Target of rapamycin (TOR), a master sensor for growth factors and nutrition availability in eukaryotic species, is a specific target protein of rapamycin. Rapamycin inhibits TOR kinase activity viaFK506 binding protein 12 kDa (FKBP12) in all examined heterotrophic eukaryotic organisms. In Arabidopsis, several independent studies have shown that AtFKBP12 is non-functional under aerobic condition, but one study suggests that AtFKBP12 is functional during anaerobic growth. However, the functions of AtFKBP12 have never been examined in parallel under aerobic and anaerobic growth conditions so far. To this end, we cloned the FKBP12 gene of humans, yeast, and Arabidopsis, respectively. Transgenic plants were generated, and pharmacological examinations were performed in parallel with Arabidopsis under aerobic and anaerobic conditions. ScFKBP12 conferred plants with the strongest sensitivity to rapamycin, followed by HsFKBP12, whereas AtFKBP12 failed to generate rapamycin sensitivity under aerobic condition. Upon submergence, yeast and human FKBP12 can significantly block cotyledon greening while Arabidopsis FKBP12 only retards plant growth in the presence of rapamycin, suggesting that hypoxia stress could partially restore the functions of AtFKBP12 to bridge the interaction between rapamycin and TOR. To further determine if communication between TOR and auxin signaling exists in plants, yeast FKBP12 was introduced into DR5::GUS homozygous plants. The transgenic plants DR5/BP12 were then treated with rapamycin or KU63794 (a new inhibitor of TOR). GUS staining showed that the auxin content of root tips decreased compared to the control. DR5/BP12 plants lost sensitivity to auxin after treatment with rapamycin. Auxin-defective phenotypes, including short primary roots, fewer lateral roots, and loss of gravitropism, occurred in DR5/BP12 plants when seedlings were treated with rapamycin+KU63794. This indicated that the combination of rapamycin and KU63794 can significantly inhibit TOR and auxin signaling in DR5/BP12 plants. These studies demonstrate that TOR is essential for auxin signaling transduction in Arabidopsis.

Target of rapamycin (TOR) acts as a master regulator to control cell growth by integrating nutrient, energy, and growth factors in all eukaryotic species. TOR plays an evolutionarily conserved role in regulating the transcription of genes associated with anabolic and catabolic processes in Arabidopsis, but little is known about the functions of TOR in photosynthesis and phytohormone signaling, which are unique features of plants. In this study, AZD8055 (AZD) was screened as the strongest active-site TOR inhibitor (asTORi) in Arabidopsis compared with TORIN1 and KU63794 (KU). Gene expression profiles were evaluated using RNA-seq after treating Arabidopsis seedlings with AZD. More than three-fold differentially expressed genes (DEGs) were identified in AZD-treated plants relative to rapamycin-treated plants in previous studies. Most of the DEGs and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways involved in cell wall elongation, ribosome biogenesis, and cell autophagy were common to both AZD- and rapamycin-treated samples, but AZD displayed much broader and more efficient inhibition of TOR compared with rapamycin. Importantly, the suppression of TOR by AZD resulted in remodeling of the expression profile of the genes associated with photosynthesis and various phytohormones, indicating that TOR plays a crucial role in modulating photosynthesis and phytohormone signaling in Arabidopsis. These newly identified DEGs expand the understanding of TOR signaling in plants. This study elucidates the novel functions of TOR in photosynthesis and phytohormone signaling and provides a platform to study the downstream targets of TOR in Arabidopsis.

Pre-B and pre-T lymphocytes must orchestrate a transition from a highly proliferative state to a quiescent one during development. Cyclin D3 is essential for these cells' proliferation, but little is known about its posttranslational regulation at this stage. Here, we show that the dual specificity tyrosine-regulated kinase 1A (DYRK1A) restrains Cyclin D3 protein levels by phosphorylating T283 to induce its degradation. Loss of DYRK1A activity, via genetic inactivation or pharmacologic inhibition in mice, caused accumulation of Cyclin D3 protein, incomplete repression of E2F-mediated gene transcription, and failure to properly couple cell cycle exit with differentiation. Expression of a nonphosphorylatable Cyclin D3 T283A mutant recapitulated these defects, whereas inhibition of Cyclin D:CDK4/6 mitigated the effects of DYRK1A inhibition or loss. These data uncover a previously unknown role for DYRK1A in lymphopoiesis, and demonstrate how Cyclin D3 protein stability is negatively regulated during exit from the proliferative phases of B and T cell development.

Therapeutic options for diffuse malignant peritoneal mesothelioma (DMPM) are limited to surgery and locoregional chemotherapy. Despite improvements in survival rates, patients eventually succumb to disease progression. We investigated splicing deregulation both as molecular prognostic factor and potential novel target in DMPM, while we tested modulators of SF3b complex for antitumor activity.

Mutations affecting RNA splicing factors are the most common genetic alterations in myelodysplastic syndrome (MDS) patients and occur in a mutually exclusive manner. The basis for the mutual exclusivity of these mutations and how they contribute to MDS is not well understood. Here we report that although different spliceosome gene mutations impart distinct effects on splicing, they are negatively selected for when co-expressed due to aberrant splicing and downregulation of regulators of hematopoietic stem cell survival and quiescence. In addition to this synthetic lethal interaction, mutations in the splicing factors SF3B1 and SRSF2 share convergent effects on aberrant splicing of mRNAs that promote nuclear factor κB signaling. These data identify shared consequences of splicing-factor mutations and the basis for their mutual exclusivity.

Estrogen has been reported to affect pain perception, although the underlying mechanisms remain unclear. In this investigation, pain behavior testing, patch clamp recording, and immunohistochemistry were used on rats and transgenic mice to determine which estrogen receptors (ERs) and the related signaling pathway are involved in the rapid modulation of estrogen on P2X3 receptor-mediated events. The results showed that 17β-estradiol (E2) rapidly inhibited pain induced by α,β-methylene ATP (α,β-me-ATP), a P2X1 and P2X3 receptor agonist in ovariectomized rats and normal rats in diestrus. The ERα agonist 4,49,499-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT) and G protein-coupled receptor 30 (GPR30) agonist G-1 mimicked the estrogen effect, whereas the ERβ agonist diarylpropionitrile (DPN) had no effect. In cultured rat dorsal root ganglion (DRG) neurons, PPT and G-1 but not DPN significantly attenuated α,β-me-ATP-mediated currents, with the dose-response curve of these currents shifted to the right. The inhibitory effect of E2 on P2X3 currents was blocked by G-15, a selective antagonist to the GPR30 estrogen receptor. E2 lacked this effect in DRG neurons from ERα-knockout mice but partly remained in those from ERβ-knockout mice. The P2X3 and GPR30 receptors were coexpressed in the rat DRG neurons. Furthermore, the ERK1/2 inhibitor U0126 reversed the inhibitory effect of E2 on α,β-me-ATP-induced pain and of PPT or G-1 on P2X3 receptor-mediated currents. The cAMP-protein kinase A (PKA) agonist forskolin, but not the PKC agonist phorbol-12-myristate-13-acetate (PMA), mimicked the estrogen-inhibitory effect on P2X3 receptor currents, which was blocked by another ERK1/2 inhibitor, PD98059. These results suggest that estrogen regulates P2X3-mediated peripheral pain by acting on ERα and GPR30 receptors expressed in primary afferent neurons, which probably involves the intracellular cAMP-PKA-ERK1/2 pathway.

Recent studies have shown that activating mutations of NOTCH1 are responsible for the majority of T cell acute lymphoblastic leukemia (T-ALL) cases. Most of these mutations truncate its C-terminal domain, a region that is important for the NOTCH1 proteasome-mediated degradation. We report that the E3 ligase FBW7 targets NOTCH1 for ubiquitination and degradation. Our studies map in detail the amino acid degron sequence required for NOTCH1-FBW7 interaction. Furthermore, we identify inactivating FBW7 mutations in a large fraction of human T-ALL lines and primary leukemias. These mutations abrogate the binding of FBW7 not only to NOTCH1 but also to the two other characterized targets, c-Myc and cyclin E. The majority of the FBW7 mutations were present during relapse, and they were associated with NOTCH1 HD mutations. Interestingly, most of the T-ALL lines harboring FBW7 mutations were resistant to gamma-secretase inhibitor treatment and this resistance appeared to be related to the stabilization of the c-Myc protein. Our data suggest that FBW7 is a novel tumor suppressor in T cell leukemia, and implicate the loss of FBW7 function as a potential mechanism of drug resistance in T-ALL.

Immune evasion is a well-recognized hallmark of cancer and recent studies with immunotherapy agents have suggested that tumors with increased numbers of neoantigens elicit greater immune responses. We hypothesized that the immune system presents a common selective pressure on high mutation burden tumors and therefore immune evasion mutations would be enriched in high mutation burden tumors. The JAK family of kinases is required for the signaling of a host of immune modulators in tumor, stromal, and immune cells. Therefore, we analyzed alterations in this family for the hypothesized signature of an immune evasion mutation. Here, we searched a database of 61,704 unique solid tumors for alterations in the JAK family kinases (JAK1/2/3, TYK2). We used The Cancer Genome Atlas and Cancer Cell Line Encyclopedia data to confirm and extend our findings by analyzing gene expression patterns. Recurrent frameshift mutations in JAK1 were associated with high mutation burden and microsatellite instability. These mutations occurred in multiple tumor types including endometrial, colorectal, stomach, and prostate carcinomas. Analyzing gene expression signatures in endometrial and stomach adenocarcinomas revealed that tumors with a JAK1 frameshift exhibited reduced expression of interferon response signatures and multiple anti-tumor immune signatures. Importantly, endometrial cancer cell lines exhibited similar gene expression changes that were expected to be tumor cell intrinsic (e.g. interferon response) but not those expected to be tumor cell extrinsic (e.g. NK cells). From these data, we derive two primary conclusions: 1) JAK1 frameshifts are loss of function alterations that represent a potential pan-cancer adaptation to immune responses against tumors with microsatellite instability; 2) The mechanism by which JAK1 loss of function contributes to tumor immune evasion is likely associated with loss of the JAK1-mediated interferon response.

Antibody-based c-mesenchymal-epithelial transition factor (c-Met) inhibition is a promising strategy for hepatocellular carcinoma (HCC) treatment, but the intrinsic agonistic activity of the anti-c-Met antibody limits its application in drug development. Constructing a monovalent one-armed antibody has been reported to be an effective way to create an inhibitory anti-c-Met antibody.

Hyaluronan accumulation in the tumor microenvironment is associated with poor prognosis in several solid human cancers. To understand the role of stromal hyaluronan in tumor progression, we engineered 3T3HAS3, a hyaluronan-producing fibroblast cell line, by lentiviral transduction of Balb/c 3T3 cells with the human hyaluronan synthase 3 (HAS3) gene. 3T3HAS3 cells significantly enhanced tumor growth when co-grafted with MDA-MB-468 cells in nude mice. Immunohistochemical analysis of the xenograft tumors showed that MDA-MB-468 cells were surrounded by hyaluronan-accumulating stroma, closely resembling the morphology observed in human breast cancer specimens. Tumor growth of MDA-MB-468 + 3T3HAS3 co-grafts was greatly reduced upon hyaluronan degradation by lentiviral transduction of a human hyaluronidase gene in 3T3HAS3 cells, or by systemic administration of pegvorhyaluronidase alfa (PEGPH20). In contrast, the growth of the co-graft tumors was not inhibited when CD44 expression was reduced or ablated by small hairpin RNA-mediated CD44 knockdown in MDA-MB-468 cells, CD44 CRISPR knockout in 3T3HAS3 cells, or by grafting these cells in CD44 knockout nude mice. Collectively, these data demonstrate that tumor growth of an engineered xenograft breast cancer model with hyaluronan-accumulating stroma can be dependent on hyaluronan and independent of CD44.

Excessive monocyte activation with the development of excessive or uncontrolled release of proinflammatory cytokines often results in host tissue injury and even death in patients with pneumonia caused by the 2019 novel coronavirus. However, the changes of cytokine profiles of coronavirus disease 2019 (COVID-19) patients, as well as the underlying mechanisms that are involved, remain unknown. Using a cytokine array containing 174 inflammation-related cytokines, we found significantly altered cytokine profiles in severe COVID-19 patients compared with those in mild patients or healthy controls, and identified leptin, CXCL-10, IL-6, IL-10, IL-12, and TNF-α as the top differentially expressed cytokines. Notably, leptin showed high consistency with CXCL-10 and TNF-α in predicting disease severity, and correlated with body mass index, decreased lymphocyte counts, and disease progression. Further analysis demonstrated that monocytes in severe patients with higher leptin levels were inclined toward M1 polarization. Mechanistic studies revealed that leptin synergistically up-regulated expression levels of inflammatory cytokines and surface markers with IL-6 in monocytes through STAT3 and NF-κB signaling pathways. Collectively, our results suggest that overweight COVID-19 patients were prone to have higher leptin levels, which further activated monocytes, resulting in amplified or dysregulated immune responses. Taken together, our findings argue that leptin correlates severity of COVID-19 and may indicate a possible mechanism by which overweight patients have a greater tendency to develop severe conditions.

Estrogen receptor beta (ERβ) agonists could inhibit inflammation in animal models of inflammatory bowel disease (IBD). However, the mechanism underlying such effect and the potential role of P2 × 7 receptor (P2X7R) remains unclear. In the present study, we examined whether the effect of ERβ activation in IBD rats was related to P2X7R. Overexpression of ERβ using a recombinant lentivirus in IBD rats improved the IBD-like symptoms, including weight loss, disease activity index (DAI) scores, and inflammatory responses. ERβ agonists DPN and ERB-041 attenuated P2X7R expression in macrophages from colitis rats and in a murine macrophage cell line (RAW264.7) in response to either lipopolysaccharide (LPS) or adenosine triphosphate (ATP). DPN and ERB-041 also blocked increased production of TNF-α, IL-6, and IL-1β in the rectocolon of colitis rats. The two ERβ agonists reversed LPS- and ATP-induced up-regulation of P2X7R and its downstream proteins, including NLRP3, ASC, caspase-1, and pro-IL-1β, in RAW264.7 cells. Also, in both the rectocolon of colitis rats and RAW264.7 cells, ERβ agonists reversed the up-regulation of extracellular regulated protein kinases (ERK), the Janus kinase 2 (JAK2), and signal transducer and activator of transcription 3 (STAT3), but not up-regulation of serine threonine kinase or cAMP-response element binding protein (CREB). Blockade of JAK2 or STAT3 phosphorylation significantly reduced the ability of DPN to down-regulate P2X7R expression and the ability of ERB-041 and DPN to inhibit IL-1β release from RAW264.7 cells. We found that ERβ and P2X7R co-localized in the macrophages of rat rectocolon and in RAW264.7 cells. Deletion of macrophages from colitis rats with clodronate abolished the inhibitory effect of DPN. These results suggest that ERβ plays an important anti-inflammatory role in IBD rats by down-regulating P2X7R expression and inhibiting IL-1β release from macrophages through the JAK2/STAT3 signaling pathway.

Ulcerative colitis (UC), one of the typical inflammatory bowel diseases caused by dysregulated immunity, still requires novel therapeutic medicine with high efficacy and low toxicity. Hericium erinaceus has been widely used to treat different health problems especially gastrointestinal sickness in China for thousands of years. Here, we isolated, purified, and characterized a novel low weight polysaccharide (HEP10, Mw: 9.9 kDa) from the mycelia of H. erinaceus in submerged culture. We explored the therapeutic effect of HEP10 on UC and explored its underlying mechanisms. On one hand, HEP10 suppressed the production of TNF-α, IL-1β, IL-6, inducible iNOS, and COX-2 in LPS challenged murine macrophage RAW264.7 cells, as well as in colons from DSS-induced colitis mice. On the other hand, HEP10 treatment markedly suppressed the activation of NLRP3 inflammasome, NF-κB, AKT, and MAPK pathways. Moreover, HEP10 reversed DSS-induced alternation of the gut community composition and structure by significantly increasing Akkermansia muciniphila and also promoting functional shifts in gut microbiota. Structural equation modeling also highlighted that HEP10 can change widely through gut microbiota. In conclusion, HEP10 has a better prebiotic effect than the crude polysaccharides of H. erinaceus, which can be used as a novel dietary supplement and prebiotic to ameliorate colitis.

Neutrophil elastase (NE) is a critical proteolytic enzyme that is involved in cancer. We previously reported high NE expression in peripheral blood neutrophils from acute promyelocytic leukemia (APL) patients. The present study aimed to elucidate the specific role and mechanisms of NE in APL development.

To study the protective effect of trimetazidine on myocardial cells in rats with myocardial infarction and explore its effect on ERK signaling pathway.

This study aimed to evaluate the safety and efficacy of chimeric antigen receptor (CAR) disialoganglioside 2 (GD2)-specific (4SCAR-GD2) T cells for treatment of refractory and/or recurrent neuroblastoma (NB) in pediatric patients.

This study aimed to develop a simple microfluidic chip analysis technology to study the inhibitory effect of protocatechuic acid on shear-induced platelet aggregation. The microfluidic chip designed in this study simulates 80% fixed narrow microchannels. This microchannel narrow model uses the finite element analysis module of the three-dimensional modeling software solidwork to analyze fluid dynamic behavior. Blood treated with protocatechuic acid at 1, 2, 4, 8, or 16 µg/mL was passed through the microchannel stenosis model at a shear rate of 10,000 s-1. The platelet adhesion and aggregation behaviors were then measured using fluorescence microscopy and observed in real time. Simultaneously, the antiplatelet aggregation effect of protocatechuic acid was analyzed using thromboelastography and photoelectric turbidimetry. The designed stenosis model of the microfluidic chip can produce a gradient of fluid shear rate, and the gradient of fluid shear rate can induce platelet aggregation. Under this model, the degree of platelet adhesion and aggregation increased as the shear rate increased. In the experimental concentration range of 0-8 µmol/mL, protocatechuic acid exerted a concentration-dependent inhibition of platelet aggregation. In contrast, thromboelastography and photoelectric turbidimetry failed to demonstrate an inhibitory effect. The microfluidic chip analysis technology developed in this study can be used to study the effect of protocatechin in inhibiting platelet aggregation induced by shear rate in vitro. This technology is simple to operate and can be used as a new type of antiplatelet aggregation analysis technology for screening studies of novel potential antiplatelet aggregation drugs.

To investigate the incidence of microvascular myocardial ischemia in diabetic patients without obstructive coronary artery disease (CAD) and its relationship with angina.

Soil salinization is a common environmental problem that seriously affects the yield and quality of crops. Sugar beet (Beta vulgaris L.), one of the main sugar crops in the world, shows a strong tolerance to salt stress. To decipher the molecular mechanism of sugar beet under salt stress, we conducted transcriptomic analyses of two contrasting sugar beet genotypes. To the best of our knowledge, this is the first comparison of salt-response transcriptomes in sugar beet with contrasting genotypes. Compared to the salt-sensitive cultivar (S710), the salt-tolerant one (T710MU) showed better growth and exhibited a higher chlorophyll content, higher antioxidant enzyme activity, and increased levels of osmotic adjustment molecules. Based on a high-throughput experimental system, 1714 differentially expressed genes were identified in the leaves of the salt-sensitive genotype, and 2912 in the salt-tolerant one. Many of the differentially expressed genes were involved in stress and defense responses, metabolic processes, signal transduction, transport processes, and cell wall synthesis. Moreover, expression patterns of several genes differed between the two cultivars in response to salt stress, and several key pathways involved in determining the salt tolerance of sugar beet, were identified. Our results revealed the mechanism of salt tolerance in sugar beet and provided potential metabolic pathways and gene markers for growing salt-tolerant cultivars.

The quality of crystalline two-dimensional (2D) polymers1-6 is intimately related to the elusive polymerization and crystallization processes. Understanding the mechanism of such processes at the (sub)molecular level is crucial to improve predictive synthesis and to tailor material properties for applications in catalysis7-10 and (opto)electronics11,12, among others13-18. We characterize a model boroxine 2D dynamic covalent polymer, by using in situ scanning tunnelling microscopy, to unveil both qualitative and quantitative details of the nucleation-elongation processes in real time and under ambient conditions. Sequential data analysis enables observation of the amorphous-to-crystalline transition, the time-dependent evolution of nuclei, the existence of 'non-classical' crystallization pathways and, importantly, the experimental determination of essential crystallization parameters with excellent accuracy, including critical nucleus size, nucleation rate and growth rate. The experimental data have been further rationalized by atomistic computer models, which, taken together, provide a detailed picture of the dynamic on-surface polymerization process. Furthermore, we show how 2D crystal growth can be affected by abnormal grain growth. This finding provides support for the use of abnormal grain growth (a typical phenomenon in metallic and ceramic systems) to convert a polycrystalline structure into a single crystal in organic and 2D material systems.