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DNA-damaging agents (DDAs) have long been used in cancer therapy. However, the precise mechanisms by which DDAs induce cell death are not fully understood and drug resistance remains a major clinical challenge. Schlafen 11 (SLFN11) was identified as the gene most strongly correlated with the sensitivity to DDAs based on mRNA expression levels. SLFN11 sensitizes cancer cells to DDAs by cleaving and downregulating tRNA Leu (TAA). Elucidating the detailed mechanism by which SLFN11 induces cell death is expected to provide insights into overcoming drug resistance. Here, we show that, upon administration of DDAs, SLFN11 cleaves tRNA Leu (TAA), leading to ER stress and subsequent cell death regulated by inositol-requiring enzyme 1 alpha (IRE1α). These responses were significantly alleviated by SLFN11 knockout or transfection of tRNA Leu (TAA). Our proteomic analysis suggests that tRNA Leu (TAA) influences proteins essential for maintaining proteostasis, especially those involved in ubiquitin-dependent proteolysis. Additionally, we identified the cleavage sites of tRNA Leu (TAA) generated by SLFN11 in cells, and revealed that tRNA fragments contribute to ER stress and cell death. These findings suggest that SLFN11 plays a crucial role in proteostasis by regulating tRNAs, and thus determines cell fate under DDA treatment. Consequently, targeting SLFN11-mediated tRNA regulation could offer a novel approach to improve cancer therapy., Competing Interests: CONFLICT OF INTEREST None declared.

Objectives: Unresectable canine hepatocellular carcinoma (HCC) has limited nonsurgical treatment options. Sorafenib is a targeted therapy for unresectable canine HCC. However, there are limited reports on the expression of target genes. Therefore, the efficacy of the targeted therapies for canine HCC remains unclear., Data Description: Liver specimens were obtained from 11 dogs with HCC and four dogs without HCC. We performed RNA seq using the mRNA extracted from the specimens. Differentially expressed genes (DEGs) between canine HCC and normal liver were explored based on previously reported molecular-targeted agents for human tumours. PARP3, DNMT1, FGF19, FGF23, and RET DEGs were upregulated, whereas KIT, FGFR2, and FGF21 DEGs were downregulated., Competing Interests: Declarations. Ethics approval and consent to participate: Informed consent for sample collection of HCC in this study was obtained from all clients at first examination of their dogs. The normal liver was a piece of liver tissue that had been collected and cryopreserved in another project that was properly conducted according to the Guidelines of Animal Care and Use in Osaka Metropolitan University. Consent for publication: Informed consent for publication of HCC in this study was obtained from all clients at first examination of their dogs. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Schlafen (SLFN) 11 enhances cellular sensitivity to various DNA-damaging anticancer agents. Among the human SLFNs (SLFN5/11/12/13/14), SLFN11 is unique in its drug sensitivity and ability to block replication under DNA damage. In biochemical analysis, SLFN11 binds single-stranded DNA (ssDNA), and this binding is enhanced by the dephosphorylation of SLFN11. In this study, human cell-based assays demonstrated that a point mutation at the ssDNA-binding site of SLFN11 or a constitutive phosphorylation mutant abolished SLFN11-dependent drug sensitivity. Additionally, we discovered that nuclear SLFN13 with a point mutation mimicking the DNA-binding site of SLFN11 was recruited to chromatin, blocked replication, and enhanced drug sensitivity. Through generating multiple mutants and structure analyses of SLFN11 and SLFN13, we identified protein phosphatase 2A as a binding partner of SLFN11 and the putative binding motif in SLFN11. These findings provide crucial insights into the unique characteristics of SLFN11, contributing to a better understanding of its mechanisms., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Although bone has an innate capacity for repair, clinical situations such as comminuted fracture, open fracture, or the surgical resection of bone tumors produce critical-sized bone defects that exceed the capacity and require external intervention. Initiating endochondral ossification (EO) by the implantation of a cartilaginous template into the bone defect is a relatively new approach to cure critical-sized bone defects. The combination of chondrogenically primed mesenchymal stromal/stem cells and artificial scaffolds has been the most extensively studied approach for inducing endochondral bone formation in bone defects. In this study, we prepared cartilage (human-induced pluripotent stem [hiPS]-Cart) from hiPS cells (hiPSCs) in a scaffoldless manner and implanted hiPS-Cart into 3.5 mm large defects created in the femurs of immunodeficient mice to examine the repair capacity. For the control, nothing was implanted into the defects. The implantation of hiPS-Cart significantly induced more new bone in the defect compared with the control. Culture periods for the chondrogenic differentiation of hiPSCs significantly affected the speed of bone induction, with less time resulting in faster bone formation. Histological analysis revealed that hiPS-Cart induced new bone formation in a manner resembling EO of the secondary ossification center, with the cartilage canal, which extended from the periphery to the center of hiPS-Cart, initially forming in unmineralized cartilage, followed by chondrocyte hypertrophy at the center. In the newly formed bone, the majority of osteocytes, osteoblasts, and adipocytes expressed human nuclear antigen (HNA), suggesting that these types of cells mainly derived from the perichondrium of hiPS-Cart. Osteoclasts and blood vessel cells did not express HNA and thus were mouse. Finally, integration between the newly formed bone and mouse femur was attained substantially. Although hiPS-Cart induced new bone that filled bone defects, the newly formed bone, which is a hybrid of human and mouse, had not remodeled to mature bone within the observation period of this study (28 weeks). Impact statement Although bone has an innate capacity for repair, critical-sized bone defects that exceed the capacity require external intervention. We prepared cartilage (human-induced pluripotent stem [hiPS]-Cart) from hiPS cells (hiPSCs) in a scaffoldless manner and examined whether implantation of hiPS-Cart heals critical-sized defects created in the femurs of immunodeficient mice. The implantation of hiPS-Cart induced new bone in the defect in a manner resembling endochondral bone formation of the secondary ossification center. Although hiPS-Cart induced new bone that filled bone defects, the newly formed bone, which is a hybrid of human and mouse, had not remodeled to mature bone within the observation period of this study (28 weeks).

Background: In dogs, hepatic lymphoma is characterized by neoplastic lymphocyte infiltration into the liver. Reports on the computed tomography (CT) findings of the liver for canine hepatic lymphoma are few, with only one study of multiple liver lesions type., Objectives: The purpose of this study was to retrospectively assess the CT findings of the liver in canine diffuse hepatic lymphoma., Methods: As control, five dogs without abnormalities in the liver were included. CT data were analysed, and the following were noted: presence of edge bluntness of the liver, presence of periportal collar sign, the liver size:body weight (BW) ratio and the mean attenuation of liver lesions on pre-contrast, arterial-phase, portal-phase and equilibrium-phase post-contrast images., Results: On CT examination, edge bluntness of the liver was significantly detected in lymphoma (4/5, 80%), as opposed to the control (0/5, 0%) (p = 0.048, φ = 0.82). The periportal collar sign was detected in lymphoma (3/5, 60%), as opposed to the control (0/5, 0%) (p = 0.17, φ = 0.65). The liver size:BW ratio of lymphoma cases was significantly higher compared to that of the control cases (p = 0.0002, r = 0.92). The mean Hounsfield unit of lymphoma cases in the pre-contrast, arterial-phase, portal-phase and equilibrium-phase images were significantly lower than in the control cases (p = 0.005, r = 0.81; p = 0.0003, r = 0.91; p = 0.01, r = 0.75 and p = 0.02, r = 0.71, respectively)., Conclusions: Hepatic lymphoma should be a differential for a blunted and enlarged liver with hypoattenuation on CT examination., (© 2021 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Objectives: Primary nasal tumours in cats are rare, with lymphoma being the most common feline nasal tumour, followed by adenocarcinoma. Although CT can reliably detect feline nasal tumours, there are no specific CT features that identify each tumour type. To our knowledge, there have been no reports describing MRI findings, including diffusion-weighted imaging (DWI), for nasal lymphomas and adenocarcinomas in cats. Therefore, this retrospective study aimed to evaluate the MRI findings of nasal lymphoma and adenocarcinoma, including qualitative and quantitative analysis of DWI., Methods: MRI examination was performed on seven cats with histologically confirmed lymphoma and on two with adenocarcinoma. The MRI protocol included T2-weighted imaging (T2WI), T1-weighted imaging (T1WI) and DWI. Apparent diffusion coefficient (ADC) values were measured using DWI. Contrast agent was not used in one cat with lymphoma., Results: Of the cats with lymphoma, three (43%) were iso- and hyperintense on T2WI, seven (100%) were isointense on T1WI, five (83%) exhibited mild heterogeneous enhancement, including a prominent region of non-enhancement on post-contrast T1WI, and seven (100%) cats exhibited hyperintensity on DWI. The median ADC values were 0.45 × 10 -3 mm 2 /s (range 0.37-0.53 × 10 -3  mm 2 /s). For adenocarcinoma, two (100%) were iso- and hyperintense on T2WI, two (100%) were isointense on T1WI, two (100%) exhibited marked heterogeneous enhancement on post-contrast T1WI and two (100%) were isointense on DWI. The median ADC values were 1.08 × 10 -3  mm 2 /s (range 0.88-1.27 × 10 -3  mm 2 /s). The median ADC values of lymphoma tended to be lower than adenocarcinoma ( P  = 0.056)., Conclusions and Relevance: Determining ADC value and tumours with a large area of non-enhancement may be helpful in differentiating nasal lymphoma from nasal adenocarcinoma.

In dogs, diagnosis of prostate cancer is often delayed because clinical signs are not pathognomonic. Although ultrasonography is mainly performed to detect prostate cancer, the ultrasonographic appearance is not specific. In humans, magnetic resonance imaging (MRI), including diffusion-weighted imaging (DWI) is used to localize the prostate tumour. To our knowledge, there are no studies of MRI findings for detecting or localizing prostate cancer. The purpose of this study was to assess MRI findings of prostate cancer. As a control, three prostate hyperplasia cases were included. MRI data were analysed, and the following parameters were noted: signal intensity (SI) of prostate lesion on T2-weighted imaging, T1-weighted imaging and DWI, enhancement pattern of prostate lesion, and relative contrast enhancement indices (RCEI) and apparent diffusion coefficient (ADC) value of prostate lesion. For MRI examination, the mean RCEI of the adenocarcinoma was significantly lower than that of hyperplasia (P = .01, r = .8). The SI of the DWI of adenocarcinoma was significantly higher compared to hyperplasia (P = .03, φ = 1). The mean ADC values of the adenocarcinoma were significantly lower than that of hyperplasia (P = .03, r = .82). Thus, less enhancement on MRI, and low ADC value on MRI may help to detect prostate adenocarcinoma., (© 2020 John Wiley & Sons Ltd.)

Achondroplasia is caused by gain-of-function mutations in FGFR3 gene and leads to short-limb dwarfism. A stabilized analogue of C-type natriuretic peptide (CNP) is known to elongate bone by interacting with FGFR3 signals and thus is a promising drug candidate. However, it needs daily administration by percutaneous injection. FGFR inhibitor compounds are other drug candidates for achondroplasia because they directly fix the mutant protein malfunction. Although FGFR inhibitors elongate the bone of model mice, their adverse effects are not well studied. In this study, we found that a new FGFR inhibitor, ASP5878, which was originally developed as an anti-cancer drug, elongated the bone of achondroplasia model male mice at the dose of 300 μg/kg, which confers an AUC of 275 ng·h/ml in juvenile mice. Although ASP5878 was less effective in bone elongation than a CNP analogue, it is advantageous in that ASP5878 can be administered orally. The AUC at which minimal adverse effects were observed (very slight atrophy of the corneal epithelium) was 459 ng·h/ml in juvenile rats. The positive discrepancy between AUCs that brought efficacy and minimal adverse effect suggests the applicability of ASP5878 to achondroplasia in the clinical setting. We also analyzed effects of ASP5878 in a patient-specific induced pluripotent stem cell (iPSC) model for achondroplasia and found the effects on patient chondrocyte equivalents. Nevertheless, cautious consideration is needed when referring to safety data obtained from its application to adult patients with cancer in clinical tests.

Introduction: In-body tissue architecture (iBTA) technology, based on cell-free tissue engineering, can produces collagenous tissues for implantation by subcutaneous embedding a designed mold. The aim of this study was to evaluate the biocompatibility of iBTA-induced "Biosheet®" collagenous sheets, as scaffold materials for bladder reconstruction., Methods: Canine Biosheet® implants were prepared by embedding molds into subcutaneous pouches in beagles for 8 weeks. A part of canine bladder wall was excised (2 × 2 cm) and repaired by patching the same sized autologous Biosheet®. The Biosheet® implants were harvested 4 weeks (n = 1) and 12 weeks (n = 3) after the implantation and evaluated histologically., Results: No disruption of the patched Biosheet® implants or urinary leakage into the peritoneal cavity was observed during the entire observation periods. There were no signs of chronic inflammation or Biosheet® rejection. The urine-contacting surface of luminal surface of the Biosheet® was covered with a multicellular layer of urothelium cells 4 weeks after implantation. α-SMA-positive muscle cells were observed at the margin of the Biosheet® implants at 12 weeks after the implantation. In addition, in the center of the Biosheet® implants, the formation of microvessels stained as α-SMA-positive was observed., Conclusion: Biosheet® implants have biocompatibility as a scaffold for bladder reconstruction, indicating that they may be applicable for full-thickness bladder wall substitution. Further studies are required for definitive evaluation as a scaffold for bladder reconstruction., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Y.N. is an employee of Biotube Co., Ltd. The other authors declare no conflicts of interest associated with this manuscript., (© 2020 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Custom-made implants have recently gained attention in veterinary medicine because of their ability to properly fit animal bones having a wide variety of shapes and sizes. The effect of custom-made implants on bone soundness and the regeneration process is not yet clear. We fabricated a 3D printed Ti-6Al-4V custom-made bone plate that fits the shape of the dog radius, and placed it into the radius where an osteotomy had been made. The preferential orientation of the apatite c-axis contributes to the mechanical integrity of the bone and is a reliable measure of bone quality. We determined this parameter as well as the bone shape and bone mineral density (BMD). The bone portion which lies parallel to the bone plate exhibited bone resorption, decreased BMD, and significant degradation of apatite orientation, relative to the portion outside the plate, at 7 months after the operation. This demonstrates the presence of stress shielding in which applied stress is not transmitted to bone due to the insertion of a stiff bone plate. This reduced stress condition clearly influences the bone regeneration process. The apatite orientation in the regenerated site remained different even after 7 months of regeneration, indicating insufficient mechanical function in the regenerated portion. This is the first study in which the apatite orientation and BMD of the radius were evaluated under conditions of stress shielding in dogs. Our results suggest that assessment of bone repair by radiography can indicate the degree of restoration of BMD, but not the apatite orientation., Competing Interests: The authors have declared that no competing interests exist.

In veterinary medicine, abdominal ultrasonography is used to rank the differential diagnosis of renal lesions. However, a conventional sonographic examination may show nonspecific findings. The purpose of this study was to assess the computed tomography (CT) findings of canine renal tumors, including renal cell carcinoma (RCC), lymphoma, and hemangiosarcoma (HSA). In this retrospective study, the following CT parameters were recorded for each dog: 1) extent of renal involvement of tumors, 2) enhancement pattern, 3) number of renal tumors, 4) renal tumor vessel enhancement in the corticomedullary phase, 5) presence of lymphadenopathy and lung metastasis, and 6) attenuation values of the renal tumors on the pre- and post-contrast corticomedullary, nephrographic, and excretory phase images. Fifteen dogs met the inclusion criteria, of which nine had RCCs, four had lymphomas, and two had HSAs. RCCs tended to show heterogeneous enhancement and unilateral renal involvement, and vessel enhancement was detected in the corticomedullary phase in dogs with RCC. Conversely, renal lymphomas showed homogeneous enhancement, bilateral renal involvement, and multiple masses; in these dogs, no vessel enhancement was detected in the corticomedullary phase, and the incidence of lymphadenopathy was low. However, in dogs with lymphadenopathy, the renal lymphoma was associated with regionally severe lymphadenopathy. Finally, renal HSAs tended to show heterogeneous enhancement with a non-enhanced area and unilateral renal involvement; in these dogs, vessel enhancement was detected in the nephrographic phase, with the enhancement expanding around the vessel. These findings had no significant differences. Further studies with a larger sample size are required to examine the association between CT and histopathological findings., Competing Interests: The authors have declared that no competing interests exist.

Apatite formation on CaSiO3 ceramics was investigated using two different simulated body fluids (SBF) proposed by Kokubo (1990) and Tas (2000) and three sample/SBF (S/S) ratios (1.0, 2.5 and 8.3 mg/ml) at 36.5 degrees C for 1-25 days. The CaSiO3 ceramic was prepared by firing coprecipitated gel with Ca/Si = 0.91 at 1400 degrees C. The bulk density was 2.14 g/cm3 and the relative density about 76%. The two SBF solutions contain different concentrations of HCO3- and Cl- ions, the concentrations of which are closer to human blood plasma in the Tas SBF formulation than in the Kokubo formulation. The pH values in the former solution are also more realistic. The CaSiO3 ceramics show apatite formation in SBF (Kokubo) after soaking for only 1 day at all S/S ratios whereas different phases were formed at each S/S ratio in SBF (Tas). The crystalline phases formed were mainly apatite at S/S = 1.0 mg/ml, carbonate-type apatite at 2.5 mg/ml and calcite at 8.3 mg/ml. At higher S/S ratios the increase in the Ca concentration became higher while the P concentration became lower in the reacted SBF. These changes in SBF concentrations and increasing pH occurred at higher S/S ratios, producing more favorable conditions in the SBF for the formation of carbonate bearing phases, finally leading to the formation of calcite instead of apatite in the higher HCO3- ion concentration SBF (Tas). Apatite is, however, formed in the lower HCO3- ion concentration SBF (Kokubo) even though the Ca and P concentrations change in a similar manner to SBF (Tas).

The effect of the solid/solution (S/S) ratio on apatite formation from CaSiO3 ceramics in simulated body fluid (SBF) was investigated. CaSiO3 ceramics with a Ca/Si ratio of 0.91 were prepared by sintering CaSiO3 powder coprecipitated from ethanol solutions of Ca(NO3)2. 4H2O and Si(OC2H5)4 using NH4OH as the precipitant. These ceramics were reacted with SBF at S/S ratios of 1.0, 2.5 and 8.3 mg/ml at 36.5 degrees C for various times. Formation of apatite was observed at all the S/S ratios after soaking for 1 day. The amount and microstructure of the apatite obtained at a S/S ratio of 8.3 mg/ml, however, differed largely from the product formed at the other two S/S ratios. The apatite formed at S/S = 8.3 mg/ml was of smaller particle size, formed in smaller amount and with less preferred orientation of the (001) of apatite crystals compared with those formed at S/S = 1.0 and 2.5 mg/ml. An increase of Ca and decrease of the P components occurred in the soaked SBF at S/S = 8.3 mg/ml, the changes being much more marked than with the other two S/S ratios. These differences in the concentration changes in SBF at different S/S ratios are attributed to the difference in the apatite formation from the CaSiO3 ceramics.

In order to understand the relationship between peroxisomal dysfunction and clinical manifestations of peroxisomal disorders, the effect of thioridazine, a peroxisomal beta-oxidation antagonist, on the differentiation, membrane lipid composition and membrane fluidity of C-6 glial cells was examined. In our study, induction of 2',3'-cyclic-nucleotide 3'-phosphohydrolase (CNP), which was considered to be a membrane-associated enzyme closely associated with myelination, was inhibited with supplementation of thioridazine, followed by an increase in the relative concentration of longer chain fatty acids in cell membrane lipids, indicating that thioridazine causes impaired differentiation in the glial stem cell system. Membrane fluidity of C-6 glial cells was examined using a fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The DPH anisotropy value was decreased in the glial cells treated with thioridazine. These results indicate that the alteration of the membrane lipid composition caused by thioridazine affects the differentiation of glial cells via the changes in membrane properties.

The inhibiting influences of some non-steroidal anti-inflammatory drugs (NSAIDs) and other agents on cyclooxygenase (CO) and lipoxygenase (LO) activities in some enzyme preparations were investigated. Washed rat platelets (CO and 12-LO), rat polymorphonuclear leukocytes (PMNs, CO and 5-LO), rat renal medulla homogenate (CO), and purified soybean LO (15-LO) were used as enzyme preparations. The IC50 values of drugs on the enzyme activities were determined in each preparation. In addition, the inhibitory activities of the drugs on the generation of chemiluminescence from PMNs stimulated by phorbol myristate acetate were tested. NSAIDs (indomethacin, ketoprofen and phenylbutazone) showed a selective inhibition of CO in each preparation, but benoxaprofen inhibited both enzymes, especially in PMNs. BW755C, 1-phenyl-3-pyrazolidone (phenidone), toluene-3,4-dithiol (dithiol), 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (ethoxyquin) and acetone phenylhydrazone (APH) inhibited both enzyme activities. Nordihydroguaiaretic acid (NDGA) showed a relatively selective inhibition of LO in all the preparations used. APH inhibited soybean 15-LO markedly compared with the other enzymes tested. Ethoxyquin inhibited COs more markedly than LOs, and with regard to LO inhibition, it inhibited 5-LO in PMNs markedly. BW755C, phenidone, ethoxyquin, NDGA, APH and dithiol, which strongly inhibited 5-LO, showed an inhibitory activity on the generation of chemiluminescence from PMNs activated with phorbol ester.

The relation of the polar head group composition of cellular phospholipids to a biochemical expression of oligodendroglial differentiation was studied in cultured C-6 glial cells. Induction of the oligodendroglial enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), was determined after alteration of the polar head group composition of phospholipids by exposure of the cells to choline analogues, especially N,N'-dimethylethanolamine. To accomplish the phospholipid alteration, cells were grown in the presence of the analogue in medium free of exogenous lipid, i.e., first for 24 h in 10% delipidated serum and then for 48 h in serum-free medium. The 48-h exposure to serum-free medium resulted in untreated C-6 cells in a several fold increase in CNP activity, but in cells treated with 2.5 mM N,N'-dimethylethanolamine, total inhibition of this induction was observed. A graded, concentration-dependent inhibitory effect of the analogue on the induction of CNP was defined. The effect of the analogue was relatively specific, e.g., the activity of another plasma membrane enzyme of C-6 cells, (Na+ + K+)-activated ATPase, was not affected. Morever, there was no evidence of a toxic effect of the analogue; thus, total protein synthesis and cell growth were not altered, and the induction of CNP in serum-free medium recurred after removal of the analogue. N,N'-Dimethylethanolamine was shown to be incorporated into cellular phospholipids, primarily at the expense of phosphatidylcholine. The data define an important role for the polar head group composition of membrane phospholipids in oligodendroglial differentiation in this model system.

Serum beta-galactosidase activity was found to be markedly increased in clotting blood from normal subjects, but patients with galactosialidosis showed only a slight increase of this enzyme activity. Consequently, the beta-galactosidase activity was low in serum after long-term clotting in patients with this disease. The mechanism of the enzyme activation is unknown. The use of anticoagulants completely inhibited the activation.