Kobe Pharmaceutical University

Disruption of osteoblast differentiation can lead to severe bone diseases, such as osteoporosis and osteosclerosis. Our previous study showed that a reduced 4S/6S ratio promotes osteoblast differentiation, linking this specific chondroitin sulfate (CS) modification to bone pathology. This study investigated the effect of forced elevation of the 4S/6S ratio on differentiation. C4ST-1 was found to exhibit overexpression, increasing the 4S/6S ratio, significantly suppressing osteoblast differentiation, as evidenced by reduced Akp2 gene expression and ALP activity. This inhibition was far more potent than that caused by the enzymatic removal of CS. Notably, treating C4ST-1-overexpressing cells with chondroitinase reduced differentiation inhibition to a level similar to that in mock cells treated with chondroitinase. These results suggest that the strong inhibition was due to the excessive 4-sulfated CS produced by C4ST-1, implying a mechanism distinct from the inhibition caused by a lack of CS. To elucidate this mechanism, we investigated the potential feedback loop. The increase in 4-sulfated CS from C4ST-1 overexpression enhanced Wnt3a expression, which upregulated p53 expression. Pharmacological inhibition of β-catenin and p53 partially restored Akp2 expression, confirming their roles as key mediators. We propose that a high 4S/6S ratio activates a Wnt/β-catenin-p53 axis, where p53 acts as a brake on differentiation. Our findings highlight the critical role of CS sulfation patterns in fine-tuning the osteoblast fate.

Bortezomib, a first-in-class proteasome inhibitor, is widely used to treat multiple myeloma and other hematological malignancies. Despite its therapeutic efficacy, bortezomib causes peripheral neuropathy (PN) in approximately 20-30% of patients, often leading to dose reduction or discontinuation. Preventive or therapeutic approaches to bortezomib-induced PN are currently unavailable, as its precise mechanism remains unclear. In this study, we compared the effects of bortezomib and the second-generation proteasome inhibitor carfilzomib on peripheral nerve cells to identify candidate molecules involved in PN development. Transcriptome profiling of differentiated F11 cells, a hybridoma of a rat embryonic dorsal root ganglion and mouse neuroblastoma cell line N18TG2, revealed that bortezomib selectively upregulated α/β-hydrolase containing domain 4 (Abhd4), whereas carfilzomib did not. This finding was confirmed by quantitative RT-PCR and immunoblotting, which demonstrated consistent increases in Abhd4 mRNA and protein levels following bortezomib treatment. Functional analysis further revealed that Abhd4 overexpression promoted early apoptosis, suggesting a mechanistic link between bortezomib-induced Abhd4 elevation and neuronal vulnerability. Therefore, these results suggest that Abhd4 represents a candidate molecular signature associated with bortezomib-induced PN. Although further in vivo validation is needed, these findings warrant further investigation of Abhd4 as a potential contributor to bortezomib-induced PN.

Abstract Many plant-derived lipophilic compounds benefit human life. For example, shikonin derivatives—lipophilic red pigments from the medicinal plant Lithospermum erythrorhizon —exhibit wound-healing and antimicrobial effects. These compounds are excreted from root epidermal cells and accumulate in the root bark. Our understanding of how lipophilic compounds are transported in plant cells remains limited, and L. erythrorhizon hairy roots serve as a good model for studying the lipid secretion. To investigate this, the shikonin secretion was induced in cultured hairy roots, which were then fixed via chemical fixation or high-pressure freezing followed by freeze-substitution and observed by transmission electron microscopy. Electron micrographs from chemically fixed samples showed multivesicular bodies (MVBs) surrounding electron-dense bodies—presumed to contain shikonin derivatives—and fusing with the plasma membrane. Similarly, freeze-substituted samples revealed MVBs fused with the plasma membrane. Additionally, some MVBs contacted plastids containing enzymes for shikonin precursor biosynthesis. These findings suggest MVBs play key roles in both synthesis and secretion of shikonin derivatives. Highlight This study reveals that multivesicular bodies mediate both synthesis and secretion of shikonin derivatives in the hairy roots of Lithospermum erythrorhizon .

In recent years, the development of nucleic acid therapeutics has gained considerable traction, with the aim of controlling the expression of disease-related genes. In particular, numerous small interfering RNA (siRNA) drugs that act through the RNA interference (RNAi) mechanism have been developed. Growing attention has also been directed toward strategies that inhibit microRNAs (miRNAs), which endogenously mediate RNAi. Both approaches involve nucleic acid drugs that modulate the RNA-induced silencing complex (RISC)—the central effector of RNAi—to control disease-related gene expression through either gene silencing or restoration. To improve the design of nucleic acid therapeutics, artificial nucleic acids have been employed to enhance enzymatic stability and target RNA recognition. Although ribose-based artificial nucleic acids have been widely adopted, researchers have also begun exploring alternatives with chemical structures that differ substantially from those of the ribose type, especially to achieve greater enzymatic resistance. Against this backdrop, we have focused on non-ribose type acyclic artificial nucleic acids with amino-acid-based backbones, serinol nucleic acid (SNA) and acyclic L-threoninol nucleic acid (L-aTNA), and have explored their use in designing nucleic acid therapeutics. This account describes designs for both siRNA and antisense oligonucleotides against miRNA (anti-miRNA), which are referred to as AMOs. The unique properties of these acyclic nucleic acids are highlighted, along with an introduction to research on siRNA designs aimed at regulating strand selection during RISC assembly—a key strategy for minimizing off-target effects. This account also includes the design and mechanism of action for our AMO. A key limitation of acyclic artificial nucleic acids is also addressed, with a chemical strategy proposed to overcome it. Ultimately, this review suggests that the unique chemical properties of acyclic artificial nucleic acids may enable the next generation of highly stable and selective nucleic acid therapeutics, paving the way for more effective and safer clinical applications.

Abstract The evolution of the microstructure in highly porous compacts was analyzed using scanning electron microscopy on yttrium iron oxide samples exhibiting densities of less than 30% of theoretical density. During grain growth in highly porous compacts, the grains initially form a mesh chain structure, followed by either elongation or the formation of ring‐like structures, and finally assembly into hollow or coarser spheres. The latter process may involve the formation of fine necks growing through the ring structure, acting as the core of a coarser sphere. Thus, YFeO3 porous materials have macroscopic and microscopic pores, and the total porosity level as well as porous cluster sizes inside granules can be controlled by adjusting temperature or dwell time.

Shikonin, a 1,4-naphthoquinone derivative produced by several Boraginaceae species, exhibits unique pharmacological properties and is used as a natural dye. The regulatory factors of shikonin production have been demonstrated using a cell culture system of Lithospermum erythrorhizon. Among these factors, copper is known to be the strongest enhancer of shikonin production. Although shikonin biosynthesis has been studied for over 40 years, the steps of naphthalene ring formation are still unknown, as is the reason for the effect of copper. In this study, we explored candidate genes associated with shikonin production using a PCR-select subtraction experiment. Polyphenol oxidase (PPO), a dicopper-dependent oxidoreductase, was highlighted because it showed synchronous expression with shikonin production. Transcriptome analysis of hairy roots and cultured cells of this plant revealed that, of the five PPO genes expressed in L. erythrorhizon, only PPO1 showed a strong correlation with shikonin production. Next, we generated genome-edited hairy roots of LePPO1 using CRISPR/Cas9-mediated mutagenesis to analyze its impact on shikonin derivative and other specialized metabolite production. The results showed that shikonin content was markedly reduced in all LePPO1-ge lines. Interestingly, the content of deoxyshikonofuran, a hydroquinone derivative and shunt product that branches after GHQ-3′′-OH in the shikonin biosynthetic pathway, remained unaffected in the LePPO1-ge lines. These findings suggest that LePPO1 participates in naphthalene ring formation and explain why a copper ion is crucial for shikonin biosynthesis.

Background: Mesenchymal stem cells (MSCs) possess an intrinsic tumor-tropic ability, and therefore, MSCs may potentially be used as biomimetic carriers for active drug delivery systems targeting tumors. We previously developed a method to efficiently load liposomes onto the surface of MSCs via electrostatic interactions. The prepared liposome-loaded MSCs (Lip-MSCs) spontaneously accumulated in solid melanoma tumors with low vascular permeability while stably carrying liposomes. Methods: To explore Lip-MSC applications in cancer chemotherapy, doxorubicin (DOX)-encapsulated liposomes (DOX-Lip) were prepared and loaded onto MSCs. The cell viability, DOX-releasing properties, tumor-homing capacity, and anti-tumor efficacy of DOX-Lip-MSCs were analyzed. Results: Small liposomes (100 nm) retained DOX, whereas significant leakage of DOX was observed from 600 nm-sized liposomes. Based on this result, we used 100 nm DOX-Lip for the preparation of DOX-Lip-MSCs. Compared with MSCs loaded with DOX by incubation with DOX solution, DOX-Lip-MSCs could load a larger amount of DOX with minimal cytotoxicity. DOX-Lip-MSCs also showed sustained DOX release. DOX-Lip-MSCs efficiently migrated toward the conditioned medium of B16/BL6 melanoma cells in vitro and accumulated in B16/BL6 tumors in vivo, leading to a significant inhibitory effect on tumor growth. Conclusions: Lip-MSCs can serve as an efficient carrier to deliver anti-cancer drugs into solid tumors.

Chronic inflammation via dysregulation of T cell immune responses is critically involved in the pathogenesis of atherosclerotic cardiovascular disease. Improving the balance between proinflammatory T cells and anti-inflammatory regulatory T cells (Tregs) may be an attractive approach for treating atherosclerosis. Although C-C chemokine receptor 4 (CCR4) has been shown to mediate the recruitment of T cells to inflamed tissues, its role in atherosclerosis is unclear. Here, we show that genetic deletion of CCR4 in hypercholesterolemic mice accelerates the development of early atherosclerotic lesions characterized by an inflammatory plaque phenotype. This was associated with the augmentation of proinflammatory T helper type 1 (Th1) cell responses in peripheral lymphoid tissues, para-aortic lymph nodes, and atherosclerotic aorta. Mechanistically, CCR4 deficiency in Tregs impaired their suppressive function and tended to inhibit their migration to the atherosclerotic aorta, and subsequently augmented Th1 cell-mediated immune responses through defective regulation of dendritic cell function, which accelerated aortic inflammation and atherosclerotic lesion development. Thus, we revealed a previously unrecognized role for CCR4 in controlling the early stage of atherosclerosis via Treg-dependent regulation of proinflammatory T cell responses. Our data suggest that CCR4 is an important negative regulator of atherosclerosis.

Acute myocardial infarction (MI) is a leading cause of morbidity and mortality, and therapeutic options remain limited. Endogenously generated nitric oxide (NO) is highly cardioprotective, but protection is not replicated by nitroso-vasodilators (e.g., nitrates, nitroprusside) used in clinical practice, highlighting specificity in NO-based signaling and untapped therapeutic potential. Signaling by NO is mediated largely by S-nitrosylation, entailing specific enzymes that form and degrade S-nitrosothiols in proteins (SNO-proteins), termed nitrosylases and denitrosylases, respectively. SNO-CoA Reductase 2 (SCoR2; product of the Akr1a1 gene) is a recently discovered protein denitrosylase. Genetic variants in SCoR2 have been associated with cardiovascular disease, but its function is unknown. Here we show that mice lacking SCoR2 exhibit robust protection in an animal model of MI. SCoR2 regulates ketolytic energy availability, antioxidant levels and polyol homeostasis via S-nitrosylation of key metabolic effectors. Human cardiomyopathy shows reduced SCoR2 expression and an S-nitrosylation signature of metabolic reprogramming, mirroring SCoR2-/- mice. Deletion of SCoR2 thus coordinately reprograms multiple metabolic pathways—ketone body utilization, glycolysis, pentose phosphate shunt and polyol metabolism— to limit infarct size, establishing SCoR2 as a novel regulator in the injured myocardium and a potential drug target.Mice lacking the denitrosylase enzyme SCoR2/AKR1A1 demonstrate robust cardioprotection resulting from reprogramming of multiple metabolic pathways, revealing widespread, coordinated metabolic regulation by SCoR2.