Apoptosis inhibitor of macrophage (AIM) produced by tissue macrophages has been implicated in a variety of pathological conditions, including acute kidney injury (AKI). AIM protects renal cells from apoptosis, modulating inflammation, and promotes tissue repair, thereby contributing to the maintenance of kidney function in AKI. However, the role of AIM in anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) remains unknown. In this study, whether AIM could be detected in the urine and kidneys of patients with AAV was investigated, and the potential of urinary AIM as a biomarker for AAV-related kidney injury was evaluated. A total of 57 patients with AAV treated in our departments between December 2011 and March 2024, together with 16 healthy adults, were enrolled. Serum and urinary AIM levels were measured by ELISA. The correlations of urinary AIM levels with renal function, urinary protein levels, clinical parameters, and established AKI biomarkers were analyzed. The intrarenal localization of AIM was examined by immunostaining using renal biopsy specimens from patients with AAV. Urinary AIM levels were significantly higher in patients with AAV than in healthy controls (18.8 ± 2.8 vs. 0.00 ± 0.0 μg/gCr, p < 0.01). Urinary AIM levels decreased significantly after immunosuppressive therapy. Patients with higher urinary AIM levels tended to show limited improvement in renal function. Urinary AIM levels were significantly correlated with serum creatinine, estimated glomerular filtration rate, urinary protein excretion, urinary kidney injury molecule-1, and urinary neutrophil gelatinase-associated lipocalin. In contrast, no significant correlations were observed between urinary AIM levels and urinary N-acetyl-β-d-glucosaminidase, MPO-ANCA titers, or C-reactive protein levels. AIM was not detected in normal kidneys, but it was detected in renal tubules of patients with AAV. AIM-positive cells expressed aquaporin-1, but not uromodulin or aquaporin-2. AIM-positive cells were partially colocalized with KIM-1-positive tubules and CD68-positive cells. These findings suggest that urinary AIM may serve as a non-invasive biomarker reflecting the severity of tubular injury in patients with AAV.

Autophagy plays a key role in cellular homeostasis, but dysregulated autophagy can lead to resistance to chemotherapeutic agents. The Ridaifen (RID) compound series comprises structural analogues of tamoxifen that exhibit more potent anticancer activity and have been implicated in modulating autophagy. Here, we investigated how the RID compounds interact with autophagy and explored the factors contributing to their enhanced cytotoxicity. We synthesized RID derivatives containing varying numbers of basic side chains and evaluated their intracellular behavior. We assessed cell viability using an MTT assay and determined lysosomal pH by flow cytometry. To visualize the subcellular distribution of the RID derivative, we employed a fluorescent dye‒conjugated form of the compound. Additionally, we monitored autophagic and apoptotic markers through immunoblotting. RID-B demonstrated potent lysosomal neutralization and inhibited autophagic flux near its half-maximal inhibitory concentration. This neutralization led to the accumulation of insoluble SQSTM1-containing aggregates, implicating proteotoxic stress in apoptosis. Confocal imaging revealed proton-dependent lysosomal localization of RID-B, followed by partial cytoplasmic translocation. Notably, co-treatment with bafilomycin A1 reduced RID-B‒induced apoptosis, underscoring lysosomal dysfunction initiated apoptotic signaling. Analyses across multiple RID derivatives showed a correlation among the number of basic side chains, lysosomal neutralization, and between lysosomal neutralization and cytotoxicity. Our findings indicate that basic side chains markedly enhance lysosomotropic behavior, enabling sustained autophagy inhibition and apoptosis induction. By revealing a strong link between lysosomal neutralization and proteotoxic cell death, the results suggest that modified tamoxifen analogues, such as RID-B, may offer a promising strategy to overcome autophagy-related drug resistance in cancer therapy.

Efficient immobilization of DNA on solid surfaces is important for the advancement of electrochemical DNA sensors, biosensors, and bioelectrodes. In this study, we established a new method for immobilizing circular DNA through the formation of triple-helical structures, enabling the high-density and oriented attachment of plasmid DNA to the substrate surface. We engineered biotinylated circular DNA containing a triple-stranded region by hybridizing a biotinylated homopyrimidine third strand with homopurine-homopyrimidine sequences in circular DNA. Subsequently, the modified circular DNA was effectively immobilized on a streptavidin-biotin-functionalized substrate. Using a cell-free protein synthesis system, the yield of Discosoma sp. red (DsRed) fluorescent protein synthesized from immobilized circular DNA was approximately 4.6 times higher than that from immobilized linear DNA. Notably, the immobilized circular DNA template was effectively reused in multiple consecutive rounds of protein synthesis, highlighting its potential for repeated application. Overall, our strategy significantly enhances protein synthesis efficiency and provides a robust platform for the development of high-performance DNA arrays, biosensors, and bioelectrodes.

This study investigated the effects of wheat lipoxygenase isozyme III (LOX III) and its truncated form, Mini-LOX III, on flour dough properties using yeast-expressed recombinant enzymes and hypothesized their potential to enhance cereal-based food quality. These enzymes actively catalyze linoleic acid, which is crucial for dough formation. The addition of recombinant LOX III and Mini-LOX III to wheat flour significantly changed glutenin protein composition. An increase in the amount of soluble glutenin and a shift in polypeptide distribution were observed, marked by a decrease in the high-molecular-weight regions and an increase in the low-molecular-weight regions. This result reflects the role of enzymes in altering the hydrophobicity of glutenin surfaces, thereby affecting the protein solubility and dough properties. Thus, recombinant LOX III and Mini-LOX III offer new avenues for enhancing the texture and quality of cereal-based foods, providing valuable insights into the role of wheat LOX in flour processing and its potential industrial applications.

We synthesized ridaifen-B boron dipyrromethene (RID-B-BODIPY) using 2-methyl-6-nitro benzoic anhydride (MNBA)-mediated dehydration condensation reaction between amino alkyl-tethered RID and BODIPY FL. Comparative experiments between dicyclohexylcarbodiimide (DCC) and MNBA for their coupling reactions demonstrated that MNBA is an effective condensation reagent for amines and BODIPY FL. A cell staining study with RID-B-BODIPY showed intracellular localization of BODIPY FL fluorescence, attributed to the RID-B structure, indicating the successful development of a tool for analyzing intracellular molecular behavior efficiently.

In this study, a double-stranded DNA (dsDNA) fluorescent labeling method was developed using the fusion proteins of fluorescent protein (FP), and 7 kDa DNA-binding family members including Sso7d from Sulfolobus solfataricus, Aho7c from Acidianus hospitalis, ATSV7 from Acidianus tailed spindle virus and Sto7 from Sulfolobus tokodaii. Using this fluorescent DNA labeling method, we succeeded in single-molecule imaging of bacteriophage λDNA molecules stretched on glass surfaces. The fluorescence of the λDNA with FP fusion proteins decayed 2.4- to 6.4-fold slower than that of the typical intercalating method with SYTOX Green (SxG). In addition, the dynamic behaviors of FP-fused Aho7c-λDNA were relaxed and stretched with and without buffer flow, respectively, in microflow channels and were similar to that with typical intercalating dye, such as YOYO-1 and SxG. this fluorescent DNA labeling method. This fluorescent DNA labeling method can solve the problem of rapid fluorescence decay due to the intercalating dyes and therefore can be expected as an alternative to compound-based fluorescent dye. Thus, this study establishes FP fusion proteins as useful fluorescent DNA probes at the single-molecule level.

Engineering the microbial production of secondary metabolites is limited by the known reactions of correctly annotated enzymes. Therefore, the machine learning discovery of specialized enzymes offers great potential to expand the range of biosynthesis pathways. Benzylisoquinoline alkaloid production is a model example of metabolic engineering with potential to revolutionize the paradigm of sustainable biomanufacturing. Existing bacterial studies utilize a norlaudanosoline pathway, whereas plants contain a more stable norcoclaurine pathway, which is exploited in yeast. However, committed aromatic precursors are still produced using microbial enzymes that remain elusive in plants, and additional downstream missing links remain hidden within highly duplicated plant gene families. In the current study, machine learning is applied to predict and select plant missing link enzymes from homologous candidate sequences. Metabolomics-based characterization of the selected sequences reveals potential aromatic acetaldehyde synthases and phenylpyruvate decarboxylases in reconstructed plant gene-only benzylisoquinoline alkaloid pathways from tyrosine. Synergistic application of the aryl acetaldehyde producing enzymes results in enhanced benzylisoquinoline alkaloid production through hybrid norcoclaurine and norlaudanosoline pathways.

BACKGROUND/AIM:Azoxystrobin (AZOX), a methoxyacrylate derivative, has potent antimicrobial and antitumor activities. Here, we report the anticancer effects of AZOX on the p53-negative human myelogenous leukemia cell line HL-60RG and the p53 positive human T-cell leukemia cell line MOLT-4F.MATERIALS AND METHODS:Using both leukemia cells, the anticancer effect of AZOX treatment was analyzed throughout the cell cycle.RESULTS:AZOX damaged both cell lines dose-dependently, and the cell damage rates were almost the same in both lines. Cell cycle distribution analysis showed that the treated MOLT-4F cells arrested at the S phase, whereas HL-60RG cells increased during the subG1 phase, suggesting that cell death was occurring. AZOX-induced cell death in HL-60RG was inhibited with the addition of uridine, which is used as a substrate for the salvage pathway of pyrimidine nucleotides.CONCLUSION:AZOX has p53-independent anticancer effects in leukemia cells, but the mechanisms underlying the damage differ between cell lines.

Tumor-specific growth signal inhibition is a major anticancer strategy. Receptor tyrosine kinases (RTKs) are the most upstream receptors for growth signaling in cancer. Therefore, inhibition of RTKs has been proposed as an efficient therapeutic target. Masitinib, a c-kit inhibitor of the c-kit RTK, was developed to treat mastocytoma in dogs. In humans, however, the antitumor efficacy of masitinib was found to be attenuated against tumor cells with mutations of the c-kit gene. Here, we report that masitinib induced cell death via the intrinsic apoptotic pathway in HepG2, a c-kit-negative hepatocellular carcinoma cell line. In masitinib-treated HepG2 cells, increases in intracellular reactive oxygen species levels, loss of mitochondrial membrane potential, and cleavage of caspase-9 were observed, activating the intrinsic apoptotic pathway. Moreover, the cytotoxicity of masitinib to HepG2 cells was suppressed by treatment with the antioxidant N-acetyl-L-cysteine or a c-Jun N-terminal kinase/stress-activated protein kinase (JNKs) inhibitor. Thus, we demonstrated that the anticancer effects of masitinib are not due to its targeting c-kit, but rather to its targeting the redox balance via the JNK pathway in HepG2 cells. These results suggest that masitinib has the potential to provide a robust antitumor effect in tumor lesions and could also be applied to a broad range of other anticancer therapies.

DNA replication, repair, and recombination in the cell play a significant role in the regulation of the inheritance, maintenance, and transfer of genetic information. To elucidate the biomolecular mechanism in the cell, some molecular models of DNA replication, repair, and recombination have been proposed. These biological studies have been conducted using bulk assays, such as gel electrophoresis. Because in bulk assays, several millions of biomolecules are subjected to analysis, the results of the biological analysis only reveal the average behavior of a large number of biomolecules. Therefore, revealing the elementary biological processes of a protein acting on DNA (e.g., the binding of protein to DNA, DNA synthesis, the pause of DNA synthesis, and the release of protein from DNA) is difficult. Single-molecule imaging allows the analysis of the dynamic behaviors of individual biomolecules that are hidden during bulk experiments. Thus, the methods for single-molecule imaging have provided new insights into almost all of the aspects of the elementary processes of DNA replication, repair, and recombination. However, in an aqueous solution, DNA molecules are in a randomly coiled state. Thus, the manipulation of the physical form of the single DNA molecules is important. In this review, we provide an overview of the unique studies on DNA manipulation and single-molecule imaging to analyze the dynamic interaction between DNA and protein.

The N-type Ca2+ channel (Cav2.2) is distributed in sympathetic nerves that innervate the tubules, the vessels, and the juxtaglomerular granular cells of the kidney. However, the role of N-type Ca2+ channels in renal disease remains unknown. To address this issue, Cav2.2 knockout mice were utilized. Immunoreactive Cav2.2 was undetectable in normal kidneys of C57BL/6N mice, but it became positive in the interstitial S100-positive nerve fibers after unilateral ureteral obstruction (UUO). There were no significant differences in mean blood pressure, heart rate, and renal function between wild-type littermates and Cav2.2-knockout mice at baseline, as well as after UUO. Cav2.2 deficiency significantly reduced the EVG-positive fibrotic area, alpha-SMA expression, the production of type I collagen, and the hypoxic area in the obstructed kidneys. The expression of tyrosine hydroxylase, a marker for sympathetic neurons, was significantly increased in the obstructed kidneys of wild-type mice, but not in Cav2.2-knockout mice. These data suggest that increased Cav2.2 is implicated in renal nerve activation leading to the progression of renal fibrosis. Blockade of Cav2.2 might be a novel therapeutic approach for preventing renal fibrosis.

Activin A, a member of the transforming growth factor-beta superfamily, is a critical modulator of inflammation and plays a key role in controlling the cytokine cascade that drives the inflammatory response. However, the role of activin A in inflammatory kidney diseases remains unknown. To address this issue, we examined here whether activin A can be detected in the kidney and/or urine from patients with antineutrophil cytoplasmic antibody (ANCA) -associated vasculitis (AAV). Fifty-one patients who had been diagnosed with AAV and were treated in our department between November 2011 to March 2018 were included in this study. Forty-one patients had renal complications (renal AAV). Serum and urinary activin A levels were measured by enzyme-linked immunosorbent assay. Correlation of urinary activin A concentration with clinical parameters was analyzed. Urinary activin A was undetectable in healthy volunteers. In contrast, urinary activin A concentration was significantly increased in patients with renal AAV but not in those with non-renal AAV. Urinary activin A concentration decreased rapidly after immunosuppressive treatment. There was a significant correlation of urinary activin A level with urinary protein, L-FABP, and NAG. Histologic evaluation revealed that urinary activin A levels were significantly higher in patients with cellular crescentic glomeruli than in those lacking this damage. In situ hybridization demonstrated that the mRNA encoding the activin A βA subunit was undetectable in normal kidneys but accumulated in the proximal tubules and crescentic glomeruli of the kidneys of patients with renal AAV. Immunostaining showed that activin A protein also was present in the proximal tubules, crescentic glomeruli, and macrophages infiltrating into the interstitium in the kidneys of patients with renal AAV. These data suggested that urinary activin A concentration reflects renal inflammation and tubular damage in AAV and may be a useful biomarker for monitoring renal AAV.

Transcutaneous energy transmission systems (TETS) that supply power to ventricular assist devices have been developed. The air-core transcutaneous transformer is coated with insulating materials, however, the high-frequency leakage current (HFLC) flows because capacitive coupling may be electrically connected between the air-core transcutaneous transformer and the human body. We proposed that high-frequency isolation transformer as a way to suppress HFLC. In this study, attenuation rate of HFLC was measured according to the change of the number of turns of the high-frequency isolation transformer (HFT). As a result, HFLC was 4.2-6.9 mA when the number of turns HFT was 6 turns. TETS which uses 6 turns of HFT satisfies the restriction value of JIS T 0601-1. However, in the 3 turns HFLC was increased to 13 mA and the temperature rise of the HFT was 20 degrees because of the core loss due to high magnetic flux density. It was confirmed that our HFT is compact, lightweight, low heat generation, and is suitable for TETS for ventricular assist device.

In the original version of this Article, the abbreviation of 3,4-dihydroxyphenylacetaldehyde synthase presented in the first paragraph of the Discussion section was given incorrectly as DYPAA. The correct abbreviation for this enzyme is DHPAAS. This error has been corrected in both the PDF and HTML versions of the Article.

Tubulogenesis, the organization of epithelial cells into tubular structures, is an essential step during renal organogenesis as well as during the regeneration process of renal tubules after injury. In the present study, endothelial cell-derived factors that modulate tubule formation were examined using an in vitro human tubulogenesis system. When human renal proximal tubular epithelial cells (RPTECs) were cultured in gels, tubular structures with lumens were induced in the presence of hepatocyte growth factor (HGF). Aquaporin 1 was localized in the apical membrane of these tubular structures, suggesting that these structures are morphologically equivalent to renal tubules in vivo. HGF-induced tubule formation was significantly enhanced when co-cultured with human umbilical vein endothelial cells (HUVECs) or in the presence of HUVEC-conditioned medium (HUVEC-CM). Co-culture with HUVECs did not induce tubular structures in the absence of HGF. A phospho-receptor tyrosine kinase array revealed that HUVEC-CM markedly enhanced phosphorylation of Ret, glial cell-derived neurotrophic factor (GDNF) receptor, in HGF-induced tubular structures compared to those without HUVEC-CM. HUVECs produced GDNF, and RPTECs expressed both Ret and GDNF family receptor alpha1 (co-receptor). HGF-induced tubule formation was significantly enhanced by addition of GDNF. Interestingly, not only HGF but also GDNF significantly induced phosphorylation of the HGF receptor, Met. These data indicate that endothelial cell-derived GDNF potentiates the tubulogenic properties of HGF and may play a critical role in the epithelial-endothelial crosstalk during renal tubulogenesis as well as tubular regeneration after injury.

Renal impairment (RI) is a common complication of multiple myeloma (MM) that significantly affects treatment efficacy and mortality. However, no useful biomarkers for early detection of renal damage in MM exist. Reports indicate that activin A, a multifunctional cytokine of the TGF-β superfamily, is involved in the development and progression of various kidney diseases. In the present study, we measured urinary activin A levels in patients with newly diagnosed MM (NDMM) (n=41), smoldering MM (SMM) (n=10), and monoclonal gammopathy of undetermined significance (MGUS) (n=28), including monoclonal gammopathy of renal significance (MGRS), and assessed the correlation between urinary activin A and several clinical parameters. Urinary activin A, undetectable in healthy volunteers, was significantly increased in NDMM patients but not in patients with SMM and MGUS (97.3, 25.0, and 6.61 mg/gCr, respectively, P<0.05). In all patients with NDMM, urinary activin A levels were significantly reduced after initial treatment regardless of the therapy regimen. There was a significant correlation of urinary activin A with spot urinary protein level (P<0.001) and serum M-protein (P=0.029) but not with estimated glomerular filtration rate (eGFR), serum creatinine (Cr), N-acetyl-glucosaminidase (NAG), and serum activin A level. Histological analysis using renal biopsy samples revealed that activin A, which was absent from normal kidneys, was detected in the renal tubular cells of patients with MGRS. These data suggest that urinary activin A reflects tubular injury in MM and might aid the early detection of RI in plasma cell neoplasms.