The study aimed to investigate whether endogenous H2S pathway was involved in high-salt-stimulated mitochondria-related vascular endothelial cell (VEC) apoptosis.

The transcriptional coactivator Yes-associated protein (YAP) plays an important role in organ-size control and tumorigenesis. However, how Yap gene expression is regulated remains unknown. This study shows that the Ets family member GABP binds to the Yap promoter and activates YAP transcription. The depletion of GABP downregulates YAP, resulting in a G1/S cell-cycle block and increased cell death, both of which are substantially rescued by reconstituting YAP. GABP can be inactivated by oxidative mechanisms, and acetaminophen-induced glutathione depletion inhibits GABP transcriptional activity and depletes YAP. In contrast, activating YAP by deleting Mst1/Mst2 strongly protects against acetaminophen-induced liver injury. Similar to its effects on YAP, Hippo signaling inhibits GABP transcriptional activity through several mechanisms. In human liver cancers, enhanced YAP expression is correlated with increased nuclear expression of GABP. Therefore, we conclude that GABP is an activator of Yap gene expression and a potential therapeutic target for cancers driven by YAP.

Previous work on gene expression analysis based on RNA sequencing identified a variety of differentially expressed cDNA fragments in the genic male sterile-fertile line 114AB of Capsicum annuum L. In this work, we examined the accumulation of one of the transcript-derived fragments (TDFs), CaMF3 (male fertile 3), in the flower buds of a fertile line. The full genomic DNA sequence of CaMF3 was 1,951 bp long and contained 6 exons and 5 introns, with the complete sequence encoding a putative 25.89 kDa protein of 234 amino acids. The predicted protein of CaMF3 shared sequence similarity with members of the isoamyl acetate-hydrolyzing esterase (IAH1) protein family. CaMF3 expression was detected only in flower buds at stages 7 and 8 and in open flowers of a male fertile line; no expression was observed in any organs of a male sterile line. Fine expression analysis revealed that CaMF3 was expressed specifically in anthers of the fertile line. These results suggest that CaMF3 is an anther-specific gene that may be essential for anther or pollen development in C. annuum.

Calpains are calcium- and thiol-dependent proteases whose overactivation and degradation of various substrates have been implicated in a number of diseases and conditions such as cardiovascular dysfunction and ischemic stroke. With increasing evidence for calpain's role in cellular damage, the development of calpain inhibitors continues to be an important objective. Previously, we identified a highly specific calcium-dependent, calpain interacting peptide L-S-E-A-L, that showed homology to domains A and C of the only known endogenous inhibitor of calpains, calpastatin. This suggested that LSEAL had a calpain inhibitory function and synthetic LSEAL inhibited calpain I and II proteolysis of two calpain substrates, tau and alpha-synuclein. In the present study, we demonstrate that synthetic LSEAL is membrane permeable and is a potent inhibitor in two established models of calpain-mediated cell death using primary rat cortical neurons and SH-SY5Y neuroblastoma cells. In addition, we show that LSEAL inhibits calpain activity towards protein substrates as detected by an antibody to a calpain-specific breakdown product of spectrin. Taken together, these results suggest that LSEAL may represent a novel calpastatin mimetic with the potential for benefit in conditions of increased calpain activity such as stroke, traumatic brain injury or heart attack.

Due to the complexity of cardiac surgery, almost all patients need to be admitted to the intensive care unit (ICU) for postoperative care after surgery. After being discharged from the ICU, some patients need to be readmitted due to disease deterioration during hospitalization. We conducted a meta-analysis of the literature to investigate the incidence of readmission to the ICU in patients undergoing cardiac surgery.

Sodium-glucose cotransporter-2 inhibitors (SGLT2is), such as empagliflozin, lower blood glucose in type 2 diabetes mellitus and improve cardiorenal outcomes regardless of diabetes presence. Whether SGLT2is exert any effects on the brain's metabolism has not been studied. We conducted a single-arm clinical trial to investigate the effects of once daily administration of oral empagliflozin (25 mg) for 14 days on systemic and brain metabolism in 21 non-diabetics aged 55 years old or older. Empagliflozin lowered circulating insulin and elevated β-hydroxybutyrate over 34-h periods, both following its first administration and after 14 days of daily administration, with minor alterations in glucose homeostasis. Levels of phosphorylated insulin-like growth factor-1 receptor (pIGF-1R), phosphorylated insulin receptor (pIR), phosphorylated-in-tyrosine insulin receptor substrate-1 (pY-IRS-1), and phosphorylated protein kinase B or AKT (pAKT) were increased in extracellular vesicles enriched for neuronal origin (NEVs) following the first empagliflozin administration, but not after 14 days. Our finding of IGF-1R upregulation in NEVs is promising because several post-mortem and epidemiological studies support the idea that upregulation of IGF signaling may protect against Alzheimer's disease (AD). Moreover, our finding showing activation of insulin signaling and, in particular, the canonical pathway (pIR, pY-IRS-1, pAKT) in NEVs is important because such changes have been repeatedly associated with neuronal survival. Using brain magnetic resonance spectroscopy (MRS), we detected decreased concentrations of the excitatory neurotransmitter glutamate and its precursor glutamine after empagliflozin administration. This finding is also encouraging since glutamatergic excitotoxicity has long been implicated in AD pathology. Overall, our findings may motivate the repurposing of SGLT2is for use in AD and other, related diseases that are characterized by downregulation of IGF-1/insulin signaling in neurons and excitotoxicity.

pRb and p53 are two major tumor suppressors. Here, we found that p53 activates expression of Pirh2 and KPC1, two of the three ubiquitin ligases for p27. Loss of p53 in the absence of Skp2, the third ubiquitin ligase for p27, shrinks the cellular pool of p27 ubiquitin ligases to accumulate p27 protein. In the absence of pRb and p53, p27 was unable to inhibit DNA synthesis in spite of its abundance, but could inhibit division of cells that maintain DNA replication with rereplication. This mechanism blocked pRb/p53 doubly deficient pituitary and prostate tumorigenesis lastingly coexistent with bromodeoxyuridine-labeling neoplastic lesions, revealing an unconventional cancer cell vulnerability when pRb and p53 are inactivated.

In vitro and in vivo studies have demonstrated that brucine is able to inhibit the proliferation of liver cancer cells and growth of animal tumors, and may be a promising anticancer drug. However, high toxicity, poor water solubility, short half-life, narrow therapeutic window, and similar therapeutic and toxic doses limit its clinical application in the treatment of malignant tumors. In our previous study, brucine immuno-nanoparticles were successfully prepared and added to the culture medium of liver cancer SMMC-7721 cells, and the results indicated that the brucine immuno-nanoparticles were able to target the cell membrane of liver cancer SMMC-7721 cells and significantly inhibit the proliferation, adhesion, invasion and metastasis of SMMC-7721 cells. The aim of the present study was to investigate the antitumor effect of brucine immuno-nanoparticles in vivo by establishing an in situ transplanted liver cancer in nude mice. The results indicated that in vivo application of the brucine immuno-nanoparticles resulted in temporary liver and kidney damage, and significantly reduced the α-fetoprotein (AFP) secretion of tumor cells (Bru-NP-MAb vs. the other groups; P<0.05). The brucine concentration of tumor tissues in the brucine immuno-nanoparticles group was significantly increased compared with that of the brucine nanoparticles group (Bru-NP-MAb vs. Bru-NP group or brucine group; P<0.05). The brucine immuno-nanoparticles were able to inhibit tumor growth and cluster of differentiation 34 expression and angiogenesis of tumor tissues, and induce the apoptosis of tumor cells (Bru-NP-MAb vs. Bru-NP group or brucine group; P<0.05). In conclusion, as a novel type of targeted drug, brucine nanoparticles combined with anti-AFP monoclonal antibodies was more effective compared with brucine nanoparticles or brucine alone in inhibiting tumor growth via the enhancement of apoptosis, and the suppression of proliferation and angiogenesis in vivo. Therefore, the brucine immuno-nanoparticle is a promising targeted drug for the treatment of hepatocellular carcinoma.

Tumor-derived extracellular vesicles are important mediators of cell-to-cell communication during tumorigenesis. Here, we demonstrated that hepatocellular carcinoma (HCC)-derived ectosomes remodel the tumor microenvironment to facilitate HCC progression in an ectosomal PKM2-dependent manner. HCC-derived ectosomal PKM2 induced not only metabolic reprogramming in monocytes but also STAT3 phosphorylation in the nucleus to upregulate differentiation-associated transcription factors, leading to monocyte-to-macrophage differentiation and tumor microenvironment remodeling. In HCC cells, sumoylation of PKM2 induced its plasma membrane targeting and subsequent ectosomal excretion via interactions with ARRDC1. The PKM2-ARRDC1 association in HCC was reinforced by macrophage-secreted cytokines/chemokines in a CCL1-CCR8 axis-dependent manner, further facilitating PKM2 excretion from HCC cells to form a feedforward regulatory loop for tumorigenesis. In the clinic, ectosomal PKM2 was clearly detected in the plasma of HCC patients. This study highlights a mechanism by which ectosomal PKM2 remodels the tumor microenvironment and reveals ectosomal PKM2 as a potential diagnostic marker for HCC.

Neonatal acute respiratory distress syndrome (ARDS) has high morbidity and mortality rates worldwide, but there is a lack of pharmacologic treatment and clinical targeted therapies. In this study, we aimed to explore the effects of Lipocalin-2 (LCN2) on ferroptosis-mediated inflammation and oxidative stress in neonatal ARDS and the potential mechanism. In this study, we established an in vivo ARDS mouse model and an in vitro ARDS cell model by LPS (Lipopolysaccharide) stimulation. Lung tissue injury was evaluated by wet/dry ratios and histopathological examination. LCN2 expression was detected by qRT-PCR and Western blot. Inflammatory factors, oxidative stress and apoptosis were also detected. Ferroptosis was identified by detection of Fe2+ level and ferroptosis-associated protein expressions. Mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) pathway signaling was examined by Western blot analysis. The data revealed that LCN2 expression was significantly upregulated in neonatal mice with ARDS. Interference with LCN2 protected LPS-induced lung in neonatal mouse by reducing the radio of wet/dry and alleviating pathological damages. In addition, LCN2 silencing repressed LPS-induced inflammation, oxidative stress in vivo and in vitro, as well as apoptosis. Meanwhile, decreased level of Fe2+ and transferrin while increased levels of ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) were observed. The expression MAPK/ERK pathway was inhibited by depletion of LCN2. The present results suggest that LCN2 knockdown protected LPS-induced ARDS model via inhibition of ferroptosis-related inflammation and oxidative stress by inhibiting the MAPK/ERK pathway, thereby presenting a novel target for the treatment of ARDS.

Mitochondria need to be juxtaposed to phagosomes for the synergistic production of ample reactive oxygen species (ROS) in phagocytes to kill pathogens. However, how phagosomes transmit signals to recruit mitochondria has remained unclear. Here we found that the kinases Mst1 and Mst2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activated the GTPase Rac to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for the recruitment of mitochondria to phagosomes. Inactive forms of Rac, including the human Rac2(D57N) mutant, disrupted the TRAF6-ECSIT complex by sequestering TRAF6 and substantially diminished ROS production and enhanced susceptibility to bacterial infection. Our findings demonstrate that the TLR-Mst1-Mst2-Rac signaling axis is critical for effective phagosome-mitochondrion function and bactericidal activity.

Human insulin (INS) gene diverged from the ancestral genes of invertebrate and mammalian species millions of years ago. We previously found that mouse insulin gene (Ins2) isoforms are expressed in brain choroid plexus (ChP) epithelium cells, where insulin secretion is regulated by serotonin and not by glucose. We further compared human INS isoform expression in postmortem ChP and islets of Langerhans. We uncovered novel INS upstream open reading frame isoforms and their protein products. In addition, we found a novel alternatively spliced isoform that translates to a 74-amino acid (AA) proinsulin containing a shorter 19-AA C-peptide sequence, herein designated Cα-peptide. The middle portion of the conventional C-peptide contains β-sheet (GQVEL) and hairpin (GGGPG) motifs that are not present in Cα-peptide. Islet amyloid polypeptide (IAPP) is not expressed in ChP, and its amyloid formation was inhibited in vitro more efficiently by Cα-peptide than by C-peptide. Of clinical relevance, the ratio of the 74-AA proinsulin to proconvertase-processed Cα-peptide was significantly increased in islets from type 2 diabetes mellitus autopsy donors. Intriguingly, 100 years after the discovery of insulin, we found that INS isoforms are present in ChP from insulin-deficient autopsy donors.

Although carcinoembryonic antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) has been viewed as a tumor suppressor, increasing clinical evidence shows that high levels of CEACAM1 expression on tumors correlates with poor prognosis and high risk of metastasis. Here, we examined the consequences of CEACAM1 expression on tumor cells. We show that tumor cell-associated CEACAM1 causes intracellular retention of various NKG2D ligands in mouse and human tumor cells. CEACAM1-silenced tumor cells expressed more cell surface NKG2D ligands and exhibited greater sensitivity to natural killer cell-mediated cytolysis in vitro and rejection in vivo. Our studies reveal a novel mechanism through which CEACAM1-bearing tumor cells may escape immune-surveillance.

One mechanism of tumour suppression by pRb is repressing E2F1. Hence, E2f1 deletion diminishes tumorigenesis following Rb1 loss. However, E2F1 promotes both proliferation and apoptosis. It therefore remains unclear how de-repressed E2F1 promotes tumorigenesis. Another mechanism of pRb function is repressing Skp2 to elevate p27 to arrest proliferation. However, Skp2 deletion induced apoptosis, not proliferation arrest, in Rb1-deficient pituitary tumorigenesis. Here we show that Rb1 deletion induces higher expression of E2F1 target genes in the absence of Skp2. E2F1 binds less cyclin A but more target promoters when Rb1 is deleted with Skp2 knockout or p27T187A knockin, suggesting that stabilized p27 prevents cyclin A from binding and inhibiting E2F1. In Rb1-deficient pituitary tumorigenesis, Skp2 deletion or p27T187A mutation converts E2F1's role from proliferative to apoptotic. These findings delineate a pRb-Skp2-p27-cyclin A-E2F1 pathway that determines whether E2F1 is proliferative or apoptotic in Rb1-deficient tumorigenesis.

The proteasome is a large intracellular protease that is responsible for a large portion of intracellular proteolysis, in particular the degradation of a majority of short-lived and oxidized proteins. Inhibition of proteasome function occurs in response to multiple stressors, with proteasome inhibition sufficient for the induction of a wide range of cytotoxic processes. Although considerable advances have been made in the understanding of the proteasome, and the effects of proteasome inhibition, our understanding of these topics in Saccharomyces cerevisiae has been slowed by the inability of proteasome inhibitors to penetrate and/or be retained in S. cerevisiae. Expression of UMP1 is necessary for proteasome assembly in S. cerevisiae, and in the present study we examined the effectiveness of RNA interference for UMP1 as a means of achieving proteasome inhibition in S. cerevisiae. Induction of RNA interference for UMP1 resulted in a dramatic decrease in UMP1 at the protein level, which was not observed in cells transformed with control vector. RNA interference caused an impairment in proteasome function, and increase in protein oxidation, with proteins involved in both stress response and energy metabolism showing increased oxidation. Interestingly, RNA interference induced cell death that seemed to be autophagic in nature, suggesting possible cross talk between the proteasome and the autophagic proteolytic pathways. Taken together, these data indicate that RNA interference may be a useful model with which to study the effects of proteasome inhibition in S. cerevisiae and demonstrate the ability of proteasome inhibition to induce cytotoxic alterations in S. cerevisiae.

Hypoxia-induced pulmonary hypertension (HPH) is a lethal cardiovascular disease with the characteristic of severe remodeling of pulmonary vascular. Although a large number of dysregulated mRNAs, lncRNAs, circRNAs, and miRNAs related to HPH have been identified from extensive studies, the competitive endogenous RNA (ceRNA) regulatory network in the pulmonary artery that responds to hypoxia remains largely unknown.

TLR4 signaling plays key roles in the innate immune response to microbial infection. Innate immune cells encounter different mechanical cues in both health and disease to adapt their behaviors. However, the impact of mechanical sensing signals on TLR4 signal-mediated innate immune response remains unclear. Here we show that TLR4 signalling augments macrophage bactericidal activity through the mechanical sensor Piezo1. Bacterial infection or LPS stimulation triggers assembly of the complex of Piezo1 and TLR4 to remodel F-actin organization and augment phagocytosis, mitochondrion-phagosomal ROS production and bacterial clearance and genetic deficiency of Piezo1 results in abrogation of these responses. Mechanistically, LPS stimulates TLR4 to induce Piezo1-mediated calcium influx and consequently activates CaMKII-Mst1/2-Rac axis for pathogen ingestion and killing. Inhibition of CaMKII or knockout of either Mst1/2 or Rac1 results in reduced macrophage bactericidal activity, phenocopying the Piezo1 deficiency. Thus, we conclude that TLR4 drives the innate immune response via Piezo1 providing critical insight for understanding macrophage mechanophysiology and the host response.

More than 60% of nonsmall cell lung cancer (NSCLC) patients show a positive response to the first ALK inhibitor, crizotinib, which has been used as the standard treatment for newly diagnosed patients with ALK rearrangement. However, most patients inevitably develop crizotinib resistance due to acquired secondary mutations in the ALK kinase domain, such as the gatekeeper mutation L1196M and the most refractory mutation, G1202R. Here, we develop XMU-MP-5 as a new-generation ALK inhibitor to overcome crizotinib resistance mutations, including L1196M and G1202R. XMU-MP-5 blocks ALK signaling pathways and inhibits the proliferation of cells harboring either wild-type or mutant EML4-ALK in vitro and suppresses tumor growth in xenograft mouse models in vivo. Structural analysis provides insights into the mode of action of XMU-MP-5. In addition, XMU-MP-5 induces significant regression of lung tumors in two genetically engineered mouse (GEM) models, further demonstrating its pharmacological efficacy and potential for clinical application. These preclinical data support XMU-MP-5 as a novel selective ALK inhibitor with high potency and selectivity. XMU-MP-5 holds great promise as a new therapeutic against clinically relevant secondary ALK mutations.

Glucose consumption is generally increased in tumor cells to support tumor growth. Interestingly, we report that glycogen accumulation is a key initiating oncogenic event during liver malignant transformation. We found that glucose-6-phosphatase (G6PC) catalyzing the last step of glycogenolysis is frequently downregulated to augment glucose storage in pre-malignant cells. Accumulated glycogen undergoes liquid-liquid phase separation, which results in the assembly of the Laforin-Mst1/2 complex and consequently sequesters Hippo kinases Mst1/2 in glycogen liquid droplets to relieve their inhibition on Yap. Moreover, G6PC or another glycogenolysis enzyme-liver glycogen phosphorylase (PYGL) deficiency in both human and mice results in glycogen storage disease along with liver enlargement and tumorigenesis in a Yap-dependent manner. Consistently, elimination of glycogen accumulation abrogates liver growth and cancer incidence, whereas increasing glycogen storage accelerates tumorigenesis. Thus, we concluded that cancer-initiating cells adapt a glycogen storing mode, which blocks Hippo signaling through glycogen phase separation to augment tumor incidence.

To assess the possible neuroprotective effects of melatonin against brain white matter damage via the Toll-like receptor 4 (TLR4)/ nuclear factor-kappa B (NF-κB) signaling pathway in focal cerebral ischemic rats.