PROKR1-CREB-NR4A2 axis for oxidative muscle fiber specification and improvement of metabolic function.

Metabolic disorders are characterized by an imbalance in muscle fiber composition, and a potential therapeutic approach involves increasing the proportion of oxidative muscle fibers. Prokineticin receptor 1 (PROKR1) is a G protein-coupled receptor that plays a role in various metabolic functions, but its specific involvement in oxidative fiber specification is not fully understood. Here, we investigated the functions of PROKR1 in muscle development to address metabolic disorders and muscular diseases. A meta-analysis revealed that the activation of PROKR1 upregulated exercise-responsive genes, particularly nuclear receptor subfamily 4 group A member 2 (NR4A2). Further investigations using ChIP-PCR, luciferase assays, and pharmacological interventions demonstrated that PROKR1 signaling enhanced NR4A2 expression by Gs-mediated phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) in both mouse and human myotubes. Genetic and pharmacological interventions showed that the PROKR1-NR4A2 axis promotes the specification of oxidative muscle fibers in both myocytes by promoting mitochondrial biogenesis and metabolic function. Prokr1-deficient mice displayed unfavorable metabolic phenotypes, such as lower lean mass, enlarged muscle fibers, impaired glucose, and insulin tolerance. These mice also exhibited reduced energy expenditure and exercise performance. The deletion of Prokr1 resulted in decreased oxidative muscle fiber composition and reduced activity in the Prokr1-CREB-Nr4a2 pathway, which were restored by AAV-mediated Prokr1 rescue. In summary, our findings highlight the activation of the PROKR1-CREB-NR4A2 axis as a mechanism for increasing the oxidative muscle fiber composition, which positively impacts overall metabolic function. This study lays an important scientific foundation for the development of effective muscular-metabolic therapeutics with unique mechanisms of action.

Benzbromarone Induces Targeted Degradation of HSP47 Protein and Improves Hypertrophic Scar Formation.

Fibrotic diseases are characterized by the abnormal accumulation of collagen in the extracellular matrix, leading to the functional impairment of various organs. In the skin, excessive collagen deposition manifests as hypertrophic scars and keloids, placing a substantial burden on patients and the healthcare system worldwide. HSP47 is essential for proper collagen assembly and contributes to fibrosis. However, identifying clinically applicable HSP47 inhibitors has been a major pharmaceutical challenge. In this study, we identified benzbromarone (BBR) as an HSP47 inhibitor for hypertrophic scarring treatment. BBR inhibited collagen production and secretion in fibroblasts from patients with keloid by binding to HSP47 and inhibiting the interaction between HSP47 and collagen. Interestingly, BBR not only inhibits HSP47 but also acts as a molecular glue degrader that promotes its proteasome-dependent degradation. Through these molecular mechanisms, BBR effectively reduced hypertrophic scarring in mini pigs and rats with burns and/or excisional skin damage. Thus, these findings suggest that BBR can be used to clinically treat hypertrophic scars and, more generally, fibrotic diseases.

Tetrodotoxin and the Geographic Distribution of the Blue-Lined Octopus Hapalochlaena fasciata on the Korean Coast.

The genus Hapalochlaena, including the blue-lined octopus Hapalochlaena fasciata (H. fasciata), is highly toxic. Venomous, blue-lined octopuses were recently found in Korea, but their toxicity, toxin composition, and distribution remain largely unknown. Here we estimated the geographic distribution of the organisms along the Korean coast and clarified their toxicity. Tetrodotoxin (TTX) was present in all three specimens of H. fasciata examined, although the toxicity varied largely between individuals. The mean TTX concentration in the whole body of the three specimens was 6.5 ± 2.2 µg/g (range 3.3-8.5 µg/g). Among the body parts examined, the salivary glands exhibited the highest concentration (22.4 ± 9.7 µg/g). From 2012 to 2021, 26 individuals were obtained nearly every month from different regions of the Korean coast. A non-fatal case of a blue-lined octopus bite was reported along the Korean coast in June 2015. This is the first report on the widespread distribution of blue-lined octopuses on the Korean coast and TTX detection. The widespread distribution of the TTX-bearing H. fasciata along the Korean coast within the temperate zone indicates that the species may soon become a serious health issue in Korea. The toxicity of this species is also a potentially significant human health risk.

PROKR1 delivery by cell-derived vesicles restores the myogenic potential of Prokr1-deficient C2C12 myoblasts.

Cell-derived vesicles (CDVs) have been investigated as an alternative to exosomes. Here, we generated CDVs from Prokineticin receptor 1 (PROKR1) overexpressing HEK293T cells using micro-extrusion. More than 60 billion PROKR1-enriched CDV (PROKR1Tg CDVs) particles with canonical exosome properties were recovered from 107 cells. With 25 µg/mL of PROKR1Tg CDVs, we observed delivery of PROKR1, significant reduction of apoptosis, and myotube formation in C2C12Prokr1-/- myoblasts that have lost their myogenic potential but underwent apoptosis following myogenic commitment. Expression levels of early and late myogenic marker genes and glucose uptake capacity were restored to equivalent levels with wild-type control. Furthermore, PROKR1Tg CDVs were accumulated in soleus muscle comparable to the liver without significant differences. Therefore, CDVs obtained from genetically engineered cells appear to be an effective method of PROKR1 protein delivery and offer promise as an alternative therapy for muscular dystrophy.

Dysregulation of the Acrosome Formation Network by 8-oxoguanine (8-oxoG) in Infertile Sperm: A Case Report with Advanced Techniques.

8-Hydroxyguanine (8-oxoG) is the most common oxidative DNA lesion and unrepaired 8-oxoG is associated with DNA fragmentation in sperm. However, the molecular effects of 8-oxoG on spermatogenesis are not entirely understood. Here, we identified one infertile bull (C14) due to asthenoteratozoospermia. We compared the global concentration of 8-oxoG by reverse-phase liquid chromatography/mass spectrometry (RP-LC/MS), the genomic distribution of 8-oxoG by next-generation sequencing (OG-seq), and the expression of sperm proteins by 2-dimensional polyacrylamide gel electrophoresis followed by peptide mass fingerprinting (2D-PAGE/PMF) in the sperm of C14 with those of a fertile bull (C13). We found that the average levels of 8-oxoG in C13 and C14 sperm were 0.027% and 0.044% of the total dG and it was significantly greater in infertile sperm DNA (p = 0.0028). Over 81% of the 8-oxoG loci were distributed around the transcription start site (TSS) and 165 genes harboring 8-oxoG were exclusive to infertile sperm. Functional enrichment and network analysis revealed that the Golgi apparatus was significantly enriched with the products from 8-oxoG genes of infertile sperm (q = 2.2 × 10-7). Proteomic analysis verified that acrosome-related proteins, including acrosin-binding protein (ACRBP), were downregulated in infertile sperm. These preliminary results suggest that 8-oxoG formation during spermatogenesis dysregulated the acrosome-related gene network, causing structural and functional defects of sperm and leading to infertility.

Cellhesion VP enhances the immunomodulating potential of human mesenchymal stem cell-derived extracellular vesicles.

Mesenchymal stem cell (MSC) transplantation is a promising therapy for regenerative medicine. However, MSCs grown under two-dimensional (2D) culture conditions differ significantly in cell shape from those in the body, with downregulated stemness genes and secretion of paracrine factors. Here, we evaluated the effect of 3D culture using Cellhesion VP, a water-insoluble material composed of chitin-based polysaccharide fibers, on the characteristics of human Wharton's jelly-derived MSCs (hMSCs). Cellhesion VP significantly increased cell proliferation after retrieval. Transcriptome analyses suggested that genes involved in cell stemness, migration ability, and extracellular vesicle (EV) production were enhanced by 3D culture. Subsequent biochemical analyses showed that the expression levels of stemness genes including OCT4, NANOG, and SSEA4 were upregulated and migration capacity was elevated in 3D-cultured hMSCs. In addition, EV production was significantly elevated in 3D cells, which contained a distinct protein profile from 2D cells. Gene and drug connectivity analyses revealed that the 2D and 3D EVs had similar functions as immunomodulators; however, 3D EVs had completely distinct therapeutic profiles for various infectious and metabolic diseases based on activation of disease-associated signaling pathways. Therefore, EVs from Cellhesion VP-primed hMSCs offer a new treatment for immune and metabolic diseases.

Prokineticin receptor 1 ameliorates insulin resistance in skeletal muscle.

Type 2 diabetes mellitus may result from insulin resistance in skeletal muscle. Prokineticin receptor 1 (Prokr1) improves metabolic phenotype in adipose tissue and the cardiovascular system; however, its effects on skeletal muscle have not been investigated. We investigated the Prokr1 signaling pathways and its metabolic function in murine myoblast, satellite cells, and their differentiated myotubes. We measured the expression levels of Prokr1 in the skeletal muscle of mice as well as human skeletal muscle cell-derived myotubes. Prokineticin 2 (PROK2), a ligand of PROKR1, induced calcium mobilization in a dose-dependent manner and altered the mRNA levels of 578 genes in PROKR1-overexpressed HEK293T cells. Functional enrichment of differentially expressed genes revealed that PROKR1 activated Gq-mediated PI3K/AKT and MAPK/ERK signaling pathways in skeletal muscle cells. Prokr1 significantly activated the PI3K/AKT signaling pathway in myotubes derived from C2C12 and satellite cells, regardless of the presence or absence of insulin. Prokr1 also promoted the translocation of glucose transporter 4 (GLUT4) into the plasma membrane. In palmitate-induced insulin-resistant myotubes, Prokr1 enhanced insulin-stimulated AKT phosphorylation, GLUT4 translocation, and glucose uptake. mRNA and protein levels of Prokr1 were significantly decreased in skeletal muscle and white adipose tissue of diet-induced obese mice, and the amount of PROKR1 protein was significantly decreased in human skeletal muscle cell-derived myotubes under insulin resistance conditions. Taken together, these results demonstrate that Prokr1 plays an important role in insulin sensitivity and is a potential therapeutic target to ameliorate insulin resistance in skeletal muscle.

Antibiotic resistance of Vibrio harveyi isolated from seawater in Korea.

Vibrio harveyi is an opportunistic human pathogen that may cause gastroenteritis, severe necrotizing soft-tissue infections, and primary septicemia, with a potentially high rate of lethality. In this study, we isolated and characterized V. harveyi from seawater collected from the West Sea in Korea, including sites located near shellfish farms. For the initial isolation of putative V. harveyi, isolates were incubated on thiosulfate citrate bile salt sucrose agar plates for 24h, followed by selection of greenish colonies. Gram-negative and oxidase-positive colonies were subsequently confirmed by biochemical assays and the API 20E kit test system. Species-specific 16S rRNA and hemolysin genes were used to design V. harveyi-specific PCR primers. From 840 seawater samples, a total of 2 strains of V. harveyi were isolated from shellfish farm seawater. The two isolates were subjected to profiling against 16 antibiotics and found to be resistant to cephalothin, vancomycin, ampicillin, cefepime, cefotetan, and streptomycin.