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© 2015, public library of science. All rights reserved.The activation of bypass signals, such as MET and AXL, has been identified as a possible mechanism of EGFR-TKI resistance. Because various oncoproteins depend on HSP90 for maturation and stability, we

Extracellular superoxide dismutase (SOD3) plays an important role in maintaining vascular redox homeostasis by eliminating superoxides. The angiotensin II (AngII) peptide mediates vasoconstriction in part via reactive oxygen species (ROS) but has pathologic effects when elevated in adults. Histone deacetylase 6 (HDAC6) modulates the acetylation of non-histone substrates and is associated with hypertensive disorders. Here, we investigated the potential regulation of SOD3 by HDAC6 in human aortic endothelial cells (HAECs) and its implications for AngII-induced oxidative stress and hypertension. HDAC6 inhibition (via the specific inhibitor tubastatin A (TubA), gene knockdown, or a deacetylase activity-deficient mutant) significantly increased SOD3 protein and mRNA expression but did not affect SOD1 or SOD2 protein levels. Conversely, AngII downregulated SOD3 levels and increased ROS and superoxide levels; these effects were antagonized by TubA. We confirmed that the transcription factor Sp1 mediates TubA-induced as well as basal SOD3 expression. Notably, TubA strongly augmented Sp1 acetylation at lysine 703, which activated Sp1 binding to the proximal SOD3 promoter region and, consequently, SOD3 expression. Alternatively, AngII decreased Sp1 acetylation, and TubA-mediated SOD3 induction was reduced upon overexpression of an acetylation-resistant Sp1 mutant (K703R) compared to that by the wild-type protein. Consistent with these findings, aortic SOD3 expression was significantly higher in HDAC6-deficient mice than in wild-type mice. Moreover, AngII infusion-mediated blood pressure elevation was reduced in HDAC6-deficient mice compared with that in wild-type mice. Collectively, our results suggest that HDAC6 inhibition leads to SOD3 upregulation by enhancing Sp1 acetylation in HAECs, thereby mitigating AngII-induced oxidative stress and hypertension., (© 2025 Federation of European Biochemical Societies.)

Rationale: Although a relationship between the Gas6/AXL pathway and pulmonary fibrosis (PF) has been suggested, the precise mechanisms and clinical implications of the AXL pathway in idiopathic pulmonary fibrosis (IPF) are still unclear., Methods: Constitutive and conditional AXL-knockout mice were generated and injected with bleomycin (BLM) to induce pulmonary fibrosis. The expression of AXL and macrophage subtypes in BLM-injected mice and patients with IPF was analysed using flow cytometry. The therapeutic effects of the AXL inhibitors were examined., Results: AXL-deficient mice were resistant to BLM-induced pulmonary fibrosis and had a lower degree of M2-like macrophage differentiation than wild-type mice. Interestingly, AXL expression in monocytes was enhanced according to the progression of BLM-induced pulmonary fibrosis (PF), and these results were especially prominent in Ly6C high monocytes. Gene silencing or inhibitor treatment with AXL inhibited the differentiation of M2-like macrophages during bone marrow-derived macrophage (BMDMs) differentiation. These results were confirmed through experiments using AXL fl/fl LysMCre+ mice and systems with depletion and reconstitution of macrophages. In line with these results, patients with severe IPF had higher AXL expression in monocytes, high GAS6 levels, and an enhanced population of M2-like macrophages than those with mild IPF. Lastly, treatment with AXL inhibitors ameliorated BLM-induced PF and improved survival rate., Conclusions: The AXL pathway in classical monocytes contributed to PF progression through the induction of M2-like macrophage differentiation. Therefore, targeting AXL may be a promising therapeutic option for PF., (Copyright ©The authors 2025.)

Background and Aim: We aimed to assess the gene expression profiles of nonlesional small bowels in patients with Crohn's disease (CD) to identify its accompanying molecular alterations., Methods: We performed RNA sequencing of the uninflamed small bowel tissues obtained from 70 patients with ileal CD and 9 patients with colon cancer (non-CD controls) during bowel resection. Differentially expressed gene (DEG) analyses were performed using DESeq2. Gene set enrichment, correlation, and cell deconvolution analyses were applied to identify modules and functionally enriched transcriptional signatures of CD., Results: A comparison of CD patients and non-CD controls revealed that of the 372 DEGs, 49 protein-coding genes and 5 long non-coding RNAs overlapped with the inflammatory bowel disease susceptibility loci. The pathways related to immune and inflammatory reactions were upregulated in CD, while metabolic pathways were downregulated in CD. Compared with non-CD controls, CD patients had significantly higher proportions of immune cells, including plasma cells (P = 1.15 × 10 -4 ), and a lower proportion of epithelial cells (P = 1.12 × 10 -4 ). Co-upregulated genes (M14 module) and co-downregulated genes (M9 module) were identified in CD patients. The M14 module was enriched in immune-related genes and significantly associated with the responses to anti-tumor necrosis factor (TNF) therapy. The core signature of the M14 module was comprised of six genes and was upregulated in nonresponders to anti-TNF therapy of five independent cohorts (n = 163), indicating acceptable discrimination ability (area under the receiver operating characteristic curve of 75-86%)., Conclusions: The differences in gene expression and cellular composition between CD patients and non-CD controls imply significant molecular alterations, which are associated with the response to anti-TNF treatment., (© 2024 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Introduction: Humanized mouse models to recapitulate human biological systems still have limitations, such as the onset of lethal graft-versus-host disease (GvHD), a variable success rate, and the low accessibility of total body irradiation (TBI). Recently, mice modified with the CD47-SIRPA axis have been studied to improve humanized mouse models. However, such trials have been rarely applied in NOD mice. In this study, we created a novel mouse strain, NOD-CD47 null Rag2 null IL-2rγ null (RTKO) mice, and applied it to generate humanized mice., Methods: Four-week-old female NOD-Rag2 null IL-2rγ null (RID) and RTKO mice pre-conditioned with TBI or busulfan (BSF) injection were used for generating human CD34+ hematopoietic stem cell (HSC) engrafted humanized mice. Clinical signs were observed twice a week, and body weight was measured once a week. Flow cytometry for human leukocyte antigens was performed at intervals of four weeks or two weeks, and mice were sacrificed at 48 weeks after HSC injection., Results: For a long period from 16 to 40 weeks post transplantation, the percentage of hCD45 was mostly maintained above 25% in all groups, and it was sustained the longest and highest in the RTKO BSF group. Reconstruction of human leukocytes, including hCD3, was also most prominent in the RTKO BSF group. Only two mice died before 40 weeks post transplantation in all groups, and there were no life-threatening GvHD lesions except in the dead mice. The occurrence of GvHD has been identified as mainly due to human T cells infiltrating tissues and their related cytokines., Discussion: Humanized mouse models under all conditions applied in this study are considered suitable models for long-term experiments based on the improvement of human leukocytes reconstruction and the stable animal health. Especially, RTKO mice pretreated with BSF are expected to be a valuable platform not only for generating humanized mice but also for various immune research fields., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kim, Lee, Baek, Song, Jo, Ryu, Ryu, Seo, Kim and Heo.)

X-linked adrenoleukodystrophy (ALD), an inherited neurometabolic disorder caused by mutations in ABCD1, which encodes the peroxisomal ABC transporter, mainly affects the brain, spinal cord, adrenal glands, and testes. In ALD patients, very-long-chain fatty acids (VLCFAs) fail to enter the peroxisome and undergo subsequent β-oxidation, resulting in their accumulation in the body. It has not been tested whether in vivo base editing or prime editing can be harnessed to ameliorate ALD. We developed a humanized mouse model of ALD by inserting a human cDNA containing the pathogenic variant into the mouse Abcd1 locus. The humanized ALD model showed increased levels of VLCFAs. To correct the mutation, we tested both base editing and prime editing and found that base editing using ABE8e(V106W) could correct the mutation in patient-derived fibroblasts at an efficiency of 7.4%. Adeno-associated virus (AAV)-mediated systemic delivery of NG-ABE8e(V106W) enabled robust correction of the pathogenic variant in the mouse brain (correction efficiency: ∼5.5%), spinal cord (∼5.1%), and adrenal gland (∼2%), leading to a significant reduction in the plasma levels of C26:0/C22:0. This established humanized mouse model and the successful correction of the pathogenic variant using a base editor serve as a significant step toward treating human ALD disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Progesterone (P4) is a crucial reproductive hormone that acts as a precursor for all other endogenous steroids. P4 modulates transcriptional activity during reproduction by binding to progesterone receptors (PR). However, the physiological role of P4 in the liver is understudied. P4-mediated lipid metabolism in the liver was investigated in this study, as P4 facilitates insulin resistance and influences energy metabolism. While exogenous lipids are mainly obtained from food, the liver synthesizes endogenous triglycerides and cholesterol from a carbohydrate diet. Hepatic de novo lipogenesis (DNL) is primarily determined by acetyl-CoA and its biosynthetic pathways, which involve fatty acid and cholesterol synthesis. While P4 increased the hepatic levels of sterol regulatory element-binding protein 1 C (SREBP-1 C), peroxisome proliferator-activated receptor-gamma (PPARγ), acetyl-CoA carboxylase (ACC), and CD36, co-treatment with the P4 receptor antagonist RU486 blocked these proteins and P4-mediated lipogenesis. RNA sequencing was used to assess the role of P4 in lipogenic events, such as fatty liver and fatty acid metabolism, lipoprotein signaling, and cholesterol metabolism. P4 induced hepatic DNL and lipid anabolism were confirmed in the liver of ovarian resection mice fed a high-fat diet or in pregnant mice. P4 increased lipogenesis directly in mice exposed to P4 and indirectly in fetuses exposed to maternal P4. The lipid balance between lipogenesis and lipolysis determines fat build-up and is linked to lipid metabolism dysfunction, which involves the breakdown and storage of fats for energy and the synthesis of structural and functional lipids. Therefore, P4 may impact the lipid metabolism and reproductive development during gestation., Competing Interests: Declaration of Competing Interest The authors have declared that no conflict of interest exists., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Proper placental development in early pregnancy ensures a positive outcome later on. The developmental relationship between the placenta and embryonic organs, such as the heart, is crucial for a normal pregnancy. However, the mechanism through which the placenta influences the development of embryonic organs remains unclear. Trophoblasts fuse to form multinucleated syncytiotrophoblasts (SynT), which primarily make up the placental materno-fetal interface. We discovered that endogenous progesterone immunomodulatory binding factor 1 (PIBF1) is vital for trophoblast differentiation and fusion into SynT in humans and mice. PIBF1 facilitates communication between SynT and adjacent vascular cells, promoting vascular network development in the primary placenta. This process affected the early development of the embryonic cardiovascular system in mice. Moreover, in vitro experiments showed that PIBF1 promotes the development of cardiovascular characteristics in heart organoids. Our findings show how SynTs organize the barrier and imply their possible roles in supporting embryogenesis, including cardiovascular development. SynT-derived factors and SynT within the placenta may play critical roles in ensuring proper organogenesis of other organs in the embryo., (© 2024. The Author(s).)

Background: Tumor-derived exosomes are critical elements of the cell-cell communication response to various stimuli. This study aims to reveal that the histone deacetylase 5 (HDAC5) and p53 interaction upon radiation in hepatocellular carcinoma intricately regulates the secretion and composition of exosomes., Methods: We observed that HDAC5 and p53 expression were significantly increased by 2 Gy and 4 Gy radiation exposure in HCC. Normal- and radiation-derived exosomes released by HepG2 were purified to investigate the exosomal components., Results: We found that in the radiation-derived exosome, exosomal Maspin was notably increased. Maspin is known as an anti-angiogenic gene. The expression of Maspin was regulated at the cellular level by HDAC5, and it was elaborately regulated and released in the exosome. Radiation-derived exosome treatment caused significant inhibition of angiogenesis in HUVECs and mouse aortic tissues. Meanwhile, we confirmed that miR-151a-3p was significantly reduced in the radiation-derived exosome through exosomal miRNA sequencing, and three HCC-specific exosomal miRNAs were also decreased. In particular, miR-151a-3p induced an anti-apoptotic response by inhibiting p53, and it was shown to induce EMT and promote tumor growth by regulating p53-related tumor progression genes. In the HCC xenograft model, radiation-induced exosome injection significantly reduced angiogenesis and tumor size., Conclusions: Our present findings demonstrated HDAC5 is a vital gene of the p53-mediated release of exosomes resulting in tumor suppression through anti-cancer exosomal components in response to radiation. Finally, we highlight the important role of exosomal Maspin and mi-151a-3p as a biomarker in enhancing radiation treatment sensitivity. Therapeutic potential of HDAC5 through p53-mediated exosome modulation in radiation treatment of hepatocellular carcinoma., (© 2023. The Author(s).)

Vascular endothelial growth factor receptor-2 (VEGFR2) plays a key role in maintaining vascular endothelial homeostasis. Here, we show that blood flows determine activation and inactivation of VEGFR2 through selective cysteine modifications. VEGFR2 activation is regulated by reversible oxidation at Cys 1206 residue. H 2 O 2 -mediated VEGFR2 oxidation is induced by oscillatory flow in vascular endothelial cells through the induction of NADPH oxidase-4 expression. In contrast, laminar flow induces the expression of endothelial nitric oxide synthase and results in the S-nitrosylation of VEGFR2 at Cys 1206 , which counteracts the oxidative inactivation. The shear stress model study reveals that disturbed blood flow operated by partial ligation in the carotid arteries induces endothelial damage and intimal hyperplasia in control mice but not in knock-in mice harboring the oxidation-resistant mutant (C1206S) of VEGFR2. Thus, our findings reveal that flow-dependent redox regulation of the VEGFR2 kinase is critical for the structural and functional integrity of the arterial endothelium., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

AXL is a member of TAM receptor family and has been highlighted as a potential target for cancer treatment. Accumulating evidence has uncovered the critical role of the AXL signaling pathway in tumor growth, metastasis, and resistance against anti-cancer drugs, as well as its association with cancer immune escape. However, the function of AXL as a manipulator of the immune system in the tumor microenvironment (TME) remains unclear. Therefore, in this study, we investigated the impact of AXL on immune cells in the TME of a syngeneic tumor model using AXL knockout (AXL -/- ) mice. Compared to AXL wild-type (AXL +/+ ) mice, tumor growth was significantly suppressed in AXL -/- mice, and an induced population of tumor-infiltrated CD8 + T cells and CD103 + dendritic cells (DCs) was observed. The change of CD8 + T cells and CD103 + DCs was also confirmed in tumor-draining lymph nodes (TdLN). In addition, the clonal expansion of OVA-specific CD8 + T cells was dominant in AXL -/- mice. Finally, anti-PD-1 treatment evidenced synergistic anti-cancer effects in AXL -/- mice. Overall, our data indicate that AXL signaling may inhibit the clonal expansion of tumor-specific CD8 + T cells through the regulation of the migration of CD8 + T cells and DCs in TME. Thus, AXL may be a powerful molecular target to improve anti-cancer effects through single or combined therapy with immune checkpoint inhibitors (ICI)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

A new target that stimulates bone formation is needed to overcome limitations of current anti-osteoporotic drugs. Myokines, factors secreted from muscles, may modulate it. In this study, we investigated the role of aortic carboxypeptidase-like protein (ACLP), which is highly expressed in skeletal muscles, on bone formation. MC3T3-E1 cells and/or calvaria osteoblasts were treated with recombinant N-terminal mouse ACLP containing a signal peptide [rmACLP (N)]. The expression and secretion of ACLP were higher in skeletal muscle and differentiated myotube than in other tissues and undifferentiated myoblasts, respectively. rmACLP (N) increased bone formation, ALP activity, and phosphorylated p38 mitogen-activated protein (MAP) kinase in osteoblasts; reversal was achieved by pre-treatment with a TGF-β receptor inhibitor. Under H 2 O 2 treatment, rmACLP (N) increased osteoblast survival, phosphorylated p38 MAP kinase, and the nuclear translocation of FoxO3a in osteoblasts. H 2 O 2 treatment caused rmACLP (N) to suppress its apoptotic, oxidative, and caspase-9 activities. rmACLP (N)-stimulated osteoblast survival was reversed by pre-treatment with a p38 inhibitor, a TGF-β-receptor II blocking antibody, and a FoxO3a shRNA. Conditioned media (CM) from muscle cells stimulated osteoblast survival under H 2 O 2 treatment, in contrast to CM from ACLP knockdown muscle cells. rmACLP (N) increased the expressions of FoxO3a target anti-oxidant genes such as Sod2, Trx2, and Prx5. In conclusion, ACLP stimulated the differentiation and survival of osteoblasts. This led to the stimulation of bone formation by the activation of p38 MAP kinase and/or FoxO3a via TGF-β receptors. These findings suggest a novel role for ACLP in bone metabolism as a putative myokine., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Alcohol-associated liver disease (ALD) is caused by excessive abuse of alcohol. One of the most representative causes of ALD is the action of acetaldehyde. Acetaldehyde is a toxic material produced when alcohol is metabolized through some enzymes, and it causes endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and tissue injury. In this study, we assessed the relationship between Progesterone receptor membrane component 1 (PGRMC1) and ALD because PGRMC1 is expressed in the ER and mitochondria in the liver. Using the chronic and binge alcohol feeding models, we assessed acetaldehyde level, liver damage, alcohol-degrading enzymes, and ER stress. Compared with wild-type (WT) mice ethanol-fed Pgrmc1 knockout (KO) mice had higher levels of alanine aminotransferase (ALT) and alcohol-degrading enzymes, and Pgrmc1 KO mice had high serum acetaldehyde and ER stress levels compared with WT mice with control and ethanol feeding. Loss of Pgrmc1 increased acetaldehyde production through increased expression of alcohol dehydrogenase and catalase, which led to increased ER stress and suggested that cell death was promoted. In conclusion, it has been proposed that the loss of PGRMC1 could promote ALD and cause liver damage in alcohol-abusing humans. NEW & NOTEWORTHY Loss of Pgrmc1 increased acetaldehyde production, and excess acetaldehyde consequently increased ER stress, which activates apoptosis. Since low expression of PGRMC1 is vulnerable to alcoholic liver damage, the loss of PGRMC1 expression may increase susceptibility to ALD.

Heart failure (HF) is an emerging epidemic with a high mortality rate. Apart from conventional treatment methods, such as surgery or use of vasodilation drugs, metabolic therapy has been suggested as a new therapeutic strategy. The heart relies on fatty acid oxidation and glucose (pyruvate) oxidation for ATP-mediated contractility; the former meets most of the energy requirement, but the latter is more efficient. Inhibition of fatty acid oxidation leads to the induction of pyruvate oxidation and provides cardioprotection to failing energy-starved hearts. One of the non-canonical types of sex hormone receptors, progesterone receptor membrane component 1 ( Pgrmc1 ), is a non-genomic progesterone receptor associated with reproduction and fertility. Recent studies revealed that Pgrmc1 regulates glucose and fatty acid synthesis. Notably, Pgrmc1 has also been associated with diabetic cardiomyopathy, as it reduces lipid-mediated toxicity and delays cardiac injury. However, the mechanism by which Pgrmc1 influences the energy-starved failing heart remains unknown. In this study, we found that loss of Pgrmc1 inhibited glycolysis and increased fatty acid/pyruvate oxidation, which is directly associated with ATP production, in starved hearts. Loss of Pgrmc1 during starvation activated the phosphorylation of AMP-activated protein kinase, which induced cardiac ATP production. Pgrmc1 loss increased the cellular respiration of cardiomyocytes under low-glucose conditions. In isoproterenol-induced cardiac injury, Pgrmc1 knockout resulted in less fibrosis and low heart failure marker expression. In summary, our results revealed that Pgrmc1 ablation in energy-deficit conditions increases fatty acid/pyruvate oxidation to protect against cardiac damage via energy starvation. Moreover, Pgrmc1 may be a regulator of cardiac metabolism that switches the dominance of glucose-fatty acid usage according to nutritional status and nutrient availability in the heart.

Thyroid hormone receptor-interacting protein 13 (TRIP13) participates in various regulatory steps related to the cell cycle, such as the mitotic spindle assembly checkpoint and meiotic recombination, possibly by interacting with members of the HORMA domain protein family. Recently, it was reported that TRIP13 could regulate the choice of the DNA repair pathway, i.e., homologous recombination (HR) or nonhomologous end-joining (NHEJ). However, TRIP13 is recruited to DNA damage sites within a few seconds after damage and may therefore have another function in DNA repair other than regulation of the pathway choice. Furthermore, the depletion of TRIP13 inhibited both HR and NHEJ, suggesting that TRIP13 plays other roles besides regulation of choice between HR and NHEJ. To explore the unidentified functions of TRIP13 in the DNA damage response, we investigated its genome-wide interaction partners in the context of DNA damage using quantitative proteomics with proximity labeling. We identified MRE11 as a novel interacting partner of TRIP13. TRIP13 controlled the recruitment of MDC1 to DNA damage sites by regulating the interaction between MDC1 and the MRN complex. Consistently, TRIP13 was involved in ATM signaling amplification. Our study provides new insight into the function of TRIP13 in immediate-early DNA damage sensing and ATM signaling activation.

C1q/tumor necrosis factor-related protein 9 (CTRP9) is an adipokine and has high potential as a therapeutic target. However, the role of CTRP9 in cardiovascular disease pathogenesis remains unclear. We found CTRP9 to induce HDAC7 and p38 MAPK phosphorylation via tight regulation of AMPK in vascular endothelial cells, leading to angiogenesis through increased MEF2 activity. The expression of CTRP9 and atheroprotective MEF2 was decreased in plaque tissue of atherosclerotic patients and the ventricle of post-infarction mice. CTRP9 treatment inhibited the formation of atherosclerotic plaques in ApoE KO and CTRP9 KO mice. In addition, CTRP9 induced significant ischemic injury prevention in the post-MI mice. Clinically, serum CTRP9 levels were reduced in patients with MI compared with healthy controls. In summary, CTRP9 induces a vasoprotective response via the AMPK/HDAC7/p38 MAPK pathway in vascular endothelial cells, whereas its absence can contribute to atherosclerosis and MI. Hence, CTRP9 may represent a valuable therapeutic target and biomarker in cardiovascular diseases.

Although rats with melanin-pigmentated retinal pigment epithelial (RPE) cells are physiologically more appropriate models for human eye research than their albino counterparts, reliable models from the former strain are not available to study retinal degeneration. Here, we describe the development of a novel Pde6b -knockout Long-Evans (LE Pde6b KO) rat model that recapitulates key features of human retinitis pigmentosa (RP). After the generation of the Pde6b -knockout Sprague-Dawley rats with the CRISPR-Cpf1 system, the LE rat was back-crossed over 5 generations to develop the pigmented LE Pde6b KO strain. Interestingly, LE Pde6b KO displayed well-developed bone-spicule pigmentation; a hallmark of fundus in patients with RP which cannot be observed in non-pigmented albino rats. Moreover, the rat model showed progressive thinning of the retina, which was evident by intravital imaging with optical coherence tomography. Histologically, significant atrophy was observed in the outer nuclear layer. Functionally, LE Pde6b KO presented a marked decrease of amplitude level during electroretinogram testing, demonstrating significant loss of visual function. Therefore, these findings suggest that the LE Pde6b KO model robustly recapitulates the hallmark phenotype of RP. We believe that the LE Pde6b KO model may be used effectively for preclinical translational research to further study retinal degeneration., Competing Interests: BK and JL are inventors on a patent application related to this work filed with the ASAN FOUNDATION and the University of Ulsan Foundation for Industry Cooperation (no. 10-2021-0067261, filed on May 25, 2021). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yang, Kim, Kwak, Lee, Kim, Baek, Sung and Lee.)

Here, we found that heterozygous null of peroxisomal Nudt7 ( Nudt7 +/- ) induced the typical NAFLD features, i.e. increased levels of hepatic triglyceride (TG) and fatty acid (FA), infiltration of inflammatory cells, impaired glucose tolerance and insulin sensitivity, and stimulation of lipolysis from adipose tissue. Particularly, in Nudt7 +/- hepatocytes, de novo lipogenesis (DNL) was significantly increased. Ingenuity pathway analysis (IPA) and KEGG pathway analysis of RNA sequencing data suggested the activation of PPAR signaling in the liver of Nudt7 +/- mice. Moreover, accumulation of palmitic acid in Nudt7 +/- hepatocyte increased the level of H3K4me3 on the promoters of PPARγ resulting in the activation of PPARγ and induced the DNL in the hepatocytes of Nudt7 +/- mice. Moreover, we found that liraglutide significantly reduced typical NAFLD features induced by NUDT7 deficiency. Our data suggest that dysregulation of peroxisomal NUDT7 is responsible for upregulation of hepatic DNL by accumulation of palmitic acid and PPARγ activation., Competing Interests: The authors declare that they have no competing interests., (© 2022 The Author(s).)

The placenta regulates maternal-fetal communication, and its defect leads to significant pregnancy complications. The maternal and embryonic circulations are primitively connected in early placentation, but the function of the placenta during this developmentally essential period is relatively unknown. We thus performed a comparative proteomic analysis of the placenta before and after primary placentation and found that the metabolism and transport of lipids were characteristically activated in this period. The placental fatty acid (FA) carriers in specific placental compartments were upregulated according to gestational age, and metabolomic analysis also showed that the placental transport of FAs increased in a time-dependent manner. Further analysis of two mutant mice models with embryonic lethality revealed that lipid-related signatures could reflect the functional state of the placenta. Our findings highlight the importance of the nutrient transport function of the primary placenta in the early gestational period and the role of lipids in embryonic development., Summary Sentence: The placenta is activated characteristically in terms of lipid transport during primary placentation, and the lipid-related signatures closely reflect the functional state of the placenta., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

An ideal cancer therapeutic strategy involves the selective killing of cancer cells without affecting the surrounding normal cells. However, researchers have failed to develop such methods for achieving selective cancer cell death because of shared features between cancerous and normal cells. In this study, we have developed a therapeutic strategy called the cancer-specific insertions-deletions (InDels) attacker (CINDELA) to selectively induce cancer cell death using the CRISPR-Cas system. CINDELA utilizes a previously unexplored idea of introducing CRISPR-mediated DNA double-strand breaks (DSBs) in a cancer-specific fashion to facilitate specific cell death. In particular, CINDELA targets multiple InDels with CRISPR-Cas9 to produce many DNA DSBs that result in cancer-specific cell death. As a proof of concept, we demonstrate here that CINDELA selectively kills human cancer cell lines, xenograft human tumors in mice, patient-derived glioblastoma, and lung patient-driven xenograft tumors without affecting healthy human cells or altering mouse growth., Competing Interests: Competing interest statement: S.W.C., T.K., H.R., and K.M. are shareholders of CasCure Therapeutics, Inc., (Copyright © 2022 the Author(s). Published by PNAS.)

Despite the strong influence of the gut microbiota on atherosclerosis, a causal relationship between atherosclerosis pathophysiology and gut microbiota is still unverified. This study was performed to determine the impact of the gut microbiota on the pathogenesis of atherosclerosis caused by genetic deficiency. To elucidate the influence of the gut microbiota on atherosclerosis pathogenesis, an atherosclerosis-prone mouse model (C1q/TNF-related protein 9-knockout (CTRP9-KO) mice) was generated. The gut microbial compositions of CTRP9-KO and WT control mice were compared. Fecal microbiota transplantation (FMT) was performed to confirm the association between gut microbial composition and the progression of atherosclerosis. FMT largely affected the gut microbiota in both CTRP9-KO and WT mice, and all transplanted mice acquired the gut microbiotas of the donor mice. Atherosclerotic lesions in the carotid arteries were decreased in transplanted CTRP9-KO mice compared to CTRP9-KO mice prior to transplantation. Conversely, WT mice transplanted with the gut microbiotas of CTRP9-KO mice showed the opposite effect as that of CTRP9-KO mice transplanted with the gut microbiotas of WT mice. Here, we show that CTRP9 gene deficiency is related to the distribution of the gut microbiota in subjects with atherosclerosis. Transplantation of WT microbiotas into CTRP9-KO mice protected against the progression of atherosclerosis. Conversely, the transplantation of CTRP9-KO microbiotas into WT mice promoted the progression of atherosclerosis. Treating atherosclerosis by restoring gut microbial homeostasis may be an effective therapeutic strategy., (© 2022. The Author(s).)

Here, in Ppara -/- mice, we found that an increased DNL stimulated the cartilage degradation and identified ACOT12 as a key regulatory factor. Suppressed level of ACOT12 was observed in cartilages of OA patient and OA-induced animal. To determine the role and association of ACOT12 in the OA pathogenesis, we generated Acot12 knockout (KO) (Acot12 -/- ) mice using RNA-guided endonuclease. Acot12 -/- mice displayed the severe cartilage degradation with the stimulation of matrix MMPs and chondrocyte apoptosis through the accumulation of acetyl CoA. Delivery of acetyl CoA-conjugated chitosan complex into cartilage stimulated DNL and cartilage degradation. Moreover, restoration of ACOT12 into human OA chondrocytes and OA-induced mouse cartilage effectively rescued the pathophysiological features of OA by regulating DNL. Taken together, our study suggested ACOT12 as a novel regulatory factor in maintaining cartilage homeostasis and targeting ACOT12 could contribute to developing a new therapeutic strategy for OA., (© 2022. The Author(s).)

Background & Aims: Sphingosine 1-phosphate receptors (S1PRs) are a group of G-protein-coupled receptors that confer a broad range of functional effects in chronic inflammatory and metabolic diseases. S1PRs also may mediate the development of nonalcoholic steatohepatitis (NASH), but the specific subtypes involved and the mechanism of action are unclear., Methods: We investigated which type of S1PR isoforms is activated in various murine models of NASH. The mechanism of action of S1PR4 was examined in hepatic macrophages isolated from high-fat, high-cholesterol diet (HFHCD)-fed mice. We developed a selective S1PR4 functional antagonist by screening the fingolimod (2-amino-2-[2-(4- n -octylphenyl)ethyl]-1,3- propanediol hydrochloride)-like sphingolipid-focused library., Results: The livers of various mouse models of NASH as well as hepatic macrophages showed high expression of S1pr4. Moreover, in a cohort of NASH patients, expression of S1PR4 was 6-fold higher than those of healthy controls. S1pr4 +/- mice were protected from HFHCD-induced NASH and hepatic fibrosis without changes in steatosis. S1pr4 depletion in hepatic macrophages inhibited lipopolysaccharide-mediated Ca ++ release and deactivated the Nod-like receptor pyrin domain-containning protein 3 (NLRP3) inflammasome. S1P increased the expression of S1pr4 in hepatic macrophages and activated NLRP3 inflammasome through inositol trisphosphate/inositol trisphosphate-receptor-dependent [Ca ++ ] signaling. To further clarify the biological function of S1PR4, we developed SLB736, a novel selective functional antagonist of SIPR4. Similar to S1pr4 +/- mice, administration of SLB736 to HFHCD-fed mice prevented the development of NASH and hepatic fibrosis, but not steatosis, by deactivating the NLRP3 inflammasome., Conclusions: S1PR4 may be a new therapeutic target for NASH that mediates the activation of NLRP3 inflammasome in hepatic macrophages., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Sarcopenia and osteoporosis frequently co-occur in the elderly and have common pathophysiological determinants. Slit guidance ligand 3 (SLIT3) has been recently discovered as a novel therapeutic factor against osteoporosis, and a SLIT3 fragment containing the second leucine-rich repeat domain (LRRD2) had a therapeutic efficacy against osteoporosis. However, a role of SLIT3 in the skeletal muscle is unknown., Methods: Skeletal muscle mass, strength, and/or physical activity were evaluated in Slit3 -/- , ovariectomized, and aged mice, based on the measurements of muscle weight and grip strength, Kondziella's inverted hanging test, and/or wheel-running test. Skeletal muscles were also histologically evaluated by haematoxylin and eosin staining and/or immunofluorescence. The ovariectomized and aged mice were intravenously injected with recombinant SLIT3 LRRD2 for 4 weeks. C2C12 cells were used to know cellular effects of SLIT3, such as in vitro myogenesis, fusion, cell viability, and proliferation, and also used to evaluate its molecular mechanisms by immunocytochemistry, immunoprecipitation, western blotting, real-time PCR, siRNA transfection, and receptor-ligand binding ELISA., Results: Slit3-deficient mice exhibited decreased skeletal muscle mass, muscle strength, and physical activity. The relative masses of gastrocnemius and soleus were lower in the Slit3 -/- mice (0.580 ± 0.039% and 0.033 ± 0.003%, respectively) than those in the WT littermates (0.622 ± 0.043% and 0.038 ± 0.003%, respectively) (all, P < 0.05). Gastrocnemius of Slit3 -/- mice showed the reduced number of Type I and Type IIa fibres (all, P < 0.05), but not of Type IIb and Type IIx fibres. SLIT3 activated β-catenin signalling by promoting its release from M-cadherin, thereby increasing myogenin expression to stimulate myoblast differentiation. In vitro experiments involving ROBO2 expression, knockdown, and interaction with SLIT3 indicated that ROBO2 functions as a SLIT3 receptor to aid myoblast differentiation. SLIT3 LRRD2 dissociated M-cadherin-bound β-catenin and up-regulated myogenin expression to increase myoblast differentiation, in a manner similar to full-length SLIT3. Systemic treatment with SLIT3 LRRD2 increased skeletal muscle mass in both ovariectomized and aged mice (all, P < 0.05). The relative masses of gastrocnemius and soleus were higher in the treated aged mice (0.548 ± 0.045% and 0.033 ± 0.005%, respectively) than in the untreated aged mice (0.508 ± 0.016% and 0.028 ± 0.003%, respectively) (all, P < 0.05). SLIT3 LRRD2 treatment increased the hanging duration of the aged mice by approximately 1.7-fold (P < 0.05)., Conclusions: SLIT3 plays a sarcoprotective role by activating β-catenin signalling. SLIT3 LRRD2 can potentially be used as a therapeutic agent against muscle loss., (© 2021 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Background: ARID2 belongs to the Switch/sucrose non-fermenting complex, in which the genetic defects have been found in patients with dysmorphism, short stature and intellectual disability (ID). As the phenotypes of patients with ARID2 mutations partially overlap with those of RASopathy, this study evaluated the biochemical association between ARID2 and RAS-MAPK pathway., Methods: The phenotypes of 22 patients with either an ARID2 heterozygous mutation or haploinsufficiency were reviewed. Comprehensive molecular analyses were performed using somatic and induced pluripotent stem cells (iPSCs) of a patient with ARID2 haploinsufficiency as well as using the mouse model of Arid2 haploinsufficiency by CRISPR/Cas9 gene editing., Results: The phenotypic characteristics of ARID2 deficiency include RASopathy, Coffin-Lowy syndrome or Coffin-Siris syndrome or undefined syndromic ID. Transient ARID2 knockout HeLa cells using an shRNA increased ERK1 and ERK2 phosphorylation. Impaired neuronal differentiation with enhanced RAS-MAPK activity was observed in patient-iPSCs. In addition, Arid2 haploinsufficient mice exhibited reduced body size and learning/memory deficit. ARID2 haploinsufficiency was associated with reduced IFITM1 expression, which interacts with caveolin-1 (CAV-1) and inhibits ERK activation., Discussion: ARID2 haploinsufficiency is associated with enhanced RAS-MAPK activity, leading to reduced IFITM1 and CAV-1 expression, thereby increasing ERK activity. This altered interaction might lead to abnormal neuronal development and a short stature., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Lipotoxic hepatocyte injury is a primary event in non-alcoholic steatohepatitis (NASH), but the mechanisms of lipotoxicity are not fully defined. Sphingolipids and free cholesterol (FC) mediate hepatocyte injury, but their link in NASH has not been explored. We examined the role of free cholesterol and sphingomyelin synthases (SMSs) that generate sphingomyelin (SM) and diacylglycerol (DAG) in hepatocyte pyroptosis, a specific form of programmed cell death associated with inflammasome activation, and NASH., Design: Wild-type C57BL/6J mice were fed a high fat and high cholesterol diet (HFHCD) to induce NASH. Hepatic SMS1 and SMS2 expressions were examined in various mouse models including HFHCD-fed mice and patients with NASH. Pyroptosis was estimated by the generation of the gasdermin-D N-terminal fragment. NASH susceptibility and pyroptosis were examined following knockdown of SMS1, protein kinase Cδ (PKCδ), or the NLR family CARD domain-containing protein 4 (NLRC4)., Results: HFHCD increased the hepatic levels of SM and DAG while decreasing the level of phosphatidylcholine. Hepatic expression of Sms1 but not Sms2 was higher in mouse models and patients with NASH. FC in hepatocytes induced Sms1 expression, and Sms1 knockdown prevented HFHCD-induced NASH. DAG produced by SMS1 activated PKCδ and NLRC4 inflammasome to induce hepatocyte pyroptosis. Depletion of Nlrc4 prevented hepatocyte pyroptosis and the development of NASH. Conditioned media from pyroptotic hepatocytes activated the NOD-like receptor family pyrin domain containing 3 inflammasome (NLRP3) in Kupffer cells, but Nlrp3 knockout mice were not protected against HFHCD-induced hepatocyte pyroptosis., Conclusion: SMS1 mediates hepatocyte pyroptosis through a novel DAG-PKCδ-NLRC4 axis and holds promise as a therapeutic target for NASH., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background and Aims: Mutations in XIAP can lead to the development of treatment-refractory severe paediatric Crohn's disease [CD], for which haematopoietic stem cell transplantation is the primary therapeutic option. The interpretation of variants of uncertain significance [VUSs] in XIAP needs to be scrutinized., Methods: Targeted next-generation sequencing was performed for 33 male paediatric patients with refractory CD admitted at a tertiary referral hospital. To obtain functional data, biomolecular cell assays and supercomputing molecular dynamics simulations were performed., Results: Nine unrelated male patients harboured hemizygous XIAP variants. Four known pathogenic variants and one novel pathogenic variant [p.Lys168Serfs*12] were identified in five patients, and two novel VUSs [p.Gly205del and p.Pro260Ser] and one known VUS [p.Glu350del] were identified in the remaining four. Among children with VUSs, only the subject with p.Gly205del exhibited defective NOD2 signalling. Using molecular dynamics simulation, we determined that the altered backbone torsional energy of C203 in XIAP of p.G205del was ~2 kcal/mol, suggesting loss of zinc binding in the mutant XIAP protein and poor coordination between the mutant XIAP and RIP2 proteins. Elevated auto-ubiquitination of zinc-depleted p.G205del XIAP protein resulted in XIAP protein deficiency., Conclusion: A high prevalence of XIAP deficiency was noted among children with refractory CD. Advanced functional studies decreased the subjectivity in the case-level interpretation of XIAP VUSs and directed consideration of haematopoietic stem cell transplantation., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In this study, we hypothesized that deregulation in the maintenance of the pool of coenzyme A (CoA) may play a crucial role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Specific deletion of Acot12 (Acot12 -/- ), the major acyl-CoA thioesterase, induced the accumulation of acetyl-CoA and resulted in the stimulation of de novo lipogenesis (DNL) and cholesterol biosynthesis in the liver. KEGG pathway analysis suggested PPARα signaling as the most significantly enriched pathway in Acot12 -/- livers. Surprisingly, the exposure of Acot12 -/- hepatocytes to fenofibrate significantly increased the accumulation of acetyl-CoA and resulted in the stimulation of cholesterol biosynthesis and DNL. Interaction analysis, including proximity-dependent biotin identification (BioID) analysis, suggested that ACOT12 may directly interact with vacuolar protein sorting-associated protein 33A (VPS33A) and play a role in vesicle-mediated cholesterol trafficking and the process of lysosomal degradation of cholesterol in hepatocytes. In summary, in this study, we found that ACOT12 deficiency is responsible for the pathogenesis of NAFLD through the accumulation of acetyl-CoA and the stimulation of DNL and cholesterol via activation of PPARα and inhibition of cholesterol trafficking., (© 2021. The Author(s).)

Pgrmc1 is a non-canonical progesterone receptor related to the lethality of various types of cancer. PGRMC1 has been reported to exist in co-precipitated protein complexes with epidermal growth factor receptor (EGFR), which is considered a useful therapeutic target in hepatocellular carcinoma (HCC). Here, we investigated whether Pgrmc1 is involved in HCC progression. In clinical datasets, PGRMC1 transcription level was positively correlated with EGFR levels; importantly, PGRMC1 level was inversely correlated with the survival duration of HCC patients. In a diethylnitrosamine (DEN)-induced murine model of HCC, the global ablation of Pgrmc1 suppressed the development of HCC and prolonged the survival of HCC-bearing mice. We further found that increases in hepatocyte death and suppression of compensatory proliferation in the livers of DEN-injured Pgrmc1 -null mice were concomitant with decreases in nuclear factor κB (NF-κB)-dependent production of interleukin-6 (IL-6). Indeed, silencing of Pgrmc1 in murine macrophages led to reductions in NF-κB activity and IL-6 production. We found that the anti-proinflammatory effect of Pgrmc1 loss was mediated by reductions in EGFR level and its effect was not observed after exposure of the EGFR inhibitor erlotinib. This study reveals a novel cooperative role of Pgrmc1 in supporting the EGFR-mediated development of hepatocellular carcinoma, implying that pharmacological suppression of Pgrmc1 may be a useful strategy in HCC treatment.

Although macroautophagy/autophagy deficiency causes degenerative diseases, the deletion of essential autophagy genes in adipocytes paradoxically reduces body weight. Brown adipose tissue (BAT) plays an important role in body weight regulation and metabolic control. However, the key cellular mechanisms that maintain BAT function remain poorly understood. in this study, we showed that global or brown adipocyte-specific deletion of pink1 , a Parkinson disease-related gene involved in selective mitochondrial autophagy (mitophagy), induced BAT dysfunction, and obesity-prone type in mice. Defective mitochondrial function is among the upstream signals that activate the NLRP3 inflammasome. NLRP3 was induced in brown adipocyte precursors (BAPs) from pink1 knockout (KO) mice. Unexpectedly, NLRP3 induction did not induce canonical inflammasome activity. Instead, NLRP3 induction led to the differentiation of pink1 KO BAPs into white-like adipocytes by increasing the expression of white adipocyte-specific genes and repressing the expression of brown adipocyte-specific genes. nlrp3 deletion in pink1 knockout mice reversed BAT dysfunction. Conversely, adipose tissue-specific atg7 KO mice showed significantly lower expression of Nlrp3 in their BAT. Overall, our data suggest that the role of mitophagy is different from general autophagy in regulating adipose tissue and whole-body energy metabolism. Our results uncovered a new mitochondria-NLRP3 pathway that induces BAT dysfunction. The ability of the nlrp3 knockouts to rescue BAT dysfunction suggests the transcriptional function of NLRP3 as an unexpected, but a quite specific therapeutic target for obesity-related metabolic diseases. Abbreviations: ACTB: actin, beta; BAPs: brown adipocyte precursors; BAT: brown adipose tissue; BMDMs: bone marrow-derived macrophages; CASP1: caspase 1; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; ChIP: chromatin immunoprecipitation; EE: energy expenditure; HFD: high-fat diet; IL1B: interleukin 1 beta; ITT: insulin tolerance test; KO: knockout; LPS: lipopolysaccharide; NLRP3: NLR family, pyrin domain containing 3; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RD: regular diet; ROS: reactive oxygen species; RT: room temperature; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); WT: wild-type.

Obesity is implicated in cardiovascular disease and heart failure. When fatty acids are transported to and not adequately oxidized in cardiac cells, they accumulate, causing lipotoxicity in the heart. Since hepatic progesterone receptor membrane component 1 (Pgrmc1) suppressed de novo lipogenesis in a previous study, it was questioned whether cardiac Pgrmc1 protects against lipotoxicity. Hence, we focused on the role of cardiac Pgrmc1 in basal (Resting), glucose-dominant (Refed) and lipid-dominant high-fat diet (HFD) conditions. Pgrmc1 KO mice showed high FFA levels and low glucose levels compared to wild-type (WT) mice. Pgrmc1 KO mice presented low number of mitochondrial DNA copies in heart, and it was concomitantly observed with low expression of TCA cycle genes and oxidative phosphorylation genes. Pgrmc1 absence in heart presented low fatty acid oxidation activity in all conditions, but the production of acetyl-CoA and ATP was in pronounced suppression only in HFD condition. Furthermore, HFD Pgrmc1 KO mice resulted in high cardiac fatty acyl-CoA levels and TG level. Accordingly, HFD Pgrmc1 KO mice were prone to cardiac lipotoxicity, featuring high levels in markers of inflammation, endoplasmic reticulum stress, oxidative stress, fibrosis, and heart failure. In vitro study, it was also confirmed that Pgrmc1 enhances rates of mitochondrial respiration and fatty acid oxidation. This study is clinically important because mitochondrial defects in Pgrmc1 KO mice hearts represent the late phase of cardiac failure.

Background: Progesterone receptor membrane component 1 (Pgrmc1) is a non-classical progesterone receptor associated with the development of the mammary gland and xenograft-induced breast cancer. Importantly, Pgrmc1 is associated with the expression of estrogen receptor alpha and can be used for predicting the prognosis of breast cancer. Whether the genetic deletion of Pgrmc1 affects the progression of breast cancer is still unclear., Methods: We used MMTV-PyMT transgenic mice that spontaneously develop breast tumors. In backcrossed FVB Pgrmc1 knockout (KO) mice, we monitored the development of the primary tumor and lung metastasis. In MCF-7 and MDA-MB-231 tumor cell lines, the migratory activity was evaluated after Pgrmc1 knockdown., Results: There was no significant difference in the development of breast cancer in terms of tumor size at 13 weeks of age between WT and Pgrmc1 KO mice. However, Pgrmc1 KO mice had a significantly longer survival duration compared with WT mice. Furthermore, Pgrmc1 KO mice exhibited a significantly lower degree of lung metastasis. Compared with those of WT mice, the tumors of Pgrmc1 KO mice had a low expression of focal adhesion kinase and epithelial-mesenchymal transition markers. PGRMC1 knockdown resulted in a significantly reduced migration rate in breast cancer cell lines., Conclusions: Pgrmc1 KO mice with breast cancer had a prolonged survival, which was accompanied by a low degree of lung metastasis. PGRMC1 showed a significant role in the migration of breast cancer cells, and may serve as a potential therapeutic target in breast cancer. Video Abstract.

Asb2, ankyrin repeat, and SOCS box protein 2 form an E3 ubiquitin ligase complex. Asb2 ubiquitin ligase activity drives the degradation of filamins, which have essential functions in humans. The placenta is a temporary organ that forms during pregnancy, and normal placentation is important for survival and growth of the fetus. Recent studies have shown that approximately 25-30% of knockout (KO) mice have non-viable offspring, and 68% of knockout lines exhibit placental dysmorphologies. There are very few studies on Asb2, with insufficient research on its role in placental development. Therefore, we generated Asb2 knockout mice and undertook to investigate Asb2 expression during organogenesis, and to identify its role in early embryonic and placental development. The external morphology of KO embryos revealed abnormal phenotypes including growth retardation, pericardial effusion, pale color, and especially heart beat defect from E 9.5. Furthermore, Asb2 expression was observed in the heart from E 9.5, indicating that it is specifically expressed during early heart formation, resulting in embryonic lethality. Histological analysis of E 10.5 KO heart showed malformations such as failure of chamber formation, reduction in trabeculated myocardium length, absence of mesenchymal cells, and destruction of myocardium wall. Moreover, the histological results of Asb2-deficient placenta showed abnormal phenotypes including small labyrinth and reduced vascular complexity, indicating that failure to establish mature circulatory pattern affects the embryonic development and results in early mortality. Collectively, our results demonstrate that Asb2 knockout mice have placental defects, that subsequently result in failure to form a normal cardiac septum, and thereby result in embryo mortality in utero at around E 9.5., (Copyright © 2021. Published by Elsevier B.V.)

CRISPR-Cas systems, including Cas9 and Cpf1 (Cas12a), are promising tools for generating gene knockout mouse models. Unlike Cas9, Cpf1 can generate multiple crRNAs from a single concatemeric crRNA precursor, which is favorable for multiplex gene editing. Recently, a hybrid guide RNA (hgRNA) system employing both Cas9 and Cpf1 was developed for multiplex gene editing. As the crRNA of Cpf1 was linked to the 3' end of the sgRNA for Cas9, it can be split into separate guide RNAs by Cpf1. To examine whether this Cas9-Cpf1 hybrid system is suitable for multiplex gene knockouts in the mouse embryo, we generated an hgRNA that simultaneously targets the mouse Il10ra gene by Cas9 and mouse Dr3 (or Tnfrsf25, death receptor3) gene by Cpf1. The expression of hgRNA from a single promoter induced significant indels at each gene in cultured mouse cells upon the co-expression of both Cas9 and Cpf1. Interestingly, the hgRNA exhibited comparable Cas9-mediated indel activity without Cpf1 expression. Similarly, when the hgRNA was co-microinjected with both Cas9 and Cpf1 mRNAs into mouse zygotes at the pronuclear stage, founder mice were generated harboring mutations in both the Il10ra and Dr3 genes. However, when Cas9 mRNA was used alone without Cpf1 mRNA, the mouse Il10ra gene targeting was significantly decreased. These results indicate that the hgRNA system is a possible tool for multiplex gene targeting in the mouse embryo., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest in relation to this study., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The physiological functions of progesterone (P4) in female reproductive organs including the mammary glands are mediated via the progesterone receptor (PR), but not all P4 functions can be explained by PR-mediated signaling. Progesterone receptor membrane component 1 (PGRMC1), a potential mediator of P4 actions, plays an important role in the ovary and uterus in maintaining female fertility and pregnancy, but its function in mammary glands has not been elucidated. This study investigated the role of PGRMC1 in mouse mammary gland development. Unlike in the uterus, exogenous estrogen (E2) and/or P4 did not alter PGRMC1 expression in the mammary gland, and Pgrmc1-knockout (KO) mice displayed reduced ductal elongation and side branching in response to hormone treatment. During pregnancy, PGRMC1 was expressed within both the luminal and basal epithelium and gradually increased with gestation and decreased rapidly after parturition. Moreover, although lactogenic capacity was normal after parturition, Pgrmc1 KO resulted in defective mammary gland development from puberty until midpregnancy, while the expression of PR and its target genes was not significantly different between wild-type and Pgrmc1-KO mammary gland. These data suggest that PGRMC1 is essential for mammary gland development during puberty and pregnancy in a PR-independent manner., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Poly(ADP-ribose) polymerase 1 (PARP1) facilitates DNA damage response (DDR). While the Ewing's sarcoma breakpoint region 1 (EWS) protein fused to FLI1 triggers sarcoma formation, the physiological function of EWS is largely unknown. Here, we investigate the physiological role of EWS in regulating PARP1. We show that EWS is required for PARP1 dissociation from damaged DNA. Abnormal PARP1 accumulation caused by EWS inactivation leads to excessive Poly(ADP-Ribosy)lation (PARylation) and triggers cell death in both in vitro and in vivo models. Consistent with previous work, the arginine-glycine-glycine (RGG) domain of EWS is essential for PAR chain interaction and PARP1 dissociation from damaged DNA. Ews and Parp1 double mutant mice do not show improved survival, but supplementation with nicotinamide mononucleotides extends Ews-mutant pups' survival, which might be due to compensatory activation of other PARP proteins. Consistently, PARP1 accumulates on chromatin in Ewing's sarcoma cells expressing an EWS fusion protein that cannot interact with PARP1, and tissues derived from Ewing's sarcoma patients show increased PARylation. Taken together, our data reveal that EWS is important for removing PARP1 from damaged chromatin., (© 2020 The Authors.)

Hepatic gluconeogenesis is the main pathway for blood glucose maintenance activated during fasting. Retardation of insulin action, such as in diabetes mellitus, activates gluconeogenesis during the fed state. While the role of progesterone (P4) in diabetes is controversial, the P4 receptor, progesterone receptor membrane component 1 (PGRMC1), is known to stimulate pancreatic insulin secretion. We investigated the role of P4, via hepatic PGRMC1, during gluconeogenesis. The PGRMC1 binding chemical, AG-205, induced PGRMC1 monomer (25 kDa) abundance, and increased PEPCK expression and glucose production in parallel with cyclic AMP (cAMP) induction in Hep3B cells. PGRMC1-mediated cyclic AMP was inhibited by an adenylate cyclase inhibitor (MDL-12,330A). PEPCK suppression in Pgrmc1 KO hepatocyte was not observed after treatment of MDL-12,330A. PGRMC1 knockdown or overexpression systems in Hep3B cells confirmed that PGRMC1 mediates PEPCK expression via phosphorylation of cAMP-response element binding protein (CREB). CREB phosphorylation and PEPCK expression in primary hepatocytes were greater than that in PGRMC1 knock-out hepatocytes. Progesterone increased PGRMC1 expression, which induced cAMP and PEPCK induction and glucose production. In vivo, P4 suppressed gluconeogenesis following plasma insulin induction under normal conditions in a mouse model. However, P4 increased blood glucose via gluconeogenesis in parallel with increases in PGRMC1 and PEPCK expression in mice in both insulin-deficient and insulin-resistant conditions. We conclude that P4 increases hepatic glucose production via PGRMC1, which may exacerbate hyperglycaemia in diabetes where insulin action is limited.

Purpose: This study evaluates the oncogenic role of PIBF1 in triple-negative breast cancer (TNBC). TNBC is considered to have a poorer prognosis than other types of breast cancer and is associated with high risk of recurrence and distant metastasis. Currently, there are no effective therapies for the TNBC patients with distant metastasis due to the lack of targeted therapeutic options., Methods: The effects of PIBF1 knockdown on the cell viability and motility of TNBC cell lines were investigated. Effects of PIBF1 overexpression on tumorigenicity and cell motility were confirmed using Ba/F3 cell line and xenograft study on BALB/c nude mice., Results: In TNBC cell lines that highly express PIBF1, knockdown of PIBF1 induces apoptosis and suppresses cell viability and motility with activation of the ATR/CHK1 signaling pathway. Moreover, the oncogenic function of PIBF1 was confirmed using the Ba/F3 cell line., Conclusion: For the first time, these findings clarify the role of PIBF1 in regulating ATR/CHK1 signaling pathway and inhibiting the proliferation and migration of TNBC cell lines. These results demonstrate the oncogenic roles of PIBF1 and provide new insights into the function and the molecular mechanism of PIBF1 in malignant TNBC.

Identification of a new agent from natural products for the protection of embryonic anomalies is potentially valuable. To investigate the protective effect exerted by lycopene against nicotine-induced malformations, mouse embryos in embryonic day 8.5 with yolk sac placentas were cocultured with 1 mM nicotine and/or lycopene (1 × 10 -6 , 1 × 10 -5   μ M) for 48 h. The morphological defects and apoptotic cell deaths in the embryo and yolk sac placenta of the nicotine group were significantly increased. Exposure to nicotine resulted in reduced superoxide dismutase (SOD) activity and cytoplasmic SOD and cytoplasmic glutathione peroxidase mRNA levels, but increased lipid peroxidation level in embryos. Moreover, treatment with nicotine resulted in aggravated expressions of the mRNA or protein level of antiapoptotic (BCL2-associated X protein, B-cell lymphoma-extralarge, and caspase 3), anti-inflammatory (nuclear factor kappa-light-chain-enhancer of activated B cells and tumor necrosis factor-alpha), and vasculogenic (vascular endothelial growth factor-alpha, insulin-like growth factor-1, alpha smooth muscle actin, transforming growth factor-beta 1, and hypoxia inducible factor-1 alpha) factors in the embryo and yolk sac placenta. However, all the parameters were significantly improved by treatment with lycopene, as compared to the nicotine group. These findings indicate the potential of lycopene as a protective agent against embryonic anomalies and yolk sac vasculogenic and placenta-forming defects induced by nicotine through modulations of oxidative, apoptotic, vasculogenic, and inflammatory activities., Competing Interests: The authors declare that they have no conflicts of interests., (Copyright © 2020 Seul Gi Park et al.)

Studies have suggested that dysregulation of peroxisomal lipid metabolism might play an important role in colorectal cancer (CRC) development. Here, we found that Kras G12D -driven CRC tumors demonstrate dysfunctional peroxisomal b-oxidation and identified Nudt7 (peroxisomal coenzyme A diphosphatase NUDT7) as one of responsible peroxisomal genes. In Kras G12D -driven CRC tumors, the expression level of Nudt7 was significantly decreased. Treatment of azoxymethane/dextran sulfate sodium (AOM/DSS) into Nudt7 knockout ( Nudt7 -/- ) mice significantly induced lipid accumulation and the expression levels of CRC-related genes whereas xenografting of Nudt7 -overexpressed LS-174T cells into mice significantly reduced lipid accumulation and the expression levels of CRC-related genes. Ingenuity pathway analysis of microarray using the colon of Nudt7 -/- and Nudt7 +/+ mice treated with AOM/DSS suggested Wnt signaling as one of activated signaling pathways in Nudt7 -/- colons. Upregulated levels of β-catenin were observed in the colons of Kras G12D and AOM/DSS - treated Nudt7 -/- mice and downstream targets of β-catenin such as Myc , Ccdn1, and Nos2 , were also significantly increased in the colon of Nudt7 -/- mice. We observed an increased level of palmitic acid in the colon of Nudt7 -/- mice and attachment of palmitic acid-conjugated chitosan patch into the colon of mice induced the expression levels of b-catenin and CRC-related genes. Overall, our data reveal a novel role for peroxisomal NUDT7 in Kras G12D -driven CRC development.

Oxidative stress is a key mediator of neuronal death in acute brain injuries, such as epilepsy, trauma, and stroke. Although it is accompanied by diverse cellular changes, increases in levels of intracellular zinc ion (Zn 2+ ) and calcium ion (Ca 2+ ) may play a critical causative role in oxidative neuronal death. However, the mechanistic link between Zn 2+ and Ca 2+ dyshomeostasis in neurons during oxidative stress is not well-understood. Here, we show that the exposure of cortical neurons to H 2 O 2 led to a zinc-triggered calcium influx, which resulted in neuronal death. The cyclin-dependent kinase inhibitor, NU6027, inhibited H 2 O 2 -induced Ca 2+ increases and subsequent cell death in cortical neurons, without affecting the early increase in Zn 2+ . Therefore, we attempted to identify the zinc-regulated Ca 2+ pathway that was inhibited by NU6027. The expression profile in cortical neurons identified transient receptor potential cation channel 5 (TRPC5) as a candidate that is known to involve in the generation of epileptiform burst firing and epileptic neuronal death (Phelan KD et al. 2012a; Phelan KD et al. 2013b). NU6027 inhibited basal and zinc-augmented TRPC5 currents in TRPC5-overexpressing HEK293 cells. Consistently, cortical neurons from TRPC5 knockout mice were highly resistant to H 2 O 2 -induced death. Moreover, NU6027 is neuroprotective in kainate-treated epileptic rats. Our results demonstrate that TRPC5 is a novel therapeutic target against oxidative neuronal injury in prolonged seizures and that NU6027 is a potent inhibitor of TRPC5.

Purpose: To describe the phenotypes of a newly developed Pde6b-deficient rat model of retinal degeneration., Methods: Pde6b knockout rats were produced by CRISPR-Cpf1 technology. Pde6b knockout rats were evaluated for ocular abnormalities by comparison with wild-type eyes. Eyes were imaged using fundus photography and optical coherence tomography (OCT), stained by hematoxylin and eosin (H&E), and examined by TUNEL assay. Finally, eyes were functionally assessed by electroretinograms (ERGs)., Results: Pde6b knockout rats exhibited visible photoreceptor degeneration at 3 weeks of postnatal age. The fundus appearance of mutants was notable for pigmentary changes, vascular attenuation with an irregular vascular pattern, and outer retinal thinning, which resembled retinitis pigmentosa (RP) in humans. OCT showed profound retinal thinning in Pde6b knockout rats; the outer nuclear layer (ONL) was significantly thinner in Pde6b knockout rats, with relative preservation of the inner retina at 3 weeks of postnatal age. H&E staining confirmed extensive degeneration of the ONL, beginning at 3 weeks of postnatal age; no ONL remained in the retina by 16 weeks of postnatal age. Retinal sections of Pde6b knockout rats were highly positive for TUNEL, specifically in the ONL. In ERGs, Pde6b knockout rats showed no detectable a- or b-waves at 8 weeks of postnatal age., Conclusions: The Pde6b knockout rat exhibits photoreceptor degeneration. It may provide a better model for experimental therapy for RP because of its slower progression and larger anatomic architecture than the corresponding mouse model. Further studies in this rat model may yield insights into effective therapies for human RP.

Type I interferon (IFN-I) plays a critical role in the antitumor immune response. In our previous study, we showed that IFN-I-inducible 2'-5' oligoadenylate synthetase-like 1 (OASL1) negatively regulated IFN-I production upon tumor challenge similar to that of viral infection. Thus, OASL1-deficient ( Oasl1 - / - ) mice were more resistant to implanted tumor growth than wild-type (WT) mice. In this study, we investigated whether targeting or suppressing OASL1 could show synergistic effects on tumor clearance with conventional cancer therapies (such as chemotherapy and radiotherapy) using Oasl1 - / - mice and a transplantable lung metastatic tumor cell model. Upon treatment with the anticancer drug cisplatin, we found that Oasl1 - / - mice showed enhanced resistance to injected tumors compared to untreated Oasl1 - / - mice. Similarly, irradiated Oasl1 - / - mice showed better resistance to tumor challenge than untreated Oasl1 - / - mice. Additionally, we found that Oasl1 - / - mice applied with both types of the cancer therapies contained more cytotoxic effector cells, such as CD8 + T cells and NK cells, and produced more cytotoxic effector cytokine IFN- γ as well as IFN-I in their tumor-containing lungs compared to untreated Oasl1 - / - mice. Collectively, these results show that targeting OASL1 together with conventional cancer therapies could be an effective strategy to enhance treatment efficacy.

The rat is a time-honored traditional experimental model animal, but its use is limited due to the difficulty of genetic modification. Although engineered endonucleases enable us to manipulate the rat genome, it is not known whether the newly identified endonuclease Cpf1 system is applicable to rats. Here we report the first application of CRISPR-Cpf1 in rats and investigate whether Apoe knockout rat can be used as an atherosclerosis model. We generated Apoe- and/or Ldlr-deficient rats via CRISPR-Cpf1 system, characterized by high efficiency, successful germline transmission, multiple gene targeting capacity, and minimal off-target effect. The resulting Apoe knockout rats displayed hyperlipidemia and aortic lesions. In partially ligated carotid arteries of rats and mice fed with high-fat diet, in contrast to Apoe knockout mice showing atherosclerotic lesions, Apoe knockout rats showed only adventitial immune infiltrates comprising T lymphocytes and mainly macrophages with no plaque. In addition, adventitial macrophage progenitor cells (AMPCs) were more abundant in Apoe knockout rats than in mice. Our data suggest that the Cpf1 system can target single or multiple genes efficiently and specifically in rats with genetic heritability and that Apoe knockout rats may help understand initial-stage atherosclerosis.

Symbionts play an indispensable role in gut homeostasis, but underlying mechanisms remain elusive. To clarify the role of lactic-acid-producing bacteria (LAB) on intestinal stem-cell (ISC)-mediated epithelial development, we fed mice with LAB-type symbionts such as Bifidobacterium and Lactobacillus spp. Here we show that administration of LAB-type symbionts significantly increased expansion of ISCs, Paneth cells, and goblet cells. Lactate stimulated ISC proliferation through Wnt/β-catenin signals of Paneth cells and intestinal stromal cells. Moreover, Lactobacillus plantarum strains lacking lactate dehydrogenase activity, which are deficient in lactate production, elicited less ISC proliferation. Pre-treatment with LAB-type symbionts or lactate protected mice in response to gut injury provoked by combined treatments with radiation and a chemotherapy drug. Impaired ISC-mediated epithelial development was found in mice deficient of the lactate G-protein-coupled receptor, Gpr81. Our results demonstrate that LAB-type symbiont-derived lactate plays a pivotal role in promoting ISC-mediated epithelial development in a Gpr81-dependent manner., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) results from triglyceride accumulation within the liver and some of them advances to non-alcoholic steatohepatitis (NASH). It is important to note that in NAFLD development, hepatic de novo lipogenesis (DNL) derives from excess carbohydrates and fats under a condition of excess energy through β-oxidation. As a main regulator for DNL, sterol regulatory element-binding protein 1 (Srebp-1) forms complex with progesterone receptor membrane component 1 (Pgrmc1). To investigate whether Pgrmc1 may have a notable effect on DNL via SREBP-1 activation, we generated Pgrmc1 knockout (KO) mice and fed a high fat diet for one month. High-fat-fed Pgrmc1 KO mice showed a substantial increase in levels of hepatic TG accumulation, and they were predisposed to NAFLD when compared to WT mice. Loss of Pgrmc1 increased mature SREBP-1 protein level, suggesting that induction of hepatic steatosis in Pgrmc1 KO mice might be triggered by de novo lipogenesis. Moreover, Pgrmc1 KO mice were also more vulnerable to early stage of NASH, showing high levels of alanine aminotransferase, obesity-linked pro-inflammatory cytokines, and fibrosis markers. This is interesting because Pgrmc1 involves with the first step in regulating the hepatic de novo lipogenesis under an excess energy condition.

The key to successful drug discovery and development is to find the most suitable animal model of human diseases for the preclinical studies. The recent emergence of engineered endonucleases is allowing for efficient and precise genome editing, which can be used to develop potentially useful animal models for human diseases. In particular, zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeat systems are revolutionizing the generation of diverse genetically-engineered experimental animals including mice, rats, rabbits, dogs, pigs, and even non-human primates that are commonly used for preclinical studies of the drug discovery. Here, we describe recent advances in engineered endonucleases and their application in various laboratory animals. We also discuss the importance of genome editing in animal models for more closely mimicking human diseases.

Osteoarthritis (OA) is the most common degenerative joint disease; however, its etiopathogenesis is not completely understood. Here we show a role for NUDT7 in OA pathogenesis. Knockdown of NUDT7 in normal human chondrocytes results in the disruption of lipid homeostasis. Moreover, Nudt7 -/- mice display significant accumulation of lipids via peroxisomal dysfunction, upregulation of IL-1β expression, and stimulation of apoptotic death of chondrocytes. Our genome-wide analysis reveals that NUDT7 knockout affects the glycolytic pathway, and we identify Pgam1 as a significantly altered gene. Consistent with the results obtained on the suppression of NUDT7, overexpression of PGAM1 in chondrocytes induces the accumulation of lipids, upregulation of IL-1β expression, and apoptotic cell death. Furthermore, these negative actions of PGAM1 in maintaining cartilage homeostasis are reversed by the co-introduction of NUDT7. Our results suggest that NUDT7 could be a potential therapeutic target for controlling cartilage-degrading disorders.

In the absence of the RNA-templated reverse transcriptase, telomerase, the predominant means of terminal addition, arises from break-induced replication (BIR) at multiple homologous subtelomeric Y' loci and among internal homeologous (imperfect) (polyG1-3T) tracts. These last tracts are interspersed between subtelomeric Y' direct repeats. One major survivor class contains very short (~50 bp) terminal telomere repeats. This size is sufficient for slow growth and partial telomere functionality and cell viability. However, in cells carrying the mre11A470T allele, adjacent to the predicted Rad50/Mre11 junction, cells thrive at wild-type rates, with small, but reproducible, increases in telomere length. We have proposed that the increase in telomere size and growth rate are causally linked. To understand the BIR process at the telomere, we initiated studies of large-tract (RAD51-sensitive) homologous BIR in MRE11 and mre11A470T cells in a model color assay coupled with CHEF gel analysis and marker retention. Wild-type and mutant homologous BIR rates are maintained at the same level as the rates between wild-type and mutant homeologous BIR. However, the fidelity of BIR products was severely altered in mre11A470T cells. We find that 95% of homologous BIR in MRE11 cells gives rise to the expected product size, while 25% of BIR products in mre11A470T cells were of unpredicted size (error-prone), most of which initiated at an aberrant site. However, ~25% of homeologous MRE11 cells and 1/7 of homeologous mre11A470T cells underwent error-prone BIR. This class is initiated erroneously, followed by secondary events that elongate or truncate the telomere. We conclude that error-prone BIRs are increased in homeologous recombination in wild-type and in mre11A470T cells. This finding may explain the bypass of senescence in telomerase-negative cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)