Atrial natriuretic peptide (ANP) is a well-established biomarker of cardiac disease; however, its clinical utility in cynomolgus monkeys is limited by its short half-life and the requirement for immediate proteolysis inhibition. This study validated a human NT-proANP assay in cynomolgus monkeys to facilitate the use of archived samples for cardiac assessment. We performed in silico amino acid sequence homology analysis and validated the assay using monkey serum and plasma. Stability under various storage conditions was also evaluated. The amino acid sequence of NT-proANP showed high conservation between humans and cynomolgus monkeys. The human NT-proANP assay demonstrated good cross-reactivity and a significant positive correlation with mature ANP levels (R[2]=0.60). Furthermore, NT-proANP remained detectable for up to 6 h post-collection at room temperature, although a gradual decline was observed. These findings indicate that NT-proANP is a highly useful surrogate marker for ANP in cynomolgus monkeys. This validation expands the potential for retrospective analysis using stored samples, thereby enhancing the value of non-human primate models in cardiovascular research.

Cordycepin exhibits anti-cancer properties; however, its exact mechanism of action in liver cancer remains to be fully elucidated. In this study, we investigated the effects of cordycepin on liver cancer. Our findings demonstrated that cordycepin effectively suppressed tumor growth in liver cancer models and induced apoptosis in HepG2 cells by upregulating caspase-3 expression and decreasing mitochondrial membrane potential. These effects were primarily associated with the inhibition total mRNA and protein expression of the YAP/TAZ, as well as the increased expression of p21 and p53. Moreover, cordycepin significantly reduced the metastatic and invasive potential of HepG2 cells and downregulated the expression of MMP-2 and MMP-9. These results suggest that YAP plays a crucial role in cordycepin-mediated suppression of HepG2 cell proliferation. In conclusion, our study indicates that cordycepin exerts its anti-tumorigenic effects by modulating cellular proliferation through the downregulation of the YAP/TAZ pathway.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the degeneration of upper and lower motor neurons, resulting in progressive paralysis and death within a few years of symptom onset. Although current treatments modestly slow the disease progression, effective disease-modifying and curative therapies remain an urgent unmet need. SOD1 mutations are one of the major genetic causes of familial ALS. The p.Leu127Ser (L126S) and p.Gly94Ser (G93S) variants are clinically relevant pathogenic variants for which appropriate animal models are needed for preclinical evaluation of gene-editing therapies. However, most existing SOD1 models rely on high copy overexpression of mutant SOD1. Therefore, animal models carrying a single copy mutant human SOD1 allele are required for evaluating the in vivo efficacy of genome editing therapies. Here, we used CRISPR/Cas9-mediated homology-directed repair to generate a knock-in mouse line at the Gt(ROSA)26Sor (Rosa26) locus carrying a single-copy, 11-kb human SOD1 genomic fragment, including all exons and introns, with the L126S mutation. The Rosa26-hSOD1[L126S] mice did not develop ALS-like phenotypes during the limited observation period. However, they faithfully retained a single-copy mutant human SOD1 genomic allele, providing a valuable preclinical platform for evaluating genome-editing therapies. We also generated Rosa26-hSOD1[G93S] mice carrying the SOD1 G93S mutation with comparable efficiency. Together, these mutant human SOD1 knock-in mouse lines provide a versatile and clinically relevant platform for the preclinical evaluation of genome-editing therapies targeting heterozygous SOD1 mutations.

This study investigated whether gasdermin E (GSDME) deficiency attenuates caerulein/lipopolysaccharide (LPS)-induced severe acute pancreatitis (SAP) in mice. Wild-type (WT) and Gsdme-knockout (KO) mice were assigned to PBS or caerulein/LPS groups. SAP was induced by repeated intraperitoneal caerulein injections for 2 consecutive days followed by a single LPS injection. Pancreatic injury was assessed by body weight change, pancreas-to-body weight ratio, serum amylase and lipase, and histopathology. Inflammatory responses were evaluated by serum cytokines, serum and pancreatic IL-33, HMGB1 subcellular distribution, and MPO/F4/80 immunohistochemistry. Cell death and gasdermin pathway activation were assessed by TUNEL staining and Western blotting. Compared with WT mice subjected to caerulein/LPS challenge, Gsdme-deficient mice showed less body weight loss, lower histopathological scores, and attenuated systemic inflammatory responses. Serum and pancreatic IL-33 levels, HMGB1 nuclear-to-cytoplasmic translocation, and circulating HMGB1 were also reduced in KO mice. Consistently, pancreatic MPO- and F4/80-positive inflammatory cell infiltration, TUNEL positivity, and the cleaved caspase-3/total caspase-3 ratio were reduced in KO mice. Cleaved and full-length GSDME were absent in KO pancreatic tissues, whereas caerulein/LPS treatment increased cleaved GSDME levels in WT mice. The cleaved caspase-1/total caspase-1 ratio and cleaved GSDMD expression increased after caerulein/LPS challenge in both genotypes, but neither was further elevated in KO mice, indicating no detectable compensatory upregulation of the canonical caspase-1/GSDMD pathway at the examined time point. Collectively, Gsdme deficiency alleviated caerulein/LPS-induced pancreatic injury and systemic inflammation, indicating that the caspase-3/GSDME axis contributes importantly, but not exclusively, to experimental SAP.

Reliable individual identification is essential for long-term tracking and reproducible laboratory animal studies. In amphibians, invasive marking methods like tags and dye injections can cause welfare concerns and may be unreliable because of tag loss or migration. We developed and evaluated a non-invasive identification system for 25 adult Pleurodeles waltl using smartphone-captured images and the pre-trained convolutional neural network EfficientNetV2. To determine the most informative imaging region, separate models were trained and tested using head and whole-body images. The head-image model achieved 95.3% accuracy on the independent test dataset (macro F1-score = 0.946; Cohen's kappa = 0.951), markedly outperforming the whole-body model (56.6% accuracy). Grad-CAM visualization showed that the model primarily focused on dorsal head spot patterns, indicating that these markings are more informative for individual recognition than whole-body patterns. Because this method requires only a smartphone and a trained model, it can be implemented without specialized marking equipment. This approach enables accurate individual identification, while avoiding invasive marking and therefore supports both animal welfare and research reproducibility. It may provide a practical basis for standardizing individual identification in future amphibian research.

The peroneal muscular atrophy (pma) mouse shows congenital clubfoot due to atrophy of the peroneal muscles caused by lack of peroneal nerve innervation. Although it was reported that the peroneal nerve was absent in pma embryos, specific analysis of motor axon development in pma embryos has not been performed. Here, we generated a transgenic pma mouse that expresses enhanced green fluorescent protein (EGFP) in the spinal motor neurons, and examined the motor nerve trajectory specifically using wholemount preparations and three-dimensional reconstruction of EGFP-immunostained fibers. At embryonic day 11.5, when the sciatic nerve began to grow out of the spinal cord, no apparent differences were observed between the control and pma mice. From embryonic days 12.0 to 13.5, in the control mouse, the peroneal and tibial nerves extended into the hindlimb bud, whereas in the pma mouse, the tibial nerve formed normally but the peroneal nerve was never observed in its proper position. Moreover, an abnormal EGFP-positive axon bundle was observed near the gluteal nerve in the hip region. These results indicate that the pma mouse has axon guidance defects in the spinal motor neurons in addition to lack of the peroneal nerve.

Spermatogenesis is a highly ordered developmental process that occurs in the seminiferous tubules and involves mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and post-meiotic spermiogenesis, ultimately producing mature spermatozoa. Defects at any stage of this process can lead to male infertility. Large-scale transcriptomic and proteomic studies have identified thousands of testis-enriched genes in humans and mice, supporting the use of mouse models to uncover genetic regulators of male fertility. In this study, we focused on transmembrane channel-like protein 7 (TMC7), which is highly expressed in the testis. To investigate its physiological function, we generated a Tmc7 knockout (KO) mouse line using the CRISPR/Cas9 system. Tmc7 KO male mice were sterile, and no spermatozoa were observed in the epididymis. Instead, multinucleated giant cells containing multiple elongating spermatids were detected in the lumens of seminiferous tubules. We found that these abnormalities were associated with defective intercellular bridge (ICB) stabilization. Transmission electron microscopy further revealed that spermatid nuclei passed through gaps within the ICBs, leading to the formation of multinucleated giant cells. Given the Golgi localization of TMC7, ICB destabilization may occur secondarily due to blood-testis barrier disruption caused by alterations in the testicular microenvironment. Importantly, ectopic expression of TMC7 in Tmc7 KO mice rescued the defective spermiogenesis phenotype. Taken together, these findings demonstrate that TMC7 plays a critical role in spermiogenesis and indirectly contributes to the maintenance of ICB integrity.

NC/Jic mice infected with the rodent malaria parasite Plasmodium chabaudi AS (P. chabaudi AS) develop membranoproliferative glomerulonephritis (MPGN), a Type III hypersensitivity reaction. MRL/MpJ-lpr/lpr (MRL/lpr) mice, a murine model of systemic lupus erythematosus, also develop nephropathy, which is classified as a Type III hypersensitivity reaction. This nephropathy in MRL/lpr mice involves cluster of differentiation 72 (CD72), a negative regulator of B cell activation. NC/Jic mice harbored the same Cd72[c] haplotype as MRL/lpr mice. This haplotype is characterized by a seven amino acid deletion and 13 amino acid substitutions in the C-type lectin-like domain (CTLD) encoded by exon 8. In the present study, we investigated the contribution of the Cd72[c] haplotype to the development of MPGN in NC/Jic mice. NC/Jic-Cd72 knock-in (NC-Cd72 KI) mice, in which exon 8 of NC/Jic mice was replaced with normal exon 8 derived from the Cd72[b] haplotype, developed MPGN to a degree comparable to that in NC/Jic mice. In contrast, NC/Jic mice lacking exon 8 of Cd72 (NC-Cd72 Δex8) exhibited significantly attenuated MPGN severity. These results suggest that exon 8 of Cd72 is involved in the development of malaria-induced MPGN in NC/Jic mice, whereas the Cd72[c] haplotype is not the primary cause.

The microbiological quality of laboratory animals evolves along with changes in the research environment. To identify recent trends in modern Japanese research facilities, this study analyzed longitudinal contamination data for 16 major pathogens (including Helicobacter hepaticus, Pseudomonas aeruginosa, Staphylococcus aureus, Mycoplasma pulmonis, Mouse hepatitis virus (MHV), Murine norovirus (MNV) and various protozoa) collected over a 14-year period from 2011 to 2024. Our analysis revealed a significant decline in the testing request-level positivity rates (the number of positive testing requests per total requests) for most pathogens, including Helicobacter hepaticus, Pasteurella pneumotropica, Staphylococcus aureus, MHV, MNV and several intestinal protozoa and pinworms. Notably, the highest positivity rate, recorded for Staphylococcus aureus, decreased from 21.45% in 2011 to 13.65% in 2024. Similarly, the rate for Entamoeba dropped from 9.75% to 5.76%. Spironucleus muris, which maintained a low prevalence, showed a slight increase from 0.05% to 0.09% during the same period. These results demonstrate that the overall microbiological quality of mice from academic institutions has improved significantly over the past 14 years. While this progress reflects the success of modern bio-exclusion strategies, the data indicate that certain microorganisms persist at low levels and are not easily eradicated. These findings suggest that maintaining this high standard of quality requires continuous vigilance and the further refinement of quality control protocols to safeguard the integrity and reproducibility of biomedical research.

Adoptive transfer of CD45RB[high] T cells into immunodeficient mice is a well-established model for inducing colitis. Here, we report for the first time that a subset of these mice unexpectedly developed paralysis, a phenotype resembling experimental autoimmune encephalomyelitis (EAE). Paralytic mice exhibit the hallmark features of central nervous system (CNS) pathology, including spinal cord demyelination and immune cell infiltration, with prominent involvement of ex-Th17 (also called Th1-like) cells. The presence of ex-Th17 cells in the CNS of the affected mice suggests their contributory role in the development of paralysis. Notably, ex-Th17 cells have also been implicated in the pathogenesis of T cell transfer-induced colitis. Given the shared involvement of ex-Th17 cells in both the colitis model and the EAE-like CNS autoimmune model, we conducted comparative single-cell RNA sequencing analyses of ex-Th17 populations from colonic samples of patients with Crohn's disease and cerebrospinal fluid samples of patients with multiple sclerosis. These analyses revealed a convergent upregulation of the IFN-γ signaling pathway in both disease contexts. Collectively, our findings establish this model as a valuable platform for investigating how identical immune challenges can lead to tissue-specific autoimmune pathology and highlight ex-Th17 cells as a potential mechanistic link between systemic and CNS autoimmunity.

The equine herpes virus 2, E8 protein is a member of the viral FLIP family, and as such, it is a potent inhibitor of death receptor-induced apoptosis in cultured cells. To extend our study of the effects of E8 to animals, we generated a mouse model in which the progeny of a cross between two transgenic mice conditionally express E8 under the control of the collagen type I α2 chain (Col1α2) promoter, allowing us to monitor and characterize the effects of E8 expression in the mesenchymal cell lineage. We observed growth defects associated with irregular bone formation during development. In addition, adult animals exhibited both lipoatrophy-like and muscular atrophy-like symptoms. These abnormal phenotypes likely arise from incomplete differentiation of mesenchymal stem cells (MSCs). To examine this hypothesis in more detail, we expressed E8 in the mouse MSC line C3H10T1/2 and performed a microarray analysis. Factors such as Nov/CCN3, STEAP4, and Ankrd1/CARP, which are involved in differentiation from MSCs to osteoblasts, adipocytes and myoblasts were affected. Taken together, our results demonstrate that the constitutive expression of herpesvirus gene products in the mesenchymal progenitors affects differentiation into multiple cell lineages.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder influenced by genetic, environmental, and immunological factors, and its prevalence shows notable sex‑associated differences. KFRS4 rats spontaneously develop AD‑like dermatitis with higher severity in females, suggesting a role for sex hormones in disease modulation. This study investigated the contribution of gonadal hormones to dermatitis development in KFRS4 rats by examining the effects of gonadectomy and topical testosterone treatment. Ovariectomy did not alter dermatitis onset or severity in female rats, whereas orchiectomy markedly exacerbated dermatitis in males. Orchiectomized males developed severe lesions characterized by crusting and erosion, and exhibited significantly higher total skin lesion severity scores (TSLSS) compared with sham controls, indicating a protective role of testicular factors. To further evaluate testosterone's contribution, we applied testosterone ointment to ovariectomized females and orchiectomized males. Testosterone treatment significantly reduced TSLSS in both sexes and prevented lesion formation in the treated dorsal neck region. Histological analysis revealed increased sebaceous gland number and epidermal thickening in testosterone‑treated skin, accompanied by robust induction of androgen receptor (AR)‑positive cells within sebaceous glands and across epidermal layers. In contrast, dermatitis‑affected skin lacked AR‑positive cells, likely due to architectural disruption. In addition, testosterone treatment significantly increased the expression of filaggrin and decreased the expression of Th2 (Il4 and Il5) and Th17 (Il17a) cytokine genes. Together, these findings suggest that testosterone ameliorates dermatitis by enhancing skin barrier function and modulating type 2 and Th17 immune responses. These findings identify testosterone as a key regulator of dermatitis in KFRS4 rats and highlight androgen-mediated barrier enhancement as a potential therapeutic strategy for AD.