Central Institute for Experimental Animals

Neurotoxicity of acrylamide has been demonstrated both in humans and animals, while the mechanisms remain largely unknown. We recently reported TNF-α deletion suppressed acrylamide-induced neurotoxicity in mice at low dose. Here we further investigated expression of antioxidant and proinflammatory cytokines to explore roles of TNF-α. Wild type and TNF-α KO mice were exposed to acrylamide at 0/12.5/25 mg/kg bw for 28 days. The results showed that acrylamide significantly decreased body weight at 12.5 and 25 mg/kg bw, but decreased brain weight only at 25 mg/kg bw. TNF-α deletion didn't alleviate the above effects. Also, TNF-α deletion didn't alleviate decrease of grip strength at 25 mg/kg bw. Immunohistochemical results showed that TNF-α deletion alleviated noradrenergic axon degeneration in cortex S1FL and S1HL regions at 12.5 mg/kg bw, but not 25 mg/kg bw. Moreover, TNF-α deletion suppressed acrylamide-induced upregulation of TGF-β and NF-κB, but didn't suppress upregulation of IL-6, suggesting possible roles of IL-6 in acrylamide-induced neurotoxicity, particularly at high concentration. Moreover, this study showed a dipolar effect of TNF-α deletion on oxidative stress pathways, i.e., at 25 mg/kg bw, TNF-α deletion suppressed upregulation of oxidative stress (Keap1/HO-1/Gclc/Gclm/Sod/Cat/Gstm/MT-1); however, at 12.5 mg/kg bw, TNF-α deletion accelerated upregulation of Nqo1/Gclm/Sod1/Cat/Gsr. Taken together, genetic TNF-α ablation, at least partially, alleviated acrylamide neurotoxicity at low concentration. Limited alleviation effects at high concentration generated a hypothesis that this may be due to IL-6 signaling and dipolar regulating effects of TNF-α deletion on oxidative stress pathway. This study provided new insights into acrylamide neurotoxicity and TNF-α-targeting strategy.

Tolbutamide is metabolized by cytochrome P450 2C9 into 4-hydroxytolbutamide (4-OH TB), which retains pharmacological activity. 4-OH TB is further oxidized to the inactive metabolite 4-carboxytolbutamide, likely via the intermediate tolbutamide aldehyde (4-CHO TB). Because the conversion of 4-OH TB to 4-CHO TB is considered the rate-limiting step, the enzyme(s) catalyzing this reaction may play a crucial role in determining drug efficacy. We aimed to identify the enzyme(s) responsible for this process in the human liver. 4-CHO TB was formed from 4-OH TB in human liver cytosol (HLC) in the presence of nicotinamide-adenine dinucleotide (NAD+). The relative activity factor approach revealed that this reaction was primarily attributed to alcohol dehydrogenase 4 (ADH4). Interestingly, 4-CHO TB was also formed in HLC in the presence of nicotinamide-adenine dinucleotide phosphate (NADP+), with a 1.6-fold higher intrinsic clearance than that of NAD+. Untargeted proteomic analysis revealed a significant correlation between aldo-keto reductase 1A1 (AKR1A1) protein levels and NADP+-dependent 4-CHO TB formation in 15 HLC samples (r = 0.627, P < .05). Recombinant AKR1A1 effectively catalyzed this reaction, contributing 92% of NADP+-dependent 4-CHO TB formation in HLC. Based on hepatic NAD+ and NADP+ concentrations and the expression levels of ADH4 and AKR1A1, AKR1A1 was estimated to contribute one-third of ADH4 to 4-CHO TB formation in the human liver. In conclusion, we demonstrated that ADH4 and AKR1A1 jointly mediate the oxidation of 4-OH TB to 4-CHO TB in the human liver, highlighting the novel role of AKR1A1 as an oxidase in drug metabolism. SIGNIFICANCE STATEMENT: This study identifies aldo-keto reductase 1A1 as a novel enzyme involved in the oxidation of 4-hydroxytolbutamide in the human liver. Alongside alcohol dehydrogenase 4, aldo-keto reductase 1A1 contributes to NADP+-dependent aldehyde formation, suggesting a previously unrecognized role in drug metabolism and variability in tolbutamide clearance.

To evaluate the safety and efficacy of multimodality treatment with vincristine, actinomycin D, and cyclophosphamide (VAC) therapy, surgery, and radiotherapy according to the US Intergroup Rhabdomyosarcoma Study IV (IRS-IV), and to establish a central review system and standard treatment for intermediate-risk pediatric rhabdomyosarcoma in Japan. The Japan Rhabdomyosarcoma Study-I (JRS-I) was a single-arm, phase II trial for intermediate-risk rhabdomyosarcoma treatment with open enrollment from June 2004 to March 2009. Patients received 12 cycles of VAC every 3 weeks for 42 weeks, with local therapy beginning after Week 12. The endpoints were progression-free survival (PFS), overall survival (OS), and incidence of hepatic veno-occlusive disease (VOD). Thirty-one eligible patients were enrolled, and at a median follow-up of 5.2 years, the 3-year PFS and OS for patients were 74.2%±7.9% (95% confidence interval [CI] 55.0%-86.2%) and 90.3%±5.3% (95% CI 72.9%-96.8%), respectively. VOD occurred in 3 (8%) of the 40 evaluable patients, but all recovered, and there were no deaths. The VAC regimen for intermediate-risk rhabdomyosarcoma with the first central review system in Japan is safe and feasible, and these findings can be positioned as basic data for improving treatment outcomes in Japan.

SUMMARY The generation of human organs in animals through blastocyst complementation offers a promising solution to the shortage of transplantable organs. While human pluripotent stem cells (hPSCs) can contribute to interspecies chimaeric embryos when injected into preimplantation embryos of mice, pigs, or monkeys, their integration is often limited due to low chimaerism and segregation from host tissues, significantly impeding progress toward exogenic organ generation. Here, we demonstrate that co-overexpression of the anti-apoptotic gene BCL2 and the proto-oncogene MYCL , along with various cell cycle regulators significantly enhances human cell chimaerism by promoting cell proliferation. This strategy facilitates the generation of viable mouse pups containing hPSC-derived tissues without tumorigenesis. scRNA-seq analysis revealed that hPSCs already exit pluripotent by early post-implantation stage yet hPSCs with enhanced proliferation were able to integrate effectively into the cardiomyocytes and vasculature of both embryonic and extraembryonic tissues with gene expression profiles reflecting their structural integration. These findings highlight the critical role of cell cycle regulation in overcoming xenogeneic barriers. Our findings offer new insights into strategies for enhancing interspecies organogenesis and advancing the field of regenerative medicine. Highlight Enhancing hPSC proliferation increases human cell contribution in interspecies chimaeras Achieving the generation of viable human-mouse chimaeras without tumour formation hPSCs in post-implantation mouse epiblast exit pluripotency but retain the capacity to integrate into mouse embryogenesis hPSCs derivatives integrate into vasculature and cardiac tissues with lineage-matched transcriptional profiles

To our knowledge, no reports have described nonhuman primate (NHP) models of frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) that do not depend on an overexpression paradigm. Based on our recent success in generating single human MAPT knock-in mouse models of FTDP-17, we describe the experimental basis for generating knock-in marmoset models of FTDP-17. In addition, successful generation of mutant PSEN1 knock-in marmoset models lacking exon 9 (PSEN1-Δ9) of Alzheimer's disease (AD) indicates that we will be able to reconstitute two major pathological features of AD, i.e., amyloid plaques and neurofibrillary tangles, in an accelerated manner by combining these models.