Split Nano luciferase complementation for probing protein‐protein interactions in plant cells

Deciphering protein‐protein interactions (PPIs) is fundamental for understanding signal transduction pathways in plants. The split firefly luciferase (Fluc) complementation (SLC) assay has been widely used for analyzing PPIs. However, concern has risen about the bulky halves of Fluc interfering with the functions of their fusion partners. Nano luciferase (Nluc) is the smallest substitute for Fluc with improved stability and luminescence. Here, we developed a dual‐use system enabling the detection of PPIs through the Nluc‐based SLC and co‐immunoprecipitation assays. This was realized by coexpression of two proteins under investigation in fusion with the HA‐ or FLAG‐tagged Nluc halves, respectively. We validated the robustness of this system by reproducing multiple previously documented PPIs in protoplasts or Agrobacterium‐transformed plants. We next applied this system to evaluate the homodimerization of Arabidopsis CERK1, a coreceptor of fungal elicitor chitin, and its heterodimerization with other homologs in the absence or presence of chitin. Moreover, split fragments of Nluc were fused to two cytosolic ends of Arabidopsis calcium channels CNGC2 and CNGC4 to help sense the allosteric change induced by the bacterial elicitor flg22. Collectively, these results demonstrate the usefulness of the Nluc‐based SLC assay for probing constitutive or inducible PPIs and protein allostery in plant cells.     

Tumour‐derived exosomal miR‐3473b promotes lung tumour cell intrapulmonary colonization by activating the nuclear factor‐κB of local fibroblasts

Tumour‐derived exosomes have been shown to induce pre‐metastatic niche formation, favoring metastatic colonization of tumour cells, but the underlying molecular mechanism is still not fully understood. In this study, we showed that exosomes derived from the LLC cells could indeed significantly enhance their intrapulmonary colonization. Circulating LLC‐derived exosomes were mainly engulfed by lung fibroblasts and led to the NF‐κB signalling activation. Further studies indicated that the exosomal miR‐3473b was responsible for that by hindering the NFKB inhibitor delta's (NFKBID) function. Blocking miR‐3473b could reverse the exosome‐mediated NF‐κB activation of fibroblasts and decrease intrapulmonary colonization of lung tumour cells. Together, this study demonstrated that the miR‐3473b in exosomes could mediate the interaction of lung tumour cells and local fibroblasts in metastatic sites and, therefore, enhance the metastasis of lung tumour cells.     

AMPH1 functions as a tumour suppressor in ovarian cancer via the inactivation of PI3K/AKT pathway

AMPH1, an abundant protein in nerve terminals, plays a critical role in the recruitment of dynamin to sites of clathrin‐mediated endocytosis. Recently, it is reported to be involved in breast cancer and lung cancer. However, the impact of AMPH1 on ovarian cancer is unclear. In this study, we used gain‐of‐function and loss‐of‐function methods to explore the role of AMPH1 in ovarian cancer cells. AMPH1 inhibited ovarian cancer cell growth and cell migration, and promoted caspase‐3 activity, resulting in the increase of cell apoptosis. In xenograft mice model, AMPH1 prevented tumour progression. The anti‐oncogene effects of AMPH1 on ovarian cancer might be partially due to the inhibition of PI3K/AKT signalling pathway after overexpression of AMPH1. Immunohistochemistry analysis showed that the staining of AMPH1 was remarkably reduced in ovarian cancer tissues compared with normal ovarian tissues. In conclusion, our study identifies AMPH1 as a tumour suppressor in ovarian cancer in vitro and in vivo. This is the first evidence that AMPH1 inhibited cell growth and migration, and induced apoptosis via the inactivation of PI3K/AKT signalling pathway on ovarian cancer, which may be used as an effective strategy.     

Triptolide prevents bone loss via suppressing osteoclastogenesis through inhibiting PI3K-AKT-NFATc1 pathway

Bone loss (osteopenia) is a common complication in human solid tumour. In addition, after surgical treatment of gynaecological tumour, osteoporosis often occurs due to the withdrawal of oestrogen. The major characteristic of osteoporosis is the low bone mass with micro-architectural deteriorated bone tissue. And the main cause is the overactivation of osteoclastogenesis, which is one of the most important therapeutic targets. Inflammation could induce the interaction of RANKL/RANK, which is the promoter of osteoclastogenesis. Triptolide is derived from the traditional Chinese herb lei gong teng, presented multiple biological effects, including anti-cancer, anti-inflammation and immunosuppression. We hypothesized that triptolide could inhibits osteoclastogenesis by suppressing inflammation activation. In this study, we confirmed that triptolide could suppress RANKL-induced osteoclastogenesis in bone marrow mononuclear cells (BMMCs) and RAW264.7 cells and inhibited the osteoclast bone resorption functions. PI3K-AKT-NFATc1 pathway is one of the most important downstream pathways of RANKL-induced osteogenesis. The experiments in vitro indicated that triptolide suppresses the activation of PI3K-AKT-NFATc1 pathway and the target point located at the upstream of AKT because both NFATc1 overexpression and AKT phosphorylation could ameliorate the triptolide suppression effects. The expression of MDM2 was elevated, which demonstrated the MDM-p53-induced cell death might contribute to the osteoclastogenesis suppression. Ovariectomy-induced bone loss and inflammation activation were also found to be ameliorated in the experiments in vivo. In summary, the new effect of anti-cancer drug triptolide was demonstrated to be anti-osteoclastogenesis, and we demonstrated triptolide might be a promising therapy for bone loss caused by tumour.     

Identification of circRNA and mRNA expression profiles and functional networks of vascular tissue in lipopolysaccharide‐induced sepsis

Sepsis is the most common cause of death in intensive care units. This study investigated the circular RNA (circRNA) and mRNA expression profiles and functional networks of the aortic tissue in sepsis. We established a lipopolysaccharide (LPS)‐induced rat sepsis model. High‐throughput sequencing was performed on the aorta tissue to identify differentially expressed (DE) circRNAs and mRNAs, which were validated by real‐time quantitative polymerase chain reaction (RT‐qPCR). Bioinformatic analysis was carried out and coding and non‐coding co‐expression (CNC) and competing endogenous RNA (ceRNA) regulatory networks were constructed to investigate the mechanisms. In total, 373 up‐regulated and 428 down‐regulated circRNAs and 2063 up‐regulated and 2903 down‐regulated mRNAs were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of mRNAs showed that the down‐regulated genes were mainly enriched in the process of energy generation. CNC and ceRNA regulatory networks were constructed with seven DE circRNAs. The results of functional enrichment analysis of CNC target genes revealed the important role of circRNAs in inflammatory response. The ceRNA network also highlighted the significant enrichment in calcium signalling pathway. Significant alterations in circRNAs and mRNAs were observed in the aortic tissue of septic rats. In addition, CNC and ceRNA networks were established.     

Inhibition of the Notch1 pathway induces peripartum cardiomyopathy

Increased expression and activity of cardiac and circulating cathepsin D and soluble fms‐like tyrosine kinase‐1 (sFlt‐1) have been demonstrated to induce and promote peripartum cardiomyopathy (PPCM) via promoting cleavage of 23‐kD prolactin (PRL) to 16‐kD PRL and neutralizing vascular endothelial growth factor (VEGF), respectively. We hypothesized that activation of Hes1 is proposed to suppress cathepsin D via activating Stat3, leading to alleviated development of PPCM. In the present study, we aimed to investigate the role of Notch1/Hes1 pathway in PPCM. Pregnant mice between prenatal 3 days and postpartum 3 weeks were fed with LY‐411575 (a notch inhibitor, 10 mg/kg/d). Ventricular function and pathology were evaluated by echocardiography and histological analysis. Western blotting analysis was used to examine the expression at the protein level. The results found that inhibition of Notch1 significantly promoted postpartum ventricular dilatation, myocardial hypertrophy and myocardial interstitial fibrosis and suppressed myocardial angiogenesis. Western blotting analysis showed that inhibition of Notch1 markedly increased cathepsin D and sFlt‐1, reduced Hes1, phosphorylated Stat3 (p‐Stat3), VEGFA and PDGFB, and promoted cleavage of 23k‐D PRL to 16‐kD PRL. Collectively, inhibition of Notch1/Hes1 pathway induced and promoted PPCM via increasing the expressions of cathepsin D and sFlt‐1. Notch1/Hes1 was a promising target for prevention and therapeutic regimen of PPCM.     

Bmp2 regulates Serpinb6b expression via cAMP/PKA/Wnt4 pathway during uterine decidualization

Serpinb6b is a novel member of Serpinb family and found in germ and somatic cells of mouse gonads, but its physiological function in uterine decidualization remains unclear. The present study revealed that abundant Serpinb6b was noted in decidual cells, and advanced the proliferation and differentiation of stromal cells, indicating a creative role of Serpinb6b in uterine decidualization. Further analysis found that Serpinb6b modulated the expression of Mmp2 and Mmp9. Meanwhile, Serpinb6b was identified as a target of Bmp2 regulation in stromal differentiation. Treatment with rBmp2 resulted in an accumulation of intracellular cAMP level whose function in this differentiation program was mediated by Serpinb6b. Addition of PKA inhibitor H89 impeded the Bmp2 induction of Serpinb6b, whereas 8‐Br‐cAMP rescued the defect of Serpinb6b expression elicited by Bmp2 knock‐down. Attenuation of Serpinb6b greatly reduced the induction of constitutive Wnt4 activation on stromal cell differentiation. By contrast, overexpression of Serpinb6b prevented this inhibition of differentiation process by Wnt4 siRNA. Moreover, blockage of Wnt4 abrogated the up‐regulation of cAMP on Serpinb6b. Collectively, Serpinb6b mediates uterine decidualization via Mmp2/9 in response to Bmp2/cAMP/PKA/Wnt4 pathway.     

Identification of small RNAs involved in nitrogen fixation in Anabaena sp. PCC 7120 based on RNA-seq under steady state conditions

The aim of the present study was to investigate the tolerance of five new Achromobacter and Pseudomonas strains to kerosene and to establish if the production of several secondary metabolites increases or not when these bacteria were grown in the presence of kerosene. The biodegradation of kerosene by isolated bacteria was also investigated in this study. Five Proteobacteria were isolated from different samples polluted with petroleum and petroleum products. Based on their morphological, biochemical, and molecular characteristics, isolated bacteria were identified as Achromobacter spanius IBBPo18 and IBBPo21, Pseudomonas putida IBBPo19, and Pseudomonas aeruginosa IBBPo20 and IBBPo22. All these bacteria were able to tolerate and degrade kerosene. Higher tolerance to kerosene and degradation rates were observed for P. aeruginosa IBBPo20 and IBBPo22, compared with that observed for A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19. All these bacteria were able to produce several secondary metabolites, such as surfactants and pigments. Glycolipid surfactants produced by P. aeruginosa IBBPo20 and IBBPo22, A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19 have a very good emulsification activity, and their activity increased when they were grown in the presence of kerosene. The production of rhamnolipid surfactants by P. aeruginosa IBBPo20 and IBBPo22 was confirmed by detection of rhlAB gene involved in their biosynthesis. Pyocyanin and pyoverdin pigments were produced only by P. aeruginosa IBBPo20 and IBBPo22, while carotenoid pigments were produced by all the isolated bacteria. Significant changes in pigments production were observed when P. aeruginosa IBBPo20 and IBBPo22, A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19 were grown in the presence of kerosene. Due to their ability to tolerate and degrade kerosene, and also to produce several secondary metabolites, the isolated bacteria could be used in the bioremediation of kerosene-polluted environments.     

IL‐17A‐producing T cells exacerbate fine particulate matter‐induced lung inflammation and fibrosis by inhibiting PI3K/Akt/mTOR‐mediated autophagy

Fine particulate matter (PM2.5) is the primary air pollutant that is able to induce airway injury. Compelling evidence has shown the involvement of IL‐17A in lung injury, while its contribution to PM2.5‐induced lung injury remains largely unknown. Here, we probed into the possible role of IL‐17A in mouse models of PM2.5‐induced lung injury. Mice were instilled with PM2.5 to construct a lung injury model. Flow cytometry was carried out to isolate γδT and Th17 cells. ELISA was adopted to detect the expression of inflammatory factors in the supernatant of lavage fluid. Primary bronchial epithelial cells (mBECs) were extracted, and the expression of TGF signalling pathway‐, autophagy‐ and PI3K/Akt/mTOR signalling pathway‐related proteins in mBECs was detected by immunofluorescence assay and Western blot analysis. The mitochondrial function was also evaluated. PM2.5 aggravated the inflammatory response through enhancing the secretion of IL‐17A by γδT/Th17 cells. Meanwhile, PM2.5 activated the TGF signalling pathway and induced EMT progression in bronchial epithelial cells, thereby contributing to pulmonary fibrosis. Besides, PM2.5 suppressed autophagy of bronchial epithelial cells by up‐regulating IL‐17A, which in turn activated the PI3K/Akt/mTOR signalling pathway. Furthermore, IL‐17A impaired the energy metabolism of airway epithelial cells in the PM2.5‐induced models. This study suggested that PM2.5 could inhibit autophagy of bronchial epithelial cells and promote pulmonary inflammation and fibrosis by inducing the secretion of IL‐17A in γδT and Th17 cells and regulating the PI3K/Akt/mTOR signalling pathway.     

Room Temperature Preparation of Surface-Clean Hydrogen-Doped Plasmonic Molybdenum Oxide as a High-Efficient and Degradable Reactive Oxygen Species Scavenger

Considerable efforts have been made to develop reactive oxygen species (ROS) scavengers for removing high level of ROS. However, most of the reported ROS scavengers are nondegradable and involve harsh reaction conditions as well as utilize various surface ligands. In order to overcome these drawbacks, in the present work, we develop a facile and mild synthesis avenue for preparation of surface-clean hydrogen-doped molybdenum oxide (H_0.34MoO₃) via simply mixing MoO₃ dispersion with aluminum foil under an acidic environment without any surface capping reagents at room temperature. The resulting H_0.34MoO₃ can act as a broad-spectrum ROS scavenger, including ^.OH, H₂O₂, O₂⁻, and ¹O₂ as well as 2, 2-diphenyl-1-picrylhydrazyl (DPPH). The free radical scavenging activity of H_0.34MoO₃ achieves as high as 71.6% and 99.1% for ^.OH and DPPH scavenging, which is comparable and superior to that of ascorbic acid that is a classic free radical scavenger. More significantly, the resulting H_0.34MoO₃ is degrade, which can be degraded into molybdate ions under a neutral environment (pH 7.4).