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This file contains Supplemental figures, tables, and legends that accompany the main figures, as indicated in each legend. There are 3 supplemental tables and 6 supplemental figures. Table S1. List of potential target of the GNS compounds that were profiled in Figure 1 of the main text. Table S2. Half-maximal inhibitor concentration of the EGFR inhibitors tested. Table S3. Blood-brain-barrier penetration of the GNS compounds as assessed by the indicated pharmacokinetic measurements. Figure S1. Structural modeling studies show the binding characteristics of the indicated EGFR inhibitors. Figure S2. The effects of each EGFR inhibitor in the cell lines expressing mutant or wild type EGFR. Figure S3. In vivo effects of the GNS compounds. Figure S4. In vivo anti-tumor effects of the GNS compounds. Figure S5. In vivo anti-tumor effects of the GNS compounds in intracranial EGFR-mutant lung cancer. Figure S6. In vivo anti-tumor effects of the indicated EGFR inhibitors in intracranial EGFR-mutant lung cancer.

The clinical utility of approved EGFR small-molecule kinase inhibitors is plagued both by toxicity against wild-type EGFR and by metastatic progression in the central nervous system, a disease sanctuary site. Here, we report the discovery and preclinical efficacy of GNS-1486 and GNS-1481, two novel small-molecule EGFR kinase inhibitors that are selective for T790M-mutant isoforms of EGFR. Both agents were effective in multiple mouse xenograft models of human lung adenocarcinoma (T790M-positive or -negative), exhibiting less activity against wild-type EGFR than existing approved EGFR kinase inhibitors (including osimertinib). In addition, GNS-1486 showed superior potency against intracranial metastasis of EGFR-mutant lung adenocarcinoma. Our results offer a preclinical proof of concept for new EGFR kinase inhibitors with the potential to improve therapeutic index and efficacy against brain metastases in patients. Cancer Res; 77(5); 1200–11. ©2017 AACR.

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

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.

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.

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., Non-alcoholic fatty liver disease: Boosting regulatory enzyme a potential treatment A deficiency in levels of a key enzyme disturbs lipid metabolism in the liver and triggers non-alcoholic fatty liver disease (NAFLD). NAFLD is a common chronic condition in which fat builds up in the liver, heightening the risk of serious health problems. However, the exact mechanisms behind NAFLD are unclear. In experiments on mice, Eun-Jung Jin at Wongwang University in Iksan, South Korea, and co-workers found that deficiencies of an enzyme called ACOT12 can induce NAFLD regardless of dietary intake. The team generated mice without the Acot12 gene, which boosted the biosynthesis of lipid and accumulation of cholesterol in liver cells. Further examination showed that ACOT12 may directly interact with a protein that prompts cholesterol trafficking and degradation. Loss of ACOT12 activated a signaling pathway that inhibited this process. Their findings may inform NAFLD therapies.

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.

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.

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.

Here, we found that heterozygous null of peroxisomal

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.

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.

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.

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.

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.

Sphingosine 1-phosphate (S1P) receptors (S1PRs) are a group of G protein-coupled receptors that confer a broad range of functional effects in chronic inflammatory diseases and metabolic diseases. S1PRs may also be involved in the development of non-alcoholic steatohepatitis (NASH), but the specific subtypes involved and the mechanism of action are unclear. Here we show that the livers of various mouse models of NASH as well as Kupffer cells had a particularly strong expression of S1pr4. Accordingly, genetic depletion of S1pr4 protected the mice against hepatic inflammation and fibrosis. Moreover, SLB736, a novel selective functional antagonist of SIPR4 prevented diet-induced NASH in mice without lymphopenia. S1P increased the expression of S1pr4 in Kupffer cells and activated the NLRP3 inflammasome through PLC/IP3/IP3R-dependent [Ca++] signaling. SLB736 treatment or S1pr4 depletion in Kupffer cells inhibited LPS-mediated Ca++ release and deactivated the NLRP3 inflammasome. S1PR4 antagonism may be a novel therapeutic strategy for NASH.

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.

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. 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. 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. 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.

BackgroundARID2 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.MethodsThe 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.ResultsThe 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.DiscussionARID2 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.

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 C¿ (PKC¿), 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 PKC¿ 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-PKC¿- NLRC4 axis and holds promise as a therapeutic target for NASH., This study was supported by grants from the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology, Korea (2017R1E1A1A01073206: KUL, 2017R1E1A1A01074207: EHK) and Asan Institute for Life Sciences, Korea (2018IP0557-1). We acknowledge the support from NIH grants R01 DK113592, R01 AA024206 to AEF; grants SAF2017- 85877R and PID2019-1116691RB from Plan Nacional de I+D, Spain and by the CIBEREHD, Instituto de Salud Carlos III, Spain; the center grant P50AA011999 Southern California Research Center for ALPD and Cirrhosis funded by NIAAA/ NIH; support from AGAUR of the Generalitat de Catalunya SGR-2017–1112 and the ’ER stress-mitochondrial cholesterol axis in obesity-associated insulin resistance and comorbidities’-Ayudas Fundación BBVA (35_2018) a Equipos de Investigación Científica 2017, the Red Nacional 2018–1 02 799 T ’Enfermedades metabólicas y cáncer’ and the Fundació Marató TV3 201916-30-31.

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 μM) 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.

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 (Zn2+) and calcium ion (Ca2+) may play a critical causative role in oxidative neuronal death. However, the mechanistic link between Zn2+ and Ca2+ dyshomeostasis in neurons during oxidative stress is not well-understood. Here, we show that the exposure of cortical neurons to H2O2 led to a zinc-triggered calcium influx, which resulted in neuronal death. The cyclin-dependent kinase inhibitor, NU6027, inhibited H2O2-induced Ca2+ increases and subsequent cell death in cortical neurons, without affecting the early increase in Zn2+. Therefore, we attempted to identify the zinc-regulated Ca2+ 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 H2O2-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.

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.

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.

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.

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.

Scrotal hyperthermia leads to oxidative stress and apoptosis in spermatogenic cells, which subsequently causes male infertility. In this study, we examined the effects ofβ-carotene and/or curcumin on heat-stress- (HS-) induced testicular injuries in mice. ICR male mice (8 weeks old) were consecutively treated withβ-carotene (10 mg/kg) and/or curcumin (20 mg/kg) orally once a day for 14 days and then subjected to single exposure with scrotal HS at 43°C for 15 min on day 7. HS induced a significant reduction in testicular weight, appearance of multinucleated giant cells, and desquamation of germ cells in destructive seminiferous tubules, as well as degenerative Leydig cells. Moreover, HS reduced the superoxide dismutase (SOD) activity and mRNA levels of mitochondrial SOD, phospholipid hydroperoxide glutathione peroxidase, B-cell lymphoma-extra-large, and 3β-hydroxysteroid dehydrogenase, with increases in lipid peroxidation levels and mRNA levels of BCL2-associated X protein and caspase-3 relative to those of the control group. However, these changes were significantly recovered by combined treatment withβ-carotene and curcumin after HS. These findings indicate that the combined treatment withβ-carotene and curcumin might be a valuable protective agent to ameliorate hyperthermic spermatogenic disorders via its potent antioxidative, antiapoptotic, and androgen synthetic effects.

Studies have suggested that dysregulation of peroxisomal lipid metabolism might play an important role in colorectal cancer (CRC) development. Here, we found that KrasG12D-driven CRC tumors demonstrate dysfunctional peroxisomal b-oxidation and identified Nudt7 (peroxisomal coenzyme A diphosphatase NUDT7) as one of responsible peroxisomal genes. In KrasG12D-driven CRC tumors, the expression level of Nudt7 was significantly decreased. Treatment of azoxymethane/dextran sulfate sodium (AOM/DSS) into Nudt7 knockout (Nudt7&minus, /&minus, ) 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&minus, and Nudt7+/+ mice treated with AOM/DSS suggested Wnt signaling as one of activated signaling pathways in Nudt7&minus, colons. Upregulated levels of &beta, catenin were observed in the colons of KrasG12D and AOM/DSS-treated Nudt7&minus, mice and downstream targets of &beta, catenin such as Myc, Ccdn1, and Nos2, were also significantly increased in the colon of Nudt7&minus, mice. We observed an increased level of palmitic acid in the colon of Nudt7&minus, 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 KrasG12D-driven CRC development.