Loss of TET Activity in the Postnatal Mouse Brain Perturbs Synaptic Gene Expression and Impairs Cognitive Function.

Conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation (TET) family proteins leads to the accumulation of 5hmC in the central nervous system; however, the role of 5hmC in the postnatal brain and how its levels and target genes are regulated by TETs remain elusive. We have generated mice that lack all three Tet genes specifically in postnatal excitatory neurons. These mice exhibit significantly reduced 5hmC levels, altered dendritic spine morphology within brain regions crucial for cognition, and substantially impaired spatial and associative memories. Transcriptome profiling combined with epigenetic mapping reveals that a subset of genes, which display changes in both 5hmC/5mC levels and expression patterns, are involved in synapse-related functions. Our findings provide insight into the role of postnatally accumulated 5hmC in the mouse brain and underscore the impact of 5hmC modification on the expression of genes essential for synapse development and function.

Lenvatinib as second-line treatment in patients with unresectable hepatocellular carcinoma: A retrospective analysis.

Objective: The purpose of this study is to determine the efficacy and safety of lenvatinib as second-line therapy in Chinese patients with unresectable hepatocellular carcinoma (HCC). Methods: We performed a retrospective analysis of Chinese patients with unresectable HCC who received second-line treatment of lenvatinib at three institutions from November 2018 to February 2022. Demographic and clinicopathologic characteristics, data on the treatment regimens were obtained from medical records. Tumor response was evaluated every 4-6 weeks by modified Response Evaluation Criteria in Solid Tumors (mRECIST). Results: In total, 50 patients with unresectable HCC who received second-line treatment of lenvatinib were enrolled in this study. The objective response rate (ORR) was 18.0% and the disease control rate (DCR) was 74.0%, respectively. The duration of response (DoR) was 6.0 months. The median progression-free survival (PFS) and overall survival (OS) were 5.0 and 8.5 months, respectively. Patients who received ICIs combined with anti-angiogenic inhibitors as first-line therapy, achieving CR/PR at first-line therapy, with PFS≥6months at first-line therapy had a higher DCR. Univariate and multivariate analysis showed that AFP (ng/ml)<400, absence of extrahepatic metastasis, Child-Pugh A, tumor number<3, ICIs combined with anti-angiogenic inhibitors as first-line therapy, CR/PR to first-line therapy, and PFS≥6months at first-line therapy were independent factors of favorable PFS. Univariate analysis showed that absence of extrahepatic metastasis, tumor number<3, ICIs combined with anti-angiogenic inhibitors as first-line therapy, and PFS≥6months at first-line therapy were significantly associated with longer OS. Multivariate analysis showed that absence of extrahepatic metastasis, Child-Pugh A, tumor number<3, CR/PR to first-line therapy and PFS≥6months at first-line therapy were independent prognostic factors of OS. The majority of AEs were grade 1-2, and were reversible. Grade 3/4 AEs occurred in 12 patients (24.0%) and were mostly connected with hand-foot skin reactions (10.0%), and 10 patients had lenvatinib dose reductions. Two toxicity-related treatment interruptions were attributed to grade 3 hand-foot skin reaction, and grade 4 proteinuria, respectively. Conclusion: This study confirms the efficacy and safety of lenvatinib as second-line therapy after progression on sorafenib or ICIs combined with anti-angiogenic inhibitors.

Design and Implementation of a New Training Flight Simulator System.

Aircraft flight simulators have good cost efficiency, high reusability, and high flight safety. All airlines and aircraft manufacturing companies choose it as sophisticated training equipment for ground simulation, effectively reducing pilot training costs, ensuring personnel safety and aircraft wear and tear. The new simulator proposed in this paper combines a digital motion-cueing algorithm, flight software and motion platform to make pilots feel as if they are in the real world. By using EtherCAT technology to drive the motion-cueing platform, it can improve the data transmission speed of the simulator as well as the strong anti-interference ability of communication and the control operation efficiency. Therefore, the simulated flight subjects can perform long-distance and large-angle training. Next, a set of measurement systems was established to provide monitoring items including attitude, velocity and acceleration, which can be displayed on the screen and recorded on the computer in real time and dynamically. Finally, seven training subjects were implemented to demonstrate the feasibility and correctness of the proposed method.

Npas3 regulates stemness maintenance of radial glial cells and neuronal migration in the developing mouse cerebral cortex.

The neuronal PAS domain 3 (NPAS3) is a member of the basic helix-loop-helix (bHLH) PAS family of transcription factors and is implicated in psychiatric and neurodevelopmental disorders. NPAS3 is robustly expressed in the cortical ventricle zone (VZ), a transient proliferative zone containing progenitor cells, mainly radial glial cells, destined to give rise to cortical excitatory neurons. However, the role of NPAS3 in corticogenesis remains largely unknown. In this study, we knocked down Npas3 expression in the neural progenitor cells residing in the cortical VZ to investigate the role of Npas3 in cerebral cortical development in mice. We demonstrated that Npas3 knockdown profoundly impaired neuronal radial migration and changed the laminar cell fate of the cells detained in the deep cortical layers. Furthermore, the downregulation of Npas3 led to the stemness maintenance of radial glial cells and increased the proliferation rate of neural progenitor cells residing in the VZ/subventricular zone (SVZ). These findings underline the function of Npas3 in the development of the cerebral cortex and may shed light on the etiology of NPAS3-related disorders.

Norcantharidin overcomes vemurafenib resistance in melanoma by inhibiting pentose phosphate pathway and lipogenesis via downregulating the mTOR pathway.

Melanoma is the most aggressive type of skin cancer with a high incidence and low survival rate. More than half of melanomas present the activating BRAF mutations, along which V600E mutant represents 70%-90%. Vemurafenib (Vem) is an FDA-approved small-molecule kinase inhibitor that selectively targets activated BRAF V600E and inhibits its activity. However, the majority of patients treated with Vem develop acquired resistance. Hence, this study aims to explore a new treatment strategy to overcome the Vem resistance. Here, we found that a potential anticancer drug norcantharidin (NCTD) displayed a more significant proliferation inhibitory effect against Vem-resistant melanoma cells (A375R) than the parental melanoma cells (A375), which promised to be a therapeutic agent against BRAF V600E-mutated and acquired Vem-resistant melanoma. The metabolomics analysis showed that NCTD could, especially reverse the upregulation of pentose phosphate pathway and lipogenesis resulting from the Vem resistance. In addition, the transcriptomic analysis showed a dramatical downregulation in genes related to lipid metabolism and mammalian target of the rapamycin (mTOR) signaling pathway in A375R cells, but not in A375 cells, upon NCTD treatment. Moreover, NCTD upregulated butyrophilin (BTN) family genes, which played important roles in modulating T-cell response. Consistently, we found that Vem resistance led to an obvious elevation of the p-mTOR expression, which could be remarkably reduced by NCTD treatment. Taken together, NCTD may serve as a promising therapeutic option to resolve the problem of Vem resistance and to improve patient outcomes by combining with immunomodulatory therapy.

The Therapeutic Role of Slit2 in Anti-fibrosis, Anti-inflammation and Anti-oxidative Stress in Rats with Coronary Heart Disease.

To investigate the efficacy of Slit2 in the rats with coronary heart disease (CHD). CHD model were constructed by feeding high-fat food and injecting with pituitrin in rat, followed by recombinant Slit2 treatment, and then the cardiac function was evaluated by echocardiography, and the indicators concerning the cardiomyocyte injury markers and lipoprotein status and oxidative stress were measured. The Slit2 expression in the heart tissues was identified by immunofluorescence. Enzyme-linked immunosorbent assay (ELISA) was carried out to detect inflammatory cytokines, H2DCFDA staining to determine the ROS generation in heart tissues, Masson trichrome staining to observe myocardial fibrosis, and qRT-PCR and Western blotting to detect gene and protein expressions. Slit2 decreased the levels of LDH, CK-MB, cTnI, TG, TC and LDL-C and increased HDL-C level in CHD rats. In the normal heart tissues, Slit2 expression was significantly lower in cardiomyocytes than cardiac fibroblasts. Furthermore, the expressions of VCAM-1, ICAM-1, fibronectin and TGF-ß1 were increased in the heart tissues of CHD rats with the obvious myocardial fibrosis, which were dose-dependently reversed by recombinant Slit2. In addition, recombinant Slit2 also dose-dependently increased the activity of NO, SOD, CAT and GSH-Px, and decreased TNF-α, IL-6, MCP-1, MDA and ROS in CHD rats. Slit2 was downregulated in myocardial tissue and plasma of CHD rats. Recombinant Slit2, by regulating the level of blood lipid, can relieve the myocardial fibrosis, inflammation and oxidative stress in CHD.

Association between cigarette smoking and colorectal cancer sidedness: A multi-center big-data platform-based analysis.

BACKGROUND: Sidedness (right/left) of colorectal cancer (CRC) is essential for treatment. Whether carcinogenesis of tobacco varies by sidedness remains unclear. The present study aims to evaluate the sidedness tendency of cigarette smoking and to explore its impact on prognosis. METHODS: In the multi-center retrospective study, data on 46 166 Chinese CRC patients were extracted from a big-data platform. Logistic regression analyses were performed to evaluate qualitative and quantitative associations between smoking and tumor sidedness. Survival analyses were conducted in metastatic CRC. RESULTS: History of smoking was associated with left-sided CRC (LSCRC; Adjusted odds ratio, 1.25; 95% CI, 1.16 - 1.34; P < .001). The sidedness tendency towards LSCRC increased from non-smokers, to ex-smokers, and to current smokers (P for trend < .001). Longer duration (P for trend < .001) and larger total amount of cigarette smoking (P for trend < .001) were more associated with LSCRC, respectively. The association was confirmed in both left-sided colon cancer and rectal cancer, but was stronger for rectal cancer (P = .016). Alcoholism significantly enhanced the association by 7% (P = .027). Furthermore, prognostic advantage of metastatic LSCRC diminished among ever-smokers, with contrary survival impacts of smoking on either side of CRC. CONCLUSIONS: History of smoking was associated with LSCRC in a positive dose-response relationship, and presented opposite prognostic impacts on right- and left-sided tumors. Smoking potentially plays an instrumental role in the mechanism for sidedness heterogeneity in CRC.

C-X-C motif chemokine 16, modulated by microRNA-545, aggravates myocardial damage and affects the inflammatory responses in myocardial infarction.

BACKGROUND: Myocardial infarction (MI), a common type of coronary heart disease, is the major cause of morbidity and mortality around the world. Chemokine-mediated inflammatory cell infiltration and local inflammatory damage response are recent research hotspots. Hence, we attempted to examine the role of C-X-C motif chemokine 16 (CXCL16) as a potential candidate in MI. METHODS: Human cardiomyocytes were treated with hypoxia/reoxygenation (H/R) to establish an in vitro cell model. GEO database provided the clinical data of MI patients and GSEA verified the relationship of chemokine and MI. CCK-8 and flow cytometry analyses were used to measure cell viability and apoptosis. Bioinformatics analysis and luciferase reporter assay were conducted to determine the correlation between CXCL16 and miR-545. qRT-PCR and western blot assays were performed to investigate the expression level of the indicated genes. The activity of lactate dehydrogenase (LDH) and the levels of TNF-α, IL-6, IL-1ß, and IL-10 were explored using ELISA assay. RESULTS: CXCL16 was increased in MI. CXCL16 knockdown can reverse the inhibitory effect of H/R treatment on cell viability, while overexpression of CXCL16 showed the opposite trend. MiR-545 directly targeted CXCL16 and negatively regulated CXCL16 levels. MiR-545 promoted cell proliferation and inhibited apoptosis in the MI cell model, which attenuated the CXCL16-induced injury on cardiomyocytes. CONCLUSION: These findings demonstrated that CXCL16 aggravated MI damage through being directly targeted by miR-545 and mediating inflammatory responses, thereby providing potential therapeutic targets for MI therapy.

An integrative pan-cancer analysis of biological and clinical impacts underlying ubiquitin-specific-processing proteases.

Ubiquitin-specific-processing proteases (USPs), the largest deubiquitinating enzyme (DUB) subfamily, play critical roles in cancer. However, clinical utility of USPs is hindered by limited knowledge about their varied and substrate-dependent actions. Here, we performed a comprehensive investigation on pan-cancer impacts of USPs by integrating multi-omics data and annotated data resources, especially a deubiquitination network. Meaningful insights into the roles of 54 USPs in 29 types of cancers were generated. Although rare mutations were observed, a majority of USPs exhibited significant expressional alterations, prognostic impacts and strong correlations with cancer hallmark pathways. Notably, from our DUB-substrate interaction prediction model, additional USP-substrate interactions (USIs) were recognized to complement knowledge gap about cancer-relevant USIs. Intriguingly, expression signatures of the USIs revealed clinically meaningful cancer subtypes, where key USPs and substrates cooperatively contributed to significant prognosis differences among subtypes. Overall, this investigation provides a valuable resource to assist mechanism research and clinical utility about USPs.

XPO1 inhibitor KPT-330 synergizes with Bcl-xL inhibitor to induce cancer cell apoptosis by perturbing rRNA processing and Mcl-1 protein synthesis.

XPO1 (exportin1) mediates nuclear export of proteins and RNAs and is frequently overexpressed in cancers. In this study, we show that the orally bioavailable XPO1 inhibitor KPT-330 reduced Mcl-1 protein level, by which it synergized with Bcl-xL inhibitor A-1331852 to induce apoptosis in cancer cells. KPT-330/A-1331852 combination disrupted bindings of Mcl-1 and Bcl-xL to Bax, Bak, and/or Bim, elicited mitochondrial outer membrane permeabilization, and triggered apoptosis. KPT-330 generally mitigated mRNA expression and protein synthesis rather than mRNA nuclear export or protein stability of Mcl-1. KPT-330 inhibited mTORC1/4E-BP1 and Mnk1/eIF4E axes, which disrupted the eIF4F translation initiation complex but was dispensable for Mcl-1 reduction and KPT-330/A-1331852 combination-induced apoptosis. Mature rRNAs are integral components of the ribosome that determines protein synthesis ability. KPT-330 impeded nucleolar rRNA processing and reduced total levels of multiple mature rRNAs. Reconstitution of XPO1 by expressing degradation-resistant C528S mutant retained rRNA amount, Mcl-1 expression, and Bcl-xL inhibitor resistance upon KPT-330 treatment. KPT-330/A-1331852 combination suppressed growth and enhanced apoptosis of non-small cell lung cancer xenografts. Therefore, we clarify the reason of apoptosis resistance of cancer cells to XPO1 inhibition and develop a potential strategy for treating solid tumors.

Targeting KPNB1 overcomes TRAIL resistance by regulating DR5, Mcl-1 and FLIP in glioblastoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine with potential anticancer effect, but innate and adaptive TRAIL resistance in majority of cancers limit its clinical application. Karyopherin ß1 (KPNB1) inhibition in cancer cells has been reported to abrogate the nuclear import of TRAIL receptor DR5 and facilitate its localization on the cell surface ready for TRAIL stimulation. However, our study reveals a more complicated mechanism. Genetic or pharmacological inhibition of KPNB1 potentiated TRAIL-induced apoptosis selectively in glioblastoma cells mainly by unfolded protein response (UPR). First, it augmented ATF4-mediated DR5 expression and promoted the assembly of death-inducing signaling complex (DISC). Second, it freed Bax and Bak from Mcl-1. Third, it downregulated FLIPL and FLIPS, inhibitors of caspase-8 cleavage, partly through upregulating ATF4-induced 4E-BP1 expression and disrupting the cap-dependent translation initiation. Meanwhile, KPNB1 inhibition-induced undesirable autophagy and accelerated cleaved caspase-8 clearance. Inhibition of autophagic flux maintained cleaved caspase-8 and aggravated apoptosis induced by KPNB1 inhibitor plus TRAIL, which were abolished by caspase-8 inhibitor. These results unveil new molecular mechanism for optimizing TRAIL-directed therapeutic efficacy against cancer.

Rg3 inhibits gemcitabine-induced lung cancer cell invasiveness through ROS-dependent, NF-κB- and HIF-1α-mediated downregulation of PTX3.

PTX3, a member of the long pentraxin subfamily, associated with innate immunity is indispensable for resistance to some cancer. Gemcitabine, an analog of cytosine arabinoside, has shown restrained benefits because of profound chemoresistance. The PTX3 expression on GEM in human lung cancer cells have not yet been clarified; the present study aimed to show reactive oxygen species (ROS) mediatory PTX3 expression through distinct mechanisms. Whereas ginsenoside Rg3 is a herbal medicine with strong antitumor activity. Furthermore, we tested the hypothesis; Rg3 abrogates GEM-induced production of ROS-mediated activation of Akt and extracellular signal-regulated kinase (ERK) pathways and inhibits nuclear piling-up of nuclear factor kappa B (NF-κB) and HIF-1α. On the basis of time and dose-dependent manner, our data demonstrated that GEM-induced PTX3 expression was dependent on ROS generation as it was abrogated by pretreatment of lung cancer cells with the free radical scavenger N-acetyl-l-cysteine. Our data demonstrated that PTX3 upregulation by GEM correlated with the time-dependent escalation of NF-κB and HIF-1α in the nucleus resulted from phosphorylation-induced degradation of IκBα, whereas HIF-1α upregulation was NF-κB-dependent. Increase in ROS expression in lung cancer cells on GEM treatment preceded the nuclear accumulation of NF-κB and HIF-1α and suppression of ROS diminished these effects. ERK1/2 and Akt activation mediated the effect of ROS on NF-κB and HIF-1α and their pharmacological inhibition suppressed GEM-induced PTX3. Our study findings reinforced the role regarding PTX3 signaling in GEM-induced resistance and pointed toward an unintended and undesired effect of chemotherapy and to get an active regimen; the synergy was associated with NF-κB downregulation in lung cancer.

KPNB1 inhibition disrupts proteostasis and triggers unfolded protein response-mediated apoptosis in glioblastoma cells.

The nuclear import receptor karyopherin ß1 (KPNB1) is involved in the nuclear import of most proteins and in the regulation of multiple mitotic events. Upregulation of KPNB1 has been observed in cancers including glioblastoma. Depletion of KPNB1 induces mitotic arrest and apoptosis in cancer cells, but the underlying mechanism is not clearly elucidated. Here, we found that downregulation and functional inhibition of KPNB1 in glioblastoma cells induced growth arrest and apoptosis without apparent mitotic arrest. KPNB1 inhibition upregulated Puma and Noxa and freed Mcl-1-sequestered Bax and Bak, leading to mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Moreover, combination of Bcl-xL inhibitors and KPNB1 inhibition enhanced apoptosis in glioblastoma cells. KPNB1 inhibition promoted cytosolic retention of its cargo and impaired cellular proteostasis, resulting in elevated polyubiquitination, formation of aggresome-like-induced structure (ALIS), and unfolded protein response (UPR). Ubiquitination elevation and UPR activation in KPNB1-deficient cells were reversed by KPNB1 overexpression or inhibitors of protein synthesis but aggravated by inhibitors of autophagy-lysosome or proteasome, indicating that rebalance of cytosolic/nuclear protein distribution and alleviation of protein overload favor proteostasis and cell survival. Chronic activation of eIF2α/ATF4 cascade of UPR was responsible for the upregulation of Puma and Noxa, apoptosis and ABT-263 sensitivity. Taken together, our findings demonstrate that KPNB1 is required for proteostasis maintenance and its inhibition induces apoptosis in glioblastoma cells through UPR-mediated deregulation of Bcl-2 family members.

PRRT2 deficiency induces paroxysmal kinesigenic dyskinesia by regulating synaptic transmission in cerebellum.

Mutations in the proline-rich transmembrane protein 2 (PRRT2) are associated with paroxysmal kinesigenic dyskinesia (PKD) and several other paroxysmal neurological diseases, but the PRRT2 function and pathogenic mechanisms remain largely obscure. Here we show that PRRT2 is a presynaptic protein that interacts with components of the SNARE complex and downregulates its formation. Loss-of-function mutant mice showed PKD-like phenotypes triggered by generalized seizures, hyperthermia, or optogenetic stimulation of the cerebellum. Mutant mice with specific PRRT2 deletion in cerebellar granule cells (GCs) recapitulate the behavioral phenotypes seen in Prrt2-null mice. Furthermore, recording made in cerebellar slices showed that optogenetic stimulation of GCs results in transient elevation followed by suppression of Purkinje cell firing. The anticonvulsant drug carbamazepine used in PKD treatment also relieved PKD-like behaviors in mutant mice. Together, our findings identify PRRT2 as a novel regulator of the SNARE complex and provide a circuit mechanism underlying the PRRT2-related behaviors.

UMI-77 primes glioma cells for TRAIL-induced apoptosis by unsequestering Bim and Bak from Mcl-1.

Malignant glioma is the most common and aggressive form of brain tumor with poor prognosis of survival. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent but is insufficient of inducing apoptosis in some types of gliomas. In this study, we showed that the small-molecule Mcl-1 inhibitor UMI-77 sensitized glioma cells to TRAIL treatment, as evidenced by cell viability assay, Annexin V staining and JC-1 staining. Combination of UMI-77 and TRAIL in glioma cells led to the activation of caspase-8 and Bid, cleavage of caspase-3 and poly-ADP ribose polymerase (PARP), accumulation of tBid in the mitochondria and release of cytochrome c into the cytosol. UMI-77 alone or in combination with TRAIL untethered pro-apoptotic Bcl-2 proteins Bim and Bak from the sequestration of Mcl-1 and promoted the conformational activation of Bak. Small hairpin RNA (shRNA) of Bid attenuated the cleavage of caspase-8, Bid, caspase-3 and PARP, and reduced the cytotoxicity of UMI-77 plus TRAIL as compared with control shRNA cells, indicating this synergy entails the crosstalk between extrinsic and intrinsic apoptotic signaling. Taken together, UMI-77 enhances TRAIL-induced apoptosis by unsequestering Bim and Bak, which provides a novel therapeutic strategy for the treatment of gliomas.

Transdermal granisetron for the prevention of nausea and vomiting following moderately or highly emetogenic chemotherapy in Chinese patients: a randomized, double-blind, phase III study.

BACKGROUND: The granisetron transdermal delivery system (GTDS) has been demonstrated effectiveness in the control of chemotherapy-induced nausea and vomiting (CINV) in previous studies. This is the first phase III study to evaluate the efficacy and tolerability of GTDS in patients receiving moderately emetogenic chemotherapy (MEC) or highly emetogenic chemotherapy (HEC) in China. METHODS: A total of 313 patients were randomized into the GTDS group (one transdermal granisetron patch, 7 days) or the oral granisetron group (granisetron oral 2 mg/day, ≥2 days). The primary endpoint was the percentage of patients achieving complete control (CC) from chemotherapy initiation until 24 h after final administration (PEEP). Chi-square test and Fisher's exact test were used for statistical analysis. RESULTS: Two hundred eighty-one patients were included in the per protocol analysis. During PEEP, CC was achieved by 67 (47.52%) patients in the GTDS group and 83 (59.29%) patients in the oral granisetron group. There was no statistical significance between the groups (P=0.0559). However, the difference of the CC percentage mainly occurred on the first day of chemotherapy between the groups. The CC was 70.13% on day 1 in the GTDS group, which was significantly lower than that of 91.03% in the oral granisetron group in the full analysis set. In the following days of chemotherapy, the CC was similar between the groups. In terms of cisplatin-contained regimen and female, there was statistical significance between the groups. Both treatments were well tolerated and safe. The most common adverse event was constipation. CONCLUSIONS: GTDS provided effective and well-tolerated control of CINV in Chinese patients, especially to non-cisplatin-contained regimen.