Age-related macular degeneration (AMD) is a progressive retinal disorder characterised by oxidative stress and inflammation. Although pyrroloquinoline quinone (PQQ) has been reported to exert neuroprotective effects, its specific efficacy in in vivo models of AMD pathophysiology has not yet been elucidated. In this study, we evaluated the protective effects of PQQ against all-trans-retinal (ATR)-induced cytotoxicity in ARPE-19 cells and light-induced photoreceptor degeneration in rats. Pretreatment of ARPE-19 cells with PQQ dose-dependently mitigated ATR-induced cytotoxicity. In the in vivo model, rats received a single intraperitoneal injection of PQQ (2 or 5 mg/kg) 1 h prior to 1000-lux light exposure. Retinal function and morphology were evaluated by electroretinography and haematoxylin-eosin staining, respectively. The 5 mg/kg PQQ group retained significantly greater retinal function than the vehicle group at 3 days postexposure and demonstrated significant preservation of the outer nuclear layer at 7 days postexposure, indicating the suppression of photoreceptor cell death. Western blot analysis detected the dose-dependent suppression of light-induced c-Fos upregulation following PQQ treatment. These findings suggest that the protective effect of PQQ against phototoxic damage is associated with the suppression of c-Fos signalling, thus lending support to the further investigation of PQQ as a potential therapeutic agent for AMD.

Adipose tissue plays a critical role in determining the characteristic palatability of Japanese Black cattle beef, including its distinctive aroma, tenderness, and flavor. These quality traits are closely associated with adipogenic differentiation and lipid metabolism; however, the underlying molecular mechanisms remain poorly understood. Adipose-derived stromal cells (ASCs) residing within adipose tissue possess the capacity to differentiate into adipocytes and other mesenchymal lineages and are, therefore, considered valuable experimental resources for adipose tissue research. Nevertheless, primary cells are subject to several limitations, including a restricted proliferative lifespan caused by culture-induced stress and reduced experimental reproducibility due to inter-individual variability, which hamper their use in long-term in vitro analyses. In this study, we immortalized ASCs derived from Japanese Black cattle using the K4DT method, which involves the introduction of the R24C mutant form of cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomerase reverse transcriptase. The established bovine ASCs (bASCs) [bASCs-K4DT] exhibited a stable proliferative capacity during long-term culture while retaining their ability to undergo adipogenic differentiation. These immortalized bASCs may serve as a useful in vitro model for elucidating the molecular mechanisms underlying adipocyte differentiation and formation of breed-specific aroma and flavor characteristics.

Advancing reproductive technologies in livestock is essential to improve both productivity and genetic potential of cattle. Despite this importance, application of reproductive biotechnologies in cattle breeding remains limited. Bovine granulosa cells (bGCs), which are key components of the ovarian follicle, are critical in female reproduction as they produce steroid hormones and growth factors necessary for oocyte development. However, primary bGCs exhibit restricted proliferative capacity in vitro, limiting their utility in large-scale studies on mechanisms related to follicular development. To address this limitation, we attempted to immortalize bGCs by co-expressing human mutant cyclin-dependent kinase 4 (CDK4R24C), cyclin D1, and telomerase reverse transcriptase (TERT) using lentiviral vectors. The resulting immortalized cells (bGCs-K4DT) displayed extended proliferative lifespans, surpassing 100 population doublings without exhibiting signs of senescence. The transduced cells demonstrated a more active cell cycle profile and higher telomerase activity relative to parental bGCs. Importantly, they retained the bGC-specific marker, aromatase, albeit at reduced expression levels. This immortalized bGC offers a promising model for investigating the role of bioactive components of platelet-rich plasma (PRP) in follicular activation and growth, thereby supporting innovations in livestock reproductive technologies.

Direct reprogramming, involving overexpression of transcription factors in combination with specific small-molecule treatments to induce cell fate conversion, represents a promising strategy for regenerative medicine and disease modeling. Monocistronic expression of four transcription factors (CRX, NEUROD, RAX, and OTX2) has been demonstrated to induce the expression of phototransduction-associated genes and confer light responsiveness in fibroblasts. In contrast, polycistronic expression of the same four factors resulted in the upregulation of only two cone-specific genes, indicating that optimization of reprogramming conditions is required to achieve complete photoreceptor differentiation. In this study, we focused on Müller glial cells, which exhibit regenerative potential in the retina of lower vertebrates, and explored their reprogramming into photoreceptor-like cells. We thus combined polycistronic expression of the aforementioned transcription factors and a pharmacological reprogramming approach utilizing a defined cocktail of small molecules. Transcriptomic analysis revealed that transcription factor introduction alone led to the upregulation of the genes related to neuronal identity and extracellular matrix components. Notably, the combination of transcription factor expression and chemical treatment induced photoreceptor-specific gene expression. However, no genes associated with phototransduction were detected, suggesting that further refinement is required to promote full differentiation into functionally mature light-responsive photoreceptors. Overall, our findings demonstrate that immortalized Müller glia can partially activate neuronal and photoreceptor genes through reprogramming, suggesting their potential for scalable drug screening and disease modeling. Clinical trial number: not applicable.

Anion channelrhodopsins (ACRs) are widely used for optical silencing of neurons. Photocurrents and kinetics are crucial for the precise control of cell hyperpolarization. In this study, we investigated the roles of amino acid residues in each transmembrane domain of the Guillardia theta-derived anion channelrhodopsins, GtACR1 and GtACR2, to develop high-performance ACRs. Several chimaera ACRs were generated by replacing the transmembrane (TM) domains of GtACR1 with homologous counterparts of GtACR2, and photocurrents in HEK cells transduced with each variant were recorded by patch-clamp. The relationship between photocurrent and protein localisation was determined. Chimaeras of TM4, TM5, and TM6 in GtACR1 replaced with the homologous counterparts of GtACR2 showed wavelength changes and fast kinetics(τon/off). V109 of TM3, the putative ion tunnel with I170, one of the key interactions for the peak photocurrent. Molecular dynamics simulations using data from the point mutation analysis in I170 showed that the distance from V109 to I170 correlated negatively with the peak photocurrents. The relationship between τ-off and the distance from E68 to N239, which formed the bottleneck of the ion tunnel, was positively correlated. These findings provide useful information for the development of ACRs with higher light sensitivities and faster kinetics(τon/off).

Background: The principal glial cells of the retina, Müller glia, play a central role in retinal regeneration in teleost fish and have recently attracted attention as potential sources of neuronal regeneration in mammals. Objectives: In this study, we examined whether SV40-immortalized rat Müller glia could be directed toward neuronal differentiation using a non-genetic approach with defined culture conditions. Methods: Comprehensive transcriptomic profiling by RNA sequencing indicated that changes in culture medium alone could induce transcriptional reprogramming toward a neuronal lineage. Results: Specifically, expression of Müller glia-related genes decreased, while a subset of photoreceptor-related transcription factors and specific genes showed altered expression, suggesting early-stage induction toward a photoreceptor-like fate. This finding suggests that even immortalized cells may exhibit activation of neuronal genes through non-genetic culture interventions. Gene set enrichment analysis further revealed upregulation of pathways related to the synaptic vesicle cycle, metabolic activation, oxidative stress defense, and lysosomal function, consistent with initiation of neuronal differentiation. Conversely, pathways associated with cell cycle regulation and stemness signaling were downregulated, reflecting a transition from a proliferative to a differentiation-prone state. Collectively, these results provide preliminary molecular markers for early neuronal induction and potential targets for chemical screening. Conclusions: Importantly, this strategy enables neuronal-like differentiation of Müller glia without genetic manipulation, offering a safe and cost-effective platform. Overall, our findings may support the development of in vitro models for retinal neuroregeneration and facilitate research toward regenerative therapies for retinal disorders.

Wasabi is a traditional food in Asian area. Wasabi leaf extract derived compounds such as 6-methylsulfinylhexyl isothiocyanate (6-MSITC) have been reported to exert anti-proliferative effects on cancer cells. The antibacterial action of wasabi leaf extract has been reported previously. However, the detailed biological effects of wasabi leaf extract are not fully understood. To get a clue for the understanding, we used next-generation sequencing to identify the upregulated or downregulated genes in immortalized human dermal papilla cells. Differentially expressed genes were identified in human papilla cells in the presence or absence of wasabi leaf extract. The effects of 6-MSITC and isosaponarin were evaluated. We found that wasabi leaf extracts upregulated or downregulated the expression of cytokine-related genes. The identified expression changes will contribute to our understanding of the biological effects of wasabi. ### Competing Interest Statement This study was conducted as a part of a collaborative project between Iwate University and Kinjirushi Co., Ltd. This study was supported in part by funding from the Kinjirushi Co., Ltd. the Kinjirushi Co., Ltd., NA

Calpains cleave proteins in a calcium concentration-dependent manner, modulating their intracellular functions. Calpain-1, a member of the calpain family, is localized in the cytosol and mitochondria. Mitochondrial calpain-1 induces mitochondrial dysfunction and apoptosis by cleaving its substrate. Thus, identifying the substrate of calpain-1 is essential to understand its function. However, little is known about the substrates of mitochondrial calpain-1. To address this issue, we screened mitochondrial proteins using bioinformatics approaches and two-dimensional gel electrophoresis. We identified ATP5B as a potential substrate of mitochondrial calpain-1. Calpeptin, a pan-calpain inhibitor, and Tat-μCL, a mitochondrial calpain-1 specific inhibitor, prevented the truncation of ATP5B during in vitro Ca2+ incubation. Using recombinant human calpain-1 and ATP5B proteins, we demonstrated that calpain-1 directly cleaved ATP5B, generating a fragment of ATP5B. Based on the predicted cleavage sites in ATP5B, this cleavage may disrupt its interaction with ATP5A1, leading to mitochondrial dysfunction in ATP production. This study identified ATP5B as a novel substrate of mitochondrial calpain-1. The results provide new insights into mitochondrial dysfunction.

Testosterone signaling mediates diseases such as androgenetic alopecia and prostate cancer and is controlled by the activation of the androgen receptor (AR) and nuclear translocation of the ligand-receptor complex. This study established an immortalized dermal papilla cell line that stably expresses the AR labeled with a monomeric green fluorescence marker. The cells expressed the histone H2B protein as visualized using a red fluorescence marker, enabling the Detection of nuclear translocation under live cell conditions using image analysis. The AR was observed to be translocated from the cytoplasm to the nucleus of cells after stimulation with dihydrotestosterone (DHT). The signal intensity of the nuclear/cytoplasm ratio was analyzed using automatic image analysis and a newly developed algorithm. The quantitation method to detect nuclear translocation revealed that the AR nuclear signal plateaued approximately 20min after DHT exposure. Our developed method has the potential to save human labor by the automatic process of the image.

Wasabi is a traditional Asian food, and it has been reported that Wasabi leaf extract derived compounds, such as 6-methylsulfinylhexyl isothiocyanate (6-MSITC), exert anti-proliferative effects on cancer cells. In addition, although the antibacterial action of wasabi leaf extract has been reported, the detailed biological effects of wasabi leaf extract are not fully understood. To obtain further associated information, we used next-generation sequencing to identify upregulated or downregulated genes in immortalized human dermal papilla cells, and differentially expressed genes in human papilla cells were identified in the presence or absence of wasabi leaf extract. The effects of 6-MSITC and isosaponarin were also evaluated. The results indicated that wasabi leaf extracts upregulated or downregulated the expression of cytokine-related genes, such as GDF15, BMP6, CXCL12. The expression changes of GDF15, BMP6 were reproduced by real time PCR, and the results will contribute to our understanding of the biological effects of wasabi.

The Egyptian Rousettus bat (Rousettus aegyptiacus) is a common fruit bat species that is distributed mainly in Africa and the Middle East. Bats serve as reservoir hosts for numerous pathogens. Human activities, such as hunting bats for food, managing vermin, and causing habitat loss, elevate the likelihood of transmission of bat pathogens to humans and other animals. Consequently, bat cell lines play a crucial role as research materials for investigating viral pathogens. However, the inherent limitation of finite cell division in primary cells necessitates the use of immortalized cells derived from various bat tissues. Herein, we successfully established six fibroblast cell lines derived from an infant bat heart and lungs and an elderly bat heart. Three of the six cell lines, called K4DT cells, were transduced by a combination of cell cycle regulators, mutant cyclin‐dependent kinase 4, cyclin D1, and human telomerase reverse transcriptase. The other three cell lines, named SV40 cells, were transfected with simian virus 40 large T antigen. Transgene protein expression was detected in the transduced cells. All three K4DT cell lines and one lung‐derived SV40 cell line were virtually immortalized and nearly maintained the normal diploid karyotypes. However, the two other heart‐derived SV40 cell lines had aberrant karyotypes and the young bat‐derived cell line stopped proliferating at approximately 40 population doublings. These bat cell lines are valuable for studying pathogen genomics and biology.

Functional maturation of the visual cortex is induced by visual experiences during critical periods. Blind animals and humans exhibit improved auditory abilities after losing their vision. Here we investigated the response of the visual cortex to white noise stimuli during the progression of photoreceptor degeneration in a rat model of blindness (Royal College of Surgeons [RCS] (rdy/rdy) rats). Optical coherence tomography of RCS (+/+) rats with normal visual function revealed normal photoreceptor cells, whereas 3-month-old RCS (rdy/rdy) rats demonstrated photoreceptor cell degeneration. Visual cortex responses (VCRs) to a single flash stimulus were negligible in 3-month-old photoreceptor-degenerated rats. However, VCRs with white noise stimuli were significantly increased in blind versus RCS rats (+/+). Slight changes in the intrinsic optical signals of the control rats were observed on the ventral side of the visual cortex. In contrast, responses were markedly increased throughout the visual cortex of RCS (rdy/rdy) rats. These results indicate that the visual cortex rapidly acquires auditory system function over the first 3 months of life and that the entire visual cortex, rather than just the portion close to the auditory cortex, responds to white noise.

Abstract Alterations in the experience-dependent and autonomous elaboration of neural circuits are assumed to underlie autism spectrum disorder (ASD), though it is unclear what synaptic traits are responsible. Here, utilizing a valproic acid–induced ASD marmoset model, which shares common molecular features with idiopathic ASD, we investigate changes in the structural dynamics of tuft dendrites of upper-layer pyramidal neurons and adjacent axons in the dorsomedial prefrontal cortex through two-photon microscopy. In model marmosets, dendritic spine turnover is upregulated, and spines are generated in clusters and survived more often than in control marmosets. Presynaptic boutons in local axons, but not in commissural long-range axons, demonstrate hyperdynamic turnover in model marmosets, suggesting alterations in projection-specific plasticity. Intriguingly, nasal oxytocin administration attenuates clustered spine emergence in model marmosets. Enhanced clustered spine generation, possibly unique to certain presynaptic partners, may be associated with ASD and be a potential therapeutic target.

Calpains are calcium-dependent cysteine proteases activated by intracellular Ca2+. Although calpains mainly exist in the cytosol, calpain-13 is present in the mitochondria in mouse brains; however, the enzymatic properties and physiological functions of calpain-13 remain unknown. Hence, in this study, we predicted and evaluated the enzymatic properties of calpain-13. Based on our bioinformatic approaches, calpain-13 possessed a catalytic triad and EF-hand domain, similar to calpain-1, a well-studied calpain. Therefore, we hypothesized that calpain-13 had calpain-1-like enzymatic properties; however, calpain-13 was not proteolyzed in C57BL/6J mouse brains. Subsequently, cerebral ischemia/reperfusion (I/R) injury caused proteolysis of mitochondrial calpain-13. Thus, our study showed that mitochondrial calpain-13 was proteolyzed in the mitochondria of the I/R injured mouse brain. This finding could be valuable in further research elucidating the involvement of calpain-13 in cell survival or death in brain diseases, such as cerebral infarction.

Background: Ischemia and reperfusion (I/R) injury exacerbate the prognosis of ischemic diseases. The cause of this exacerbation is partly a mitochondrial cell death pathway. Mitochondrial calpain-5 is proteolyzed/autolyzed under endoplasmic reticulum stress, resulting in inflammatory caspase-4 activation. However, the role of calpain5 in I/R injury remains unclear. We hypothesized that calpain-5 is involved in ischemic brain disease. Methods: Mitochondria from C57BL/6J mice were extracted via centrifugation with/without proteinase K treatment. The expression and proteolysis/autolysis of calpain-5 were determined using western blotting. The mouse and human brains with I/R injury were analyzed using hematoxylin and eosin staining and immunohistochemistry. HT22 cells were treated with tunicamycin and CAPN5 siRNA. Results: Calpain-5 was expressed in the mitochondria of mouse tissues. Mitochondrial calpain-5 in mouse brains was responsive to calcium earlier than cytosolic calpain-5 in vitro calcium assays and in vivo bilateral common carotid artery occlusion model mice. Immunohistochemistry revealed that neurons were positive for calpain-5 in the normal brains of mice and humans. The expression of calpain-5 was increased in reactive astrocytes at human infarction sites. The knockdown of calpain-5 suppressed of cleaved caspase-11. Conclusions: The neurons of human and mouse brains express calpain-5, which is proteolyzed/autolyzed in the mitochondria in the early stage of I/R injury and upregulated in reactive astrocytes in the end-stage. General significance: Our results provide a comprehensive understanding of the mechanisms underlying I/R injury. Targeting the expression or activity of mitochondrial calpain-5 may suppress the inflammation during I/R injuries such as cerebrovascular diseases.

Sheep are important domestic animals for the production of wool and meat. Although numerous cultured cell lines from humans and mice have been established, the number of cell lines derived from sheep is limited. To overcome this issue, the efficient establishment of a sheep-derived cell line and its biological characterization is reported. Mutant cyclin-dependent kinase 4, cyclin D1, and telomerase reverse transcriptase were introduced into sheep muscle-derived cells in an attempt to immortalize primary cells using the K4DT method. Furthermore, the SV40 large T oncogene was introduced into the cells. The successful immortalization of sheep muscle-derived fibroblasts was shown using the K4DT method or SV40 large T antigen. Furthermore, the expression profile of established cells showed close biological characteristics of ear-derived fibroblasts. This study provides a useful cellular resource for veterinary medicine and cell biology.

Glycation, caused by reactive dicarbonyls, plays a role in various diseases by forming advanced glycation end products. In live cells, reactive dicarbonyls such as glyoxal (GO) and methylglyoxal (MGO) are produced during cell metabolism, and these should be removed consistently. However, the dicarbonyl metabolic system in the mitochondria remains unclear. It has been speculated that the mammalian mitochondrial protein ES1 is a homolog of bacterial elbB possessing glyoxalase III (GLO3) activity. Therefore, in this study, to investigate ES1 functions and GLO3 activity, we generated ES1-knockout (KO) mice and recombinant mouse ES1 protein and investigated the biochemical and histological analyses. In the mitochondrial fraction obtained from ES1-KO mouse brains, the GO metabolism and cytochrome c oxidase activity were significantly lower than those in the mitochondrial fraction obtained from wildtype (WT) mouse brains. However, the morphological features of the mitochondria did not change noticeably in the ES1-KO mouse brains compared with those in the WT mouse brains. The mitochondrial proteome analysis showed that the MGO degradation III pathway and oxidative phosphorylation-related proteins were increased. These should be the response to the reduced GO metabolism caused by ES1 deletion to compensate for the dicarbonyl metabolism and damaged cytochrome c oxidase by elevated GO. Recombinant mouse ES1 protein exhibited catalytic activity of converting GO to glycolic acid. These results indicate that ES1 possesses GLO3 activity and modulates the metabolism of GO in the mitochondria. To our knowledge, this is the first study to show a novel metabolic pathway for reactive dicarbonyls in mitochondria.

Primary cultured cells cannot proliferate infinite. The overcoming of this limit can be classified as immortalization. Bypass of p16 senescence protein induces efficient immortalization various types of mammalians is previously reported. However, the Cetacea species is not known. Here, that common minke whale-derived cells can be immortalized with a combination of human genes, mutant cyclin-dependent kinase 4 (CDK4R24C ), cyclin D1, and Telomerase Reverse Transcriptase (TERT) is reported. These results indicate that the function of cell cycle regulators in premature senescence is evolutionarily conserved. This study describes the conserved roles of cell cycle regulators in the immortalization of cells from humans to Cetacea species. Furthermore, using RNA-seq based on next-generation sequencing, the gene expression profiles of immortalized cells are compared with parental cells as well as those immortalized with SV40 large T antigen, which is once a popular method for cellular immortalization. The profiling results show that newly established common minke-whale-derived immortaliozed cells have completely different profiles from SV40 cells. This result indicates that the expression of mutant CDK4, cyclin D1, and TERT enables to establish immortalized cell lines with different biological nature from SV40 expressing cells.

Electroretinograms (ERGs) are often used to evaluate retinal function. However, assessing local retinal function can be challenging; therefore, photopic and scotopic ERGs are used to record whole-retinal function. This study evaluated focal retinal function in rats exposed to continuous light using a multifocal ERG (mfERG) system. The rats were exposed to 1000 lux of fluorescent light for 24 h to induce photoreceptor degeneration. After light exposure, the rats were reared under cyclic light conditions (12 h: 5 lux, 12 h: dark). Photopic and multifocal ERGs and single-flash and multifocal visual evoked potentials (mfVEPs) were recorded 7 days after light exposure. Fourteen days following light exposure, paraffin-embedded sections were prepared from the eyes for histological evaluation. The ERG and VEP responses dramatically decreased after 24 h of light exposure, and retinal area-dependent decreases were observed in mfERGs and mfVEPs. Histological assessment revealed severe damage to the superior retina and less damage to the inferior retina. Considering the recorded visual angles of mfERGs and mfVEPs, the degenerated area shown on the histological examinations correlates well with the responses from multifocal recordings.