Affinity-Directed Site-Specific Protein Labeling and Its Application to Antibody-Drug Conjugates.

Chemically modified proteins have diverse applications; however, conventional chemo-selective methods often yield heterogeneously labeled products. To address this limitation, site-specific protein labeling holds significant potential, driving extensive research in this area. Nevertheless, site-specific modification of native proteins remains challenging owing to the complexity of their functional groups. Therefore, a method for site-selective labeling of intact proteins is aimed to design. In this study, a novel approach to traceless affinity-directed intact protein labeling is established, which leverages small binding proteins and genetic code expansion technology. By applying this method, a site-specific antibody labeling with a drug, which leads to the production of highly effective antibody-drug conjugates specifically targeting breast cancer cell lines is achieved. This approach enables traceless conjugation of intact target proteins, which is a critical advantage in pharmaceutical applications. Furthermore, small helical binding proteins can be easily engineered for various target proteins, thereby expanding their potential applications in diverse fields. This innovative approach represents a significant advancement in site-specific modification of native proteins, including antibodies. It also bears immense potential for facilitating the development of therapeutic agents for various diseases.

Evaluation of Silk Fibroin/Gellan Gum Hydrogels with Controlled Molecular Weight through Silk Fibroin Hydrolysis for Tissue Engineering Application.

Hydrogel is a versatile material that can be manipulated to achieve the desired physicochemical properties, such as stiffness, pore size, and viscoelasticity. Traditionally, these properties have been controlled through parameters such as concentration and pH adjustments. In this study, we focused on exploring the potential of hydrolyzed silk fibroin (HSF) as a molecular weight-modulating agent to control the physicochemical properties of double-composite hydrogels. We developed a synergistic dual-crosslinked hydrogel by combining ionically crosslinked silk fibroin with gellan gum (GG). The hydrolysis of silk fibroin not only enhanced its hydrophilicity but also enabled adjustments in its mechanical properties, including the pore size, initial modulus elasticity, and relaxation time. Moreover, biocompatibility assessments based on cell viability tests confirmed the potential of these hydrogels as biocompatible materials. By highlighting the significance of developing an HSF/GG dual-crosslinked hydrogel, this study contributes to the advancement of novel double-composite hydrogels with remarkable biocompatibility. Overall, our findings demonstrate the capability of controlling the mechanical properties of hydrogels through molecular weight modulation via hydrolysis and highlight the development of a biocompatible HSF/GG dual-crosslinked hydrogel with potential biomedical applications.

Development of Gelatin-Based Shape-Memory Polymer Scaffolds with Fast Responsive Performance and Enhanced Mechanical Properties for Tissue Engineering Applications.

Shape-memory polymers (SMPs) can be defined as a reversibly changing form through deformation and recovery by external stimuli. However, there remain application limitations of SMPs, such as complicated preparation processes and slow shape recovery. Here, we designed gelatin-based shape-memory scaffolds by a facile dipping method in tannic acid solution. The shape-memory effect of scaffolds was attributed to the hydrogen bond between gelatin and tannic acid, which acts as the net point. Moreover, gelatin (Gel)/oxidized gellan gum (OGG)/calcium chloride (Ca) was intended to induce faster and more stable shape-memory behavior through the introduction of a Schiff base reaction. The chemical, morphological, physicochemical, and mechanical properties of the fabricated scaffolds were evaluated, and those results showed that the Gel/OGG/Ca had improved mechanical properties and structural stability compared with other scaffold groups. Additionally, Gel/OGG/Ca exhibited excellent shape-recovery behavior of 95.8% at 37 °C. As a consequence, the proposed scaffolds can be fixed to the temporary shape at 25 °C in just 1 s and recovered to the original shape at 37 °C within 30 s, implying a great potential for minimally invasive implantation.

Engineering Translation Components for Genetic Code Expansion.

The expansion of the genetic code consisting of four bases and 20 amino acids into diverse building blocks has been an exciting topic in synthetic biology. Many biochemical components are involved in gene expression; therefore, adding a new component to the genetic code requires engineering many other components that interact with it. Genetic code expansion has advanced significantly for the last two decades with the engineering of several components involved in protein synthesis. These components include tRNA/aminoacyl-tRNA synthetase, new codons, ribosomes, and elongation factor Tu. In addition, biosynthesis and enhanced uptake of non-canonical amino acids have been attempted and have made meaningful progress. This review discusses the efforts to engineer these translation components, to improve the genetic code expansion technology.

Conversion of Racemic Unnatural Amino Acids to Optically Pure Forms by a Coupled Enzymatic Reaction.

Genetic code expansion (GCE) technology is a useful tool for the site-specific modification of proteins. An unnatural amino acid (UAA) is one of the essential components of this technique, typically required at high concentration (1 mM or higher) in growth medium. The supply of UAAs is an important limitation to the application of GCE technology, as many UAAs are either expansive or commercially unavailable. In this study, two UAAs in a racemic mixture were converted into optically pure forms using two enzymes, the d-amino acid oxidase (RgDAAO) from Rhodotorula gracilis and the aminotransferase (TtAT) from Thermus thermophilus. In the coupled enzyme system, RgDAAO oxidizes the d-form of UAAs in a stereospecific manner and produces the corresponding α-keto acids, which are then converted into the l-form of UAAs by TtAT, resulting in the quantitative and stereospecific conversion of racemic UAAs to optically pure forms. The genetic incorporation of the optically pure UAAs into a target protein produced a better protein yield than the same experiments using the racemic mixtures of the UAAs. This method could not only be used for the preparation of optically pure UAAs from racemic mixtures, but also the broad substrate specificity of both enzymes would allow for its expansion to structurally diverse UAAs.

Protein arginine methyltransferase 1 contributes to the development of allergic rhinitis by promoting the production of epithelial-derived cytokines.

BACKGROUND: Arginine methylation is a posttranslational modification mediated by protein arginine methyltransferases (PRMTs). Although previous studies have shown that PRMT1 contributes to the severity of allergic airway inflammation or asthma, the underlying mechanism is poorly understood. OBJECTIVE: This study aimed to explore the role of PRMT1 and its relevant mechanism in the development of allergic rhinitis (AR). METHODS: The expression levels of PRMTs and cytokines were determined by RT-PCR, and the localization of PRMT1 was determined by immunohistochemistry and confocal microscopy. The levels of house dust mite (HDM)-specific immunoglobulins in serum and of cytokines in nasal lavage fluids were determined by ELISA. PRMT1 inhibition was achieved by siRNA and treatment with the pan PRMT inhibitor arginine N-methyltransferase inhibitor-1. RESULTS: PRMT1 expression was significantly increased in the nasal mucosa of patients and mice with AR. The degree of eosinophilic infiltration in the nasal mucosa was reduced in PRMT1+/- AR mice compared with wild-type mice. PRMT1 haploinsufficiency reduced the levels of HDM-specific immunoglobulins in serum and those of TH2 (IL-4, IL-5, and IL-13) and epithelial (thymic stromal lymphopoietin [TSLP], IL-25, and IL-33) cytokines in the nasal lavage fluids of AR mice. In nasal epithelial cells, HDM and IL-4 cooperate to enhance PRMT1 expression through a mitogen-activated protein kinase-dependent pathway. In addition, PRMT1 was essential for the production of TSLP, IL-25, and IL-33 in response to HDM and IL-4. Arginine N-methyltransferase inhibitor-1 treatment alleviated AR in the mouse model. CONCLUSIONS: PRMT1 plays an important role in AR development by regulating epithelial-derived cytokine production and might be a new therapeutic target for AR.

Metal-Mediated Protein Assembly Using a Genetically Incorporated Metal-Chelating Amino Acid.

Many natural proteins function in oligomeric forms, which are critical for their sophisticated functions. The construction of protein assemblies has great potential for biosensors, enzyme catalysis, and biomedical applications. In designing protein assemblies, a critical process is to create protein-protein interaction (PPI) networks at defined sites of a target protein. Although a few methods are available for this purpose, most of them are dependent on existing PPIs of natural proteins to some extent. In this report, a metal-chelating amino acid, 2,2'-bipyridylalanine (BPA), was genetically introduced into defined sites of a monomeric protein and used to form protein oligomers. Depending on the number of BPAs introduced into the protein and the species of metal ions (Ni2+ and Cu2+), dimers or oligomers with different oligomerization patterns were formed by complexation with a metal ion. Oligomer sizes could also be controlled by incorporating two BPAs at different locations with varied angles to the center of the protein. When three BPAs were introduced, the monomeric protein formed a large complex with Ni2+. In addition, when Cu2+ was used for complex formation with the protein containing two BPAs, a linear complex was formed. The method proposed in this report is technically simple and generally applicable to various proteins with interesting functions. Therefore, this method would be useful for the design and construction of functional protein assemblies.

Generation of a High-Growth Influenza Vaccine Strain in MDCK Cells for Vaccine Preparedness.

As shown during the 2009 pandemic H1N1 (A(H1N1)pdm09) outbreak, egg-based influenza vaccine production technology is insufficient to meet global demands during an influenza pandemic. Therefore, there is a need to adapt cell culture-derived vaccine technology using suspended cell lines for more rapid and larger-scale vaccine production. In this study, we attempted to generate a high-growth influenza vaccine strain in MDCK cells using an A/Puerto/8/1934 (H1N1) vaccine seed strain. Following 48 serial passages with four rounds of virus plaque purification in MDCK cells, we were able to select several MDCK-adapted plaques that could grow over 108 PFU/ml. Genetic characterization revealed that these viruses mainly had amino acid substitutions in internal genes and exhibited higher polymerase activities. By using a series of Rg viruses, we demonstrated the essential residues of each gene and identified a set of high-growth strains in MDCK cells (PB1D153N, M1A137T, and NS1N176S). In addition, we confirmed that in the context of the high-growth A/PR/8/34 backbone, A/California/7/2009 (H1N1), A/Perth/16/2009 (H3N2), and A/environment/Korea/deltaW150/2006 (H5N1) also showed significantly enhanced growth properties (more than 107 PFU/ml) in both attached- and suspended-MDCK cells compared with each representative virus and the original PR8 vaccine strain. Taken together, this study demonstrates the feasibility of a cell culture-derived approach to produce seed viruses for influenza vaccines that are cheap and can be grown promptly and vigorously as a substitute for egg-based vaccines. Thus, our results suggest that MDCK cell-based vaccine production is a feasible option for producing large-scale vaccines in case of pandemic outbreaks.

Simiduia aestuariiviva sp. nov., a gammaproteobacterium isolated from a tidal flat sediment.

A Gram-stain negative, aerobic, motile and rod-shaped bacterial strain, designated J-MY2(T), was isolated from a tidal flat sediment of the South Sea, South Korea. Strain J-MY2(T) was found to grow optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain J-MY2(T) forms a cluster with the type strains of Simiduia species. Strain J-MY2(T) exhibited 16S rRNA gene sequence similarity values of 97.62-98.77 % to the type strains of four Simiduia species and of <92.95 % sequence similarity to the type strains of the other recognized species. Strain J-MY2(T) was found to contain Q-8 as the predominant ubiquinone and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16:0, C18:1 ω7c and C17:1 ω8c as the major fatty acids. The major polar lipids of strain J-MY2(T) were identified as phosphatidylethanolamine, phosphatidylglycerol, three unidentified glycolipids and one unidentified lipid. The DNA G+C content of strain J-MY2(T) was determined to be 54.8 mol% and its mean DNA-DNA relatedness values with the type strains of the four Simiduia species were in the range 21-34 %. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain J-MY2(T) is separated from other Simiduia species. On the basis of the data presented, strain J-MY2(T) is considered to represent a novel species of the genus Simiduia, for which the name Simiduia aestuariiviva sp. nov. is proposed. The type strain is J-MY2(T) ( = KCTC 42073(T) = CECT 8571(T)).

Simiduia curdlanivorans sp. nov., a curdlan-degrading bacterium isolated from the junction between the ocean and a freshwater spring, and emended description of the genus Simiduia.

A Gram-stain-negative, aerobic, non-spore-forming, non-flagellated and rod-shaped bacterial strain, designated DMCK3-4(T), was isolated from the zone where the ocean and a freshwater spring meet at Jeju island, South Korea. Strain DMCK3-4(T) grew optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2.0% (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain DMCK3-4(T) clustered with the strains of three members of the genus Simiduia, with which it exhibited 97.0-99.0% sequence similarity. Sequence similarities to the type strains of the other species with validly published names were less than 92.2%. Strain DMCK3-4(T) contained Q-8 as the predominant ubiquinone and summed feature 3 (C(16:1)ω7c and/or C(16:1)ω6c), C(17:1)ω8c, C(16:0), C(17:0) and C(18:1)ω7c as the major fatty acids. The major polar lipids of strain DMCK3-4(T) were phosphatidylethanolamine, phosphatidylglycerol, two unidentified glycolipids, one unidentified lipid and one unidentified aminolipid. The DNA G+C content of strain DMCK3-4(T) was 51.8 mol% and its mean DNA-DNA relatedness values with Simiduia agarivorans KCTC 23176(T), Simiduia areninigrae KCTC 23293(T) and Simiduia litorea NRIC 0917(T) were 23-34%, respectively. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain DMCK3-4(T) is distinct from other species of the genus Simiduia. On the basis of the data presented, strain DMCK3-4(T) is considered to represent a novel species of the genus Simiduia, for which the name Simiduia curdlanivorans sp. nov. is proposed. The type strain is DMCK3-4(T) ( = KCTC 42075(T) =CECT 8570(T)). An emended description of the genus Simiduia is also proposed.

Sungkyunkwania multivorans gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from seawater from a seaweed farm.

A Gram-staining-negative, non-flagellated, non-gliding and rod-shaped bacterial strain, designated PDB-16(T), was isolated from seawater from a seaweed farm on the South Sea in Korea, and its taxonomic position was investigated using a polyphasic approach. Strain PDB-16(T) grew optimally at 30 °C, at pH 7.0-7.5 and in the presence of 2% (w/v) NaCl. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain PDB-16(T) formed an independent lineage within the evolutionary radiation encompassed by the family Flavobacteriaceae. Strain PDB-16(T) contained MK-6 as the predominant menaquinone and iso-C15:0, iso-C15:1 G and iso-C17:0 3-OH as the major fatty acids. The major polar lipids detected in strain PDB-16(T) were phosphatidylethanolamine and two unidentified lipids. The DNA G+C content of strain PDB-16(T) was 42.1 mol%. Strain PDB-16(T) exhibited very low 16S rRNA gene sequence similarities of less than 89.7% to the type strains of any bacterial species with validly published names and less than 90.1% to uncultured bacteria clones. The 16S rRNA gene sequence similarity values and the differences in phenotypic properties between strain PDB-16(T) and some phylogenetically related genera were sufficient to support the proposal that strain PDB-16(T) should be distinguished from previously known genera of the family Flavobacteriaceae. On the basis of the data presented, strain PDB-16(T) is considered to represent a new genus and novel species, for which the name Sungkyunkwania multivorans gen. nov., sp. nov. is proposed. The type strain of Sungkyunkwania multivorans is PDB-16(T) (=KCTC 32138(T)=CCUG 62952(T)).

Thalassococcus lentus sp. nov., an alphaproteobacterium isolated from seawater of a seaweed farm.

A Gram-negative, non-motile and rod- or ovoid-shaped bacterial strain, designated YCS-24(T), was isolated from seawater of a seaweed farm in the South Sea, South Korea. Strain YCS-24(T) grew optimally at 25-28 °C, at pH 7.0-7.5 and in the presence of 2 % (w/v) NaCl. Strain YCS-24(T) exhibited the highest 16S rRNA gene sequence similarity values of 97.5 and 97.1 % to the type strains of Thalassobius maritimus and Thalassococcus halodurans, respectively. The neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain YCS-24(T) clustered with the type strain of T. halodurans. The DNA G+C content of strain YCS-24(T) was 58.0 mol% and its DNA-DNA relatedness values with T. halodurans JCM 13833(T) and T. maritimus GSW-M6(T) were 17 ± 6.2 and 23 ± 9.2 %, respectively. The predominant ubiquinone found in strain YCS-24(T) was Q-10 and the predominant fatty acid of strain YCS-24(T) was C(18:1) ω7c. The major polar lipids of strain YCS-24(T) were phosphatidylcholine, phosphatidylglycerol, one unidentified aminolipid and one unidentified lipid. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, demonstrated that strain YCS-24(T) is distinguished from T. halodurans. On the basis of the data presented, strain YCS-24(T) (= KCTC 32084(T) = CCUG 62791(T)) represents a novel species of the genus Thalassococcus, for which the name Thalassococcus lentus sp. nov. is proposed.

Inhibition of lytic reactivation of Kaposi's sarcoma-associated herpesvirus by alloferon.

BACKGROUND: Alloferon, an immunomodulatory peptide, has antiviral capability against herpesvirus. In this research, we aimed to investigate the effect of alloferon on the regulation of the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), and its mechanisms. We also assessed the antiviral activity of alloferon on natural killer (NK) cells as an early antiviral immune responder. METHODS: We first examined the change in cell proliferation and the expression of the viral genes in a KSHV-infected cell line, body-cavity-based B lymphoma (BCBL)-1, under the lytic cycle by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment. To elucidate the antiviral mechanism of alloferon, we tested calcium influx and the activation of the extracellular signal-regulated kinase (ERK) pathway. Furthermore, we evaluated the cytotoxicity of NK cells against BCBL-1 by alloferon. RESULTS: Alloferon effectively recovered the suppressed proliferation of BCBL-1 by TPA, which was achieved by the down-regulation of lytic-cycle-related viral genes, RTA, K8 and vIRF2. To clarify the signal transduction pathways related to the regulation of the viral genes by alloferon, we confirmed that the calcium influx into BCBL-1 was apparently inhibited by alloferon, which preceded the suppression of the phosphorylation of ERK and the activation of AP-1 by TPA. Moreover, when NK cells were exposed to alloferon, their cytolytic activity was improved, and this was mediated by the enhancement of perforin/granzyme secretion. CONCLUSIONS: The results of this study suggest that alloferon can be used as an effective antiviral agent for the regulation of the KSHV life cycle by the down-regulation of AP-1 activity and for the the enhancement of antiviral immunity by up-regulation of NK cell cytotoxicity.

A Study on Correlation between Anxiety Symptoms and Suicidal Ideation.

OBJECTIVE: In South Korea, the number of deaths from suicide has increased in the last two decades, and suicide has become both a social and political problem. In this study, after controlling the variables influencing suicidal ideation, it was expected that it would be determined if anxiety symptoms are independently related to suicidal ideation. METHODS: Data were obtained from 327 psychiatric outpatients accomplished a self-reported questionnaire that included sociodemographic characteristics and clinical variables as well as self-rating scales for measuring the severity of one's anxiety, depression, and suicidal ideation. Logistic-regression analyses were used to determine the correlation between anxiety symptoms and significant suicidal ideation, adjusting for covariates. RESULTS: The patients with significant suicidal ideation were shown to be less educated, unemployed, never married, divorced, or separated by death, or living alone, and were shown to have a lower income, a drinking habit, a higher number of past suicide attempts, and more family members who committed suicide, than the patients without significant suicidal ideation. After adjusting the covariates influencing significant suicidal ideation, anxiety symptoms were associated with significant suicidal ideation. However, after adjusting for depressive symptoms, only the trait anxiety was associated with significant suicidal ideation. CONCLUSION: These findings suggest that anxiety symptoms are an independent risk factor for suicidal ideation. Clinicians may thus use anxiety symptoms for the screening examination when evaluating suicidal ideation and risk, and will have to actively evaluate and treat the anxiety symptoms of patients with suicidal tendencies.

A hantavirus causing hemorrhagic fever with renal syndrome requires gC1qR/p32 for efficient cell binding and infection.

Hantaan virus (HTNV) is a pathogenic hantavirus that causes hemorrhagic fever with renal syndrome (HFRS). HTNV infection is mediated by alpha v beta3 integrin. We used protein blots of Vero E6 cell homogenates to demonstrate that radiolabeled HTNV virions bind to gC1qR/p32, the acidic 32-kDa protein known as the receptor for the globular head domain of complement C1q. RNAi-mediated suppression of gC1qR/p32 markedly reduced HTNV binding and infection in human lung epithelial A549 cells. Conversely, transient expression of either simian or human gC1qR/p32 rendered non-permissive CHO cells susceptible to HTNV infection. These results suggest an important role for gC1qR/p32 in HTNV infection and pathogenesis.