Efficacy of early administration of sacubitril/valsartan after coronary artery revascularization in patients with acute myocardial infarction complicated by moderate-to-severe mitral regurgitation: a randomized controlled trial.

Effects of angiotensin receptor/neprilysin inhibitors (ARNI) on ventricular remodeling in patients with heart failure, especially heart failure with reduced ejection fraction (HFrEF), are better than those of angiotensin-converting enzyme inhibitors (ACEI). Acute myocardial infarction (AMI) complicated by mitral regurgitation exacerbates ventricular remodeling and increases the risk of heart failure. There is limited evidence on the effects of early administration of ARNI in patients with AMI complicated by mitral regurgitation. The aim of this trial was to examine the effectiveness and the safety of early administration of sacubitril/valsartan after coronary artery revascularization in patients with AMI complicated by moderate-to-severe mitral regurgitation. This was a randomized, single-blind, parallel-group, controlled trial. From June 2021 to June 2022, we enrolled 142 consecutive patients with AMI complicated by moderate-to-severe mitral regurgitation and followed them for 12 months. The patients received standard treatment for AMI and were randomly assigned to receive ARNI or benazepril. The primary efficacy end points were the differences in mitral regurgitant jet area (MRJA), mitral regurgitant volume (MRV), concentration of n-terminal pro-brain natriuretic peptide (NT-proBNP), left ventricular ejection fraction (LVEF), and left ventricular end-diastolic volume and end-systolic volume (LVEDV and LVESV) between groups and within groups at baseline, 1, 3, 6, and 12 months. Secondary end points included the rates of heart failure hospitalization, all-cause mortality, refractory angina, malignant arrhythmias, recurrent myocardial infarction, and stroke. Safety end points included the rates of hyperkalemia, renal dysfunction, hypotension, angioedema, and cough. The ARNI group had significantly lower NT-proBNP levels than the benazepril group at 1 month and later (P < 0.001). MRJA and MRV significantly improved in the ARNI group compared with the benazepril group at 12 months (MRJA: - 3.21 ± 2.18 cm2 vs. - 1.83 ± 2.81 cm2, P < 0.05; MRV: - 27.22 ± 15.22 mL vs. - 13.67 ± 21.02 mL, P < 0.001). The ARNI group also showed significant reductions in LVEDV and LVESV (P < 0.05) and improvement in LVEF (P < 0.05). Secondary end point analysis showed a significantly higher rate of heart failure hospitalization in the benazepril group compared with the ARNI group (HR = 2.03, 95% CI 1.12-3.68, P = 0.021). Safety end point analysis showed a higher rate of hypotension in the ARNI group (P < 0.05). Early use of sacubitril/valsartan after coronary artery revascularization in patients with AMI complicated by moderate-to-severe mitral regurgitation can significantly reduce mitral regurgitation, improve ventricular remodeling, and decrease heart failure hospitalization. Nevertheless, caution is needed to avoid hypotension. Chinese Clinical Trial Registry (ChiCTR2100054255) registered on December 11, 2021.

An unexpected role of neutrophils in clearing apoptotic hepatocytes in vivo.

Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here, we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis, and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver, and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.

Every day, the immune cells clears the remains of billions of old and damaged cells that have undergone a controlled form of death. Removing them quickly helps to prevent inflammation or the development of autoimmune diseases. While immune cells called neutrophils are generally tasked with removing invading bacteria, macrophages are thought to be responsible for clearing dead cells. However, in healthy tissue, the process occurs so efficiently that it can be difficult to confirm which cells are responsible. To take a closer look, Cao et al. focused on the liver by staining human samples to identify both immune and dead cells. Unexpectedly, there were large numbers of neutrophils visible inside dead liver cells. Further experiments in mice revealed that after entering the dead cells, neutrophils engulfed the contents and digested the dead cell from the inside out. This was a surprising finding because not only are neutrophils not usually associated with dead cells, but immune cells usually engulf cells and bacteria from the outside rather than burrowing inside them. The importance of this neutrophil behaviour was shown when Cao et al. studied samples from patients with an autoimmune disease where immune cells attack the liver. In this case, very few dead liver cells contained neutrophils, and the neutrophils themselves did not seem capable of removing the dead cells, leading to inflammation. This suggests that defective neutrophil function could be a key contributor to this autoimmune disease. The findings identify a new role for neutrophils in maintaining healthy functioning of the liver and reveal a new target in the treatment of autoimmune diseases. In the future, Cao et al. plan to explore whether compounds that enhance clearance of dead cells by neutrophils can be used to treat autoimmune liver disease in mouse models of the disease.

WNT2-SOX4 positive feedback loop promotes chemoresistance and tumorigenesis by inducing stem-cell like properties in gastric cancer.

Gastric cancer (GC) is characterized by its vigorous chemoresistance to current therapies, which is attributed to the highly heterogeneous and immature phenotype of cancer stem cells (CSCs) during tumor initiation and progression. The secretory WNT2 ligand regulates multiple cancer pathways and has been demonstrated to be a potential therapeutic target for gastrointestinal tumors; however, its role involved in gastric CSCs (GCSCs) remains unclear. Here, we found that overexpression of WNT2 enhanced stemness properties to promote chemoresistance and tumorigenicity in GCSCs. Mechanistically, WNT2 was positively regulated by its transcription factor SOX4, and in turn, SOX4 was upregulated by the canonical WNT2/FZD8/ß-catenin signaling pathway to form an auto-regulatory positive feedback loop, resulting in the maintenance of GCSCs self-renewal and tumorigenicity. Furthermore, simultaneous overexpression of both WNT2 and SOX4 was correlated with poor survival and reduced responsiveness to chemotherapy in clinical GC specimens. Blocking WNT2 using a specific monoclonal antibody significantly disrupted the WNT2-SOX4 positive feedback loop in GCSCs and enhanced the chemotherapeutic efficacy when synergized with the chemo-drugs 5-fluorouracil and oxaliplatin in a GCSC-derived mouse xenograft model. Overall, this study identified a novel WNT2-SOX4 positive feedback loop as a mechanism for GCSCs-induced chemo-drugs resistance and suggested that the WNT2-SOX4 axis may be a potential therapeutic target for gastric cancer treatment.

Targeting LGSN restores sensitivity to chemotherapy in gastric cancer stem cells by triggering pyroptosis.

Gastric cancer (GC) is notoriously resistant to current therapies due to tumor heterogeneity. Cancer stem cells (CSCs) possess infinite self-renewal potential and contribute to the inherent heterogeneity of GC. Despite its crucial role in chemoresistance, the mechanism of stemness maintenance of gastric cancer stem cells (GCSCs) remains largely unknown. Here, we present evidence that lengsin, lens protein with glutamine synthetase domain (LGSN), a vital cell fate determinant, is overexpressed in GCSCs and is highly correlated with malignant progression and poor survival in GC patients. Ectopic overexpression of LGSN in GCSC-derived differentiated cells facilitated their dedifferentiation and treatment resistance by interacting with vimentin and inducing an epithelial-to-mesenchymal transition. Notably, genetic interference of LGSN effectively suppressed tumor formation by inhibiting GCSC stemness maintenance and provoking gasdermin-D-mediated pyroptosis through vimentin degradation/NLRP3 signaling. Depletion of LGSN combined with the chemo-drugs 5-fluorouracil and oxaliplatin could offer a unique and promising approach to synergistically rendering this deadly cancer eradicable in vivo. Our data place focus on the role of LGSN in GCSC regeneration and emphasize the critical importance of pyroptosis in battling GCSC.

Construction of a pancreatic cancer nerve invasion system using brain and pancreatic cancer organoids.

Pancreatic cancer (PC) is a fatal malignancy in the human abdominal cavity that prefers to invade the surrounding nerve/nerve plexus and even the spine, causing devastating and unbearable pain. The limitation of available in vitro models restricts revealing the molecular mechanism of pain and screening pain-relieving strategies to improve the quality of life of end-stage PC patients. Here, we report a PC nerve invasion model that merged human brain organoids (hBrO) with mouse PC organoids (mPCO). After merging hBrOs with mPCOs, we monitored the structural crosstalk, growth patterns, and mutual interaction dynamics of hBrO with mPCOs for 7 days. After 7 days, we also analyzed the pathophysiological statuses, including proliferation, apoptosis and inflammation. The results showed that mPCOs tend to approximate and intrude into the hBrOs, merge entirely into the hBrOs, and induce the retraction/shrinking of neuronal projections that protrude from the margin of the hBrOs. The approximating of mPCOs to hBrOs accelerated the proliferation of neuronal progenitor cells, intensified the apoptosis of neurons in the hBrOs, and increased the expression of inflammatory molecules in hBrOs, including NLRP3, IL-8, and IL-1ß. Our system pathophysiologically replicated the nerve invasions in mouse GEMM (genetically engineered mouse model) primary and human PCs and might have the potential to be applied to reveal the molecular mechanism of nerve invasion and screen therapeutic strategies in PCs.

IDO-1 inhibitor INCB24360 elicits distant metastasis of basal extruded cancer cells in pancreatic ductal adenocarcinoma.

Neoplastic cells of non-immunogenic pancreatic ductal adenocarcinoma (PDAC) express indoleamine 2,3-dioxygenase 1 (IDO-1), an immunosuppressive enzyme. The metabolites of IDO-1 in cancers provide one-carbon units that annihilate effector T cells, and recruit immunosuppressive cells. In this study we investigated how IDO-1 affected the neoplastic cell behaviors in PDACs. Using multiple markers co-labeling method in 45-µm-thick tissue sections, we showed that IDO-1 expression was uniquely increased in the neoplastic cells extruded from ducts' apical or basal domain, but decreased in lymph metastatic cells. IDO-1+ extruding neoplastic cells displayed increased vimentin expression and decreased cytokeratin expression in PDACs, characteristics of epithelial-mesenchymal transition (EMT). However, IDO-1 expression was uncorrelated with immunosuppressive infiltrates and clinicopathological characteristics of grim outcome. We replicated basal extrusion with EMT in murine KPIC PDAC organoids by long-term IFN-γ induction; application of IDO-1 inhibitor INCB24360 or 1-MT partially reversed basal extrusion coupled EMT. Ido-1 deletion in KPIC cells deprived its tumorigenicity in immunocompetent mice, decreased cellular proliferation and macropinocytic ability, and increased immunogenicity. KPIC organoids with IFN-γ-induced basal extrusion did not accelerate distant metastasis, whereas inhibition IFN-γ-induced IDO-1 with INB24360 but not 1-MT in KPIC organoids elicited liver metastasis of subcutaneous KPIC organoid tumors, suggesting that lower IDO-1 activity accelerated distant metastasis, whereas IDO-1 was indispensable for tumorigenicity of PDAC cells and supports the survival of extruding cells.

[Research Progress in Immunosuppressive Tumor Microenvironment of Gastrointestinal Cancer].

Gastrointestinal (GI) cancer, a common malignant tumor with a high incidence in China, is showing a trend of rising incidence and is afflicting increasingly younger patients. Meanwhile, there have been constant development and innovations in new therapeutic technologies, among which, immunotherapy is now leading in a new era in the treatment of GI cancer. However, the complexity and diversity of immunosuppressive tumor microenvironment (TME) bring many obstacles to the immunotherapy of solid tumors in the GI tract. In this paper, focusing on solid tumors in the GI tract, we reviewed the main factors affecting the formation of immunosuppressive TME, and summarized strategies for targeted immunosuppressive TME-based therapies. Moreover, we analyzed the synergistic mechanism of various combination immunotherapies and reported on the latest progress in and future direction of immunotherapy for GI cancer, intending to provide new perspectives for treating solid tumors in the GI tract with immumotherapy.

Characterizing the distributions of IDO-1 expressing macrophages/microglia in human and murine brains and evaluating the immunological and physiological roles of IDO-1 in RAW264.7/BV-2 cells.

Indoleamine 2,3-dioxygenase 1 (IDO-1) is an immunosuppressive enzyme expressed in the placenta, neoplastic cells, and macrophages to reject T cells by converting tryptophan into kynurenine. However, the role of IDO-1 in brain immunity, especially in the meninges, is unclear. We aim to elucidate the distribution pattern of IDO-1+ macrophages/microglia in the human brain tissues, human glioblastoma, APP/PS1 mouse brains, and quinolinic acid model brains and explore the physiological and immunological roles of IDO-1+ macrophages/microglia. Here, we find that both human and mouse macrophages/microglia of the perivascular and subarachnoid space and in glioblastoma (GBM) expressed IDO-1 but not macrophages/microglia of parenchyma. Using IDO-1 inhibitors including 1-MT and INCB24360, we observed that inhibiting IDO-1 reduced the cellular size and filopodia growth, fluid uptake, and the macropinocytic and phagocytic abilities of human blood monocytes and RAW264.7/BV-2 cells. Inhibiting IDO-1 with 1-MT or INCB24360 increased IL-1ß secretion and suppressed NLRP3 expression in RAW264.7/BV-2 cells. Our data collectively show that IDO-1 expression in perivascular and meninges macrophages/microglia increases cellular phagocytic capacity and might suppress overactivation of inflammatory reaction.

Basal microvilli define the metabolic capacity and lethal phenotype of pancreatic cancer.

Apical microvilli of polarized epithelial cells govern the absorption of metabolites and the transport of fluid in tissues. Previously, we reported that tall and dense basal microvilli present on the endothelial cells of pancreatic cancers, a lethal malignancy with a high metabolism and unusual hypomicrovascularity, contain nutrient trafficking vesicles and glucose; their length and density were related to the glucose uptake of pancreatic cancers in a small-scale analysis. However, the implications of basal microvilli on pancreatic cancers are unknown. Here, we evaluated the clinical implications of basal microvilli in 106 pancreatic cancers. We found that basal microvilli are a dominant change in pancreatic cancers. The presence of longer and denser basal microvilli on the microvessels in pancreatic cancer tissues positively correlated with increased glucose uptake and higher metastatic (or invasive) and proliferative potentials of neoplastic cells and vice versa. Clinically, postoperative patients with longer and denser basal microvilli were more prone to unfavorable pathological characteristics and dismal prognoses. They were even more refractory to adjuvant therapy than those with shorter and thinner basal microvilli were. Our findings show that basal microvilli define the metabolic capacity and lethal phenotype of pancreatic cancers. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

The physiological characteristics of the basal microvilli microvessels in pancreatic cancers.

Pancreatic cancer (PC) is a highly lethal tumor with controversial high glucose uptake and hypomicrovascularity, and the hypomicrovasculature, which is considered to have poor perfusion, blocks the delivery of drugs to tumors. The preferential existence of a novel endothelial projection with trafficking vesicles in PCs, referring to basal microvilli, was described previously. However, the perfusion and nutrients delivering status of the basal microvilli microvessels are unknown. Here, we used the perfusion of fluorescently labeled CD31 antibody, lectin, and 2-NBDG to autochthonous PC-bearing mice, immunostaining, probe-based confocal laser endoscopy and three-dimensional (3D) reconstruction to study the nutrient trafficking, and perfusion status of the basal microvilli microvasculature in PC. Our data showed that the coperfusion of lectin and CD31 is an efficient way to show the microcirculation in most healthy organs. However, coperfusion with lectin and CD31 is inefficient for showing the microcirculation in PCs compared with that in healthy organs and immunostaining. This method does not reflect the nutrient trafficking status in the microvessels, especially in basal microvilli microvessels of PCs. In basal microvilli microvessels that were poorly labeled by lectin, we observed large vesicle-like structures with 2-NBDG preferentially located at the base of the basal microvilli or in basal microvilli, and there were long filopodia on the luminal surface of the human PC microvasculature. Our observations suggest that the PC microvasculature, especially basal microvilli microvessels, is well perfused and might be highly efficient in the trafficking of glucose or other nutrients, indicating that macropinocytosis might participate in the nutrient trafficking.

MicroRNA-760-mediated low expression of DUSP1 impedes the protective effect of NaHS on myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury (MIRI) is the leading cause of the poor prognosis for patients undergoing clinical cardiac surgery. Micro-RNAs are involved in MIRI; however, the effect of miR-760 on MIRI and the molecular mechanisms behind it have not yet been described. For our in-vivo experiments, 20 rats were randomly distributed between 2 groups (n = 10): the sham-treatment group and the ischemia-reperfusion (I/R) group. For our in-vitro experiments, H9C2 cells were subjected to hypoxia for 6 h, and then reoxygenated to establish an hypoxia-reoxygenation (H/R) model. High expression levels of of miR-760 were observed in the rats subjected to MIRI and the H9C2 cells subjected to H/R. Further, the levels of lactate dehydrogenase (LDH) and malonaldehyde (MDA) were increased, and the size of the myocardial infarct was notably greater in the rats subjected to MIRI, suggesting that miR-760 worsens the effects of MIRI. The inhibitory effects from NaHS on apoptosis were enhanced, as were the expression levels of cleaved caspase 3 and cleaved PARP in H9C2 cells exposed to H/R, and with low-expression levels of miR-760. TargetScan and dual luciferase reporter assays further confirmed the targeted relationship between dual-specificity protein phosphatase (DUSP1) and miR-760. Additionally, miR-760 overexpression and H/R treatment of H9C2 cells inhibited the expression of DUSP1, which further promoted apoptosis. Furthermore, DUSP1 enhanced the anti-apoptotic effects of NaHS in rats subjected to MIRI. Taken together, these findings suggest that miR-760 inhibits the protective effect of NaHS against MIRI.

HuR stabilizes HTT mRNA via interacting with its exon 11 in a mutant HTT-dependent manner.

Huntington's Disease (HD) is a monogenetic neurodegenerative disorder mainly caused by the cytotoxicity of the mutant HTT protein (mHTT) encoded by the mutant HTT gene. Lowering HTT mRNA has been extensively studied as a potential therapeutic strategy, but how its level is regulated endogenously has been unclear. Here we report that the RNA-binding protein (RBP) HuR interacts with and stabilizes HTT mRNA in an mHTT-dependent manner. In HD cells but not wild-type cells, siRNA knockdown or CRISPR-induced heterozygous knockout of HuR decreased HTT mRNA stability. HuR interacted with HTT mRNA at a conserved site in exon 11 rather than the 3'-UTR region of the mRNA. Interestingly, this interaction was dependent on the presence of mHTT, likely via the activation of MAPK11, which enhanced cytosolic localization of the HuR protein. Thus, mHTT, MAPK11 and HuR may form a positive feedback loop that stabilizes HTT mRNA and enhances mHTT accumulation, which may contribute to HD progression. Our data reveal a novel regulatory mechanism of HTT mRNA via non-canonical binding of HuR.

Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds.

Accumulation of mutant proteins is a major cause of many diseases (collectively called proteopathies), and lowering the level of these proteins can be useful for treatment of these diseases. We hypothesized that compounds that interact with both the autophagosome protein microtubule-associated protein 1A/1B light chain 3 (LC3)1 and the disease-causing protein may target the latter for autophagic clearance. Mutant huntingtin protein (mHTT) contains an expanded polyglutamine (polyQ) tract and causes Huntington's disease, an incurable neurodegenerative disorder2. Here, using small-molecule-microarray-based screening, we identified four compounds that interact with both LC3 and mHTT, but not with the wild-type HTT protein. Some of these compounds targeted mHTT to autophagosomes, reduced mHTT levels in an allele-selective manner, and rescued disease-relevant phenotypes in cells and in vivo in fly and mouse models of Huntington's disease. We further show that these compounds interact with the expanded polyQ stretch and could lower the level of mutant ataxin-3 (ATXN3), another disease-causing protein with an expanded polyQ tract3. This study presents candidate compounds for lowering mHTT and potentially other disease-causing proteins with polyQ expansions, demonstrating the concept of lowering levels of disease-causing proteins using autophagosome-tethering compounds.

Involvement of the microglial NLRP3 inflammasome in the anti-inflammatory effect of the antidepressant clomipramine.

BACKGROUND: Depression has recently been referred to as a neuroimmune disease because it is characterized by inflammatory changes in the cerebral cortex and hippocampus. Studies have demonstrated that microglial activation plays a crucial role in releasing inflammatory cytokines in the central nervous system (CNS), thereby contributing to depression, the mechanism underlying which remains unclear. METHODS: First, we examined microglial activation and inflammatory changes in C57BL/6 male mice injected with lipopolysaccharide (LPS; 1 mg/kg), which leads to depressive behaviors in mice that were attenuated by the antidepressant clomipramine. Second, we utilized a BV2 cell line and primary microglial cultures to determine the inflammatory response in vitro, and the effects of clomipramine exerted on the inflammatory response using real-time polymerase chain reaction and ELISA. Third, we utilized NLRP3 (NOD-like receptor protein 3) knock-out (KO) mice to prove that NLRP3 is involved in the effects of clomipramine. RESULTS: The results showed that LPS injection induced depressive-like behaviors in mice, as assessed using several behavioral tests including body weight, and forced swimming and tail suspension tests. The LPS-induced expression of interleukin-1beta (IL-1ß), IL-6, and tumor necrosis factor alpha could be downregulated by clomipramine pre-treatment both in vivo and in vitro. The inhibitory effect of clomipramine on the LPS-induced increase in cytokines was found at both the protein and gene levels. Clomipramine significantly reduced the LPS-induced increase in NLRP3 gene expression in BV2 cells. Furthermore, we utilized NLRP3 KO mice to explore whether NLPP3 was involved in this process and found that clomipramine treatment inhibits the LPS-induced increased expression of IL-1ß. CONCLUSION: These results imply that clomipramine could attenuate depressive behaviors and neuroinflammation induced by LPS via partial regulation of NLRP3.

Transplantation of Retinal Progenitor Cells from Optic Cup-Like Structures Differentiated from Human Embryonic Stem Cells In Vitro and In Vivo Generation of Retinal Ganglion-Like Cells.

Human embryonic stem cells (hESCs) have the potential to differentiate along the retinal lineage. We have efficiently differentiated human pluripotent stem cells into optic cup-like structures by using a novel retinal differentiation medium (RDM). The purpose of this study was to determine whether the retinal progenitor cells (RPCs) derived from hESCs can integrate into the host retina and differentiate into retinal ganglion cells (RGCs) in vivo. In this study, hESCs (H9-GFP) were induced to differentiate into optic cup-like structures by using our novel differentiation system. The RPCs extracted from the optic cup-like structures were transplanted into the vitreous cavity of N-methyl-d-aspartic acid-treated mice. Sham-treated eyes received the same amount of RDM. The host retinas were analyzed by triple immunofluorescence on the fourth and fifth weeks after transplantation. The optic cup-like structures were efficiently differentiated from hESCs by using our novel differentiation system in vitro for 6-8 weeks. The RPCs extracted from the optic cup-like structures migrated and integrated into the ganglion cell layer (GCL) of the host retina. Furthermore, the remaining transplanted cells were spread over the GCL and had a complementary distribution with host residual RGCs in the GCL of the mouse retina. Surprisingly, some of the transplanted cells expressed the RGC-specific marker Brn3a. These findings demonstrated that the RPCs derived from hESCs could integrate into the host GCL and differentiate into retinal ganglion-like cells in vivo, suggesting that RPCs can be used as an ideal source in supplying countless RGC and embryonic stem cell-based replacement therapies may be a promising treatment to restore vision in patients with degenerative retinal diseases.

Induction of human somatostatin and parvalbumin neurons by expressing a single transcription factor LIM homeobox 6.

Human GABAergic interneurons (GIN) are implicated in normal brain function and in numerous mental disorders. However, the generation of functional human GIN subtypes from human pluripotent stem cells (hPSCs) has not been established. By expressing LHX6, a transcriptional factor that is critical for GIN development, we induced hPSCs to form GINs, including somatostatin (SST, 29%) and parvalbumin (PV, 21%) neurons. Our RNAseq results also confirmed the alteration of GIN identity with the overexpression of LHX6. Five months after transplantation into the mouse brain, the human GABA precursors generated increased population of SST and PV neurons by overexpressing LHX6. Importantly, the grafted human GINs exhibited functional electrophysiological properties and even fast-spiking-like action potentials. Thus, expression of the single transcription factor LHX6 under our GIN differentiation condition is sufficient to robustly induce human PV and SST subtypes.

Modeling Down Syndrome with Patient iPSCs Reveals Cellular and Migration Deficits of GABAergic Neurons.

The brain of Down syndrome (DS) patients exhibits fewer interneurons in the cerebral cortex, but its underlying mechanism remains unknown. By morphometric analysis of cortical interneurons generated from DS and euploid induced pluripotent stem cells (iPSCs), we found that DS GABA neurons are smaller and with fewer neuronal processes. The proportion of calretinin over calbindin GABA neurons is reduced, and the neuronal migration capacity is decreased. Such phenotypes were replicated following transplantation of the DS GABAergic progenitors into the mouse medial septum. Gene expression profiling revealed altered cell migratory pathways, and correction of the PAK1 pathway mitigated the cell migration deficit in vitro. These results suggest that impaired migration of DS GABAergic neurons may contribute to the reduced number of interneurons in the cerebral cortex and hippocampus in DS patients.

LSL-KrasG12D; LSL-Trp53R172H/+; Ink4flox/+; Ptf1/p48-Cre mice are an applicable model for locally invasive and metastatic pancreatic cancer.

Pancreatic cancer (PC) accumulates multiple genetic mutations, including activating KRAS mutations and inactivating TP53, SMAD4 and CDKN2A mutations, during progression. The combination of mutant KRAS with a single inactivating TP53, SMAD4 or CDKN2A mutation in genetically engineered mouse models (GEMMs) showed that these mutations exert different synergistic effects in PC. However, the effect of the combination of TP53, CDKN2A and KRAS mutations on the trajectory of PC progression is unknown. Here, we report a GEMM that harbors KRAS (KrasG12D), TP53 (Trp53R172H/+), CDKN2A (Ink4flox/+) and Ptf1/p48-Cre (KPIC) mutations. Histopathology showed that KPIC mice developed adenocarcinoma that strongly resembled the pathology of human PC, characterized by rich desmoplastic stroma and low microvascularity. The median survival of KPIC mice was longer than that of LSL-KrasG12D; Ink4flox/flox; Ptf1/p48-Cre mice (KIC) (89 vs 62 days) and shorter than that of KRAS (KrasG12D), TP53 (Trp53R172H/+) and Ptf1/p48-Cre (KPC) mice. Moreover, the neoplastic cells of KPIC mice were epithelial, highly proliferative tumor cells that exhibited ERK and MAPK pathway activation and high glucose uptake. Isolated neoplastic cells from spontaneous KPIC tumors showed all molecular profiles and cellular behaviors of spontaneous KPIC tumors, including epithelial-mesenchymal transition (EMT) under drug stress as well as tumorigenic, metastatic and invasive abilities in immunocompetent mice. Furthermore, orthotopic and metastatic tumors of KPIC cells almost recapitulated the pathology of spontaneous KPIC tumors. These data show that in addition to spontaneous KPIC tumors, KPIC cells are a valuable tool for preclinical studies of locally invasive and metastatic PC.

Urine-derived induced pluripotent stem cells as a modeling tool for paroxysmal kinesigenic dyskinesia.

Paroxysmal kinesigenic dyskinesia (PKD) is a monogenic movement disorder with autosomal dominant inheritance. We previously identified the proline-rich transmembrane protein 2 (PRRT2) as a causative gene of PKD. However, the pathogenesis of PKD remains largely unknown so far. In addition, applicable modeling tools to investigate the underlying mechanisms of PKD are still lacking. The combination of disease-specific human induced pluripotent stem cells (iPSCs) and directed cell differentiation offers an ideal platform for disease modeling. In this study, we generated two iPSC lines from the renal epithelial cells of one PKD patient with the hotspot c.649dupC mutation (PKD-iPSCs). These cell lines were positive for alkaline phosphatase Nanog, Tra-1-80, Tra-1-60, SSEA-3 and SSEA-4. Teratomas with three blastoderms including ectoderm, mesoderm, and endoderm were obtained two months after injection of PKD-iPSCs into NOD/SCID mice. The expression of PRRT2 mRNA was decreased in PKD-iPSCs compared with that of the control iPSCs. Furthermore, PKD-iPSCs possessed the differentiation potential of functional glutamatergic, dopaminergic and motor neurons in vitro. Electrophysiological examinations revealed that the current densities of fast activated and deactivated sodium channels as well as voltage gated potassium channels were not different between the neurons from PKD-iPSCs and control iPSCs. Thus, PKD-iPSCs are a feasible modeling tool to investigate the pathogenic mechanisms of PKD.

Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation.

DNMT1 is an important epigenetic regulator that plays a key role in the maintenance of DNA methylation. Here we determined the crystal structure of DNMT1 in complex with USP7 at 2.9 Å resolution. The interaction between the two proteins is primarily mediated by an acidic pocket in USP7 and Lysine residues within DNMT1's KG linker. This intermolecular interaction is required for USP7-mediated stabilization of DNMT1. Acetylation of the KG linker Lysine residues impair DNMT1-USP7 interaction and promote the degradation of DNMT1. Treatment with HDAC inhibitors results in an increase in acetylated DNMT1 and decreased total DNMT1 protein. This negative correlation is observed in differentiated neuronal cells and pancreatic cancer cells. Our studies reveal that USP7-mediated stabilization of DNMT1 is regulated by acetylation and provide a structural basis for the design of inhibitors, targeting the DNMT1-USP7 interaction surface for therapeutic applications.