized RNA and disturb the transmittance of genetic information