Genome-wide characterization of Remorin gene family and their responsive expression to abiotic stresses and plant hormone in Brassica napus.

KEY MESSAGE: Remorin proteins could be positively related to salt and osmotic stress resistance in rapeseed. Remorins (REMs) play a crucial role in adaptations to adverse environments. However, their roles in abiotic stress and phytohormone responses in oil crops are still largely unknown. In this study, we identified 47 BnaREM genes in the B.napus genome. Phylogenetic relationship and synteny analysis revealed that they were categorized into 5 distinct groups and have gone through 55 segmental duplication events under purifying selection. Gene structure and conserved domains analysis demonstrated that they were highly conserved and all BnaREMs contained a conserved Remorin_C domain, with a variable N-terminal region. Promoter sequence analysis showed that BnaREM gene promoters contained various hormones and stress-related cis-acting elements. Transcriptome data from BrassicaEDB database exhibited that all BnaREMs were ubiquitously expressed in buds, stamens, inflorescences, young leaves, mature leaves, roots, stems, seeds, silique pericarps, embryos and seed coats. The qRT-PCR analysis indicated that most of them were responsive to ABA, salt and osmotic treatments. Further mutant complementary experiments revealed that the expression of BnaREM1.3-4C-1 in the Arabidopsis rem1.3 mutant restored the retarded growth phenotype and the ability to resistance to salt and osmotic stresses. Our findings provide fundamental information on the structure and evolutionary relationship of the BnaREM family genes in rapeseed, and reveal the potential function of BnaREM1.3-4C-1 in stress and hormone response.

Genome-wide analysis of sugar transporter genes in maize (Zea mays L.): identification, characterization and their expression profiles during kernel development.

Sugar transporters (STs) play a crucial role in the development of maize kernels. However, very limited information about STs in maize is known. In this study, sixty-eight ZmST genes were identified from the maize genome and classified into eight major groups based on phylogenetic relationship. Gene structure analysis revealed that members within the same group shared similar exon numbers. Synteny analysis indicated that ZmSTs underwent 15 segmental duplication events under purifying selection. Three-dimensional structure of ZmSTs demonstrated the formation of a compact helix bundle composed of 8-13 trans-membrane domains. Various development-related cis-acting elements, enriched in promoter regions, were correlated with the transcriptional response of ZmSTs during kernel development. Transcriptional expression profiles exhibited expression diversity of various ZmST genes in roots, stems, leaves, tassels, cobs, embryos, endosperms and seeds tissues. During kernel development, the expression of 24 ZmST genes was significantly upregulated in the early stage of grain filling. This upregulation coincided with the sharply increased grain-filling rate observed in the early stage. Overall, our findings shed light on the characteristics of ZmST genes in maize and provide a foundation for further functional studies.

Characterization of RING-type ubiquitin SINA E3 ligases and their responsive expression to salt and osmotic stresses in Brassica napus.

SINA (Seven in absentia) proteins in the subtype of E3 ubiquitin ligase family play a crucial role in plant growth and development. However, their functions in response to salt and osmotic stresses in oil crops are still largely unknown. In this study, a total number of 23 BnaSINAs were identified in the rapeseed genome. Chromosome location and collinear relationship analyses revealed that they were unevenly distributed on 13 chromosomes, and have gone through 22 segmental duplication events under purifying selection. Phylogenetic and gene structural analyses indicated that they belonged to five main groups, and those in the same subgroup showed similar gene structure. All BnaSINAs were predicted to form homo- or heterodimers. Except BnaSINA7, BnaSINA11, BnaSINA17 and BnaSINA18, which lacked the N-terminal RING finger, all BnaSINAs contained a conserved C-terminal SINA domain, a typical structural feature of the RING-type E3 ligase family. Transcriptional expression analyses demonstrated that most BnaSINAs were ubiquitously expressed in roots, stems, leaves, flowers, pods and seeds, and all were responsive to salt and osmotic stresses. Further, yeast two-hybrid and Arabidopsis mutant complementation analyses demonstrated that BnaSINA4 interacted with BnaSINA17 to form heterodimer, and expression of BnaSINA17 in the Arabidopsis sina2 mutant restored its growth resistance to salt and osmotic stresses. Our findings provide an important genetic foundation for the functional elucidation of BnaSINAs and a novel gene resource for the breeding of new oil crop cultivars with improved abiotic stress resistance.

A simple, rapid and low-cost qPCR assay for evaluating the severity of exosomal PD-L1-mediated T cell exhaustion in blood samples.

A novel dual-aptamer activated proximity-induced qPCR assay was developed for the quantitative analysis of exosomal PD-L1 on T cell-exosome complexes in blood samples.

Real-time in situ monitoring of Lon and Caspase-3 for assessing the state of cardiomyocytes under hypoxic conditions via a novel Au-Se fluorescent nanoprobe.

Myocardial dysfunction caused by cardiomyocyte apoptosis under ischemic and hypoxic conditions is the pathological basis of most cardiovascular diseases. Current diagnosis of myocardial dysfunction still focuses on the symptomatic stage, usually after the occurrence of the irreversible remodelling and functional impairment. Thus, early stage identification of the apoptotic cardiomyocytes induced by hypoxia is highly significant for preventing the onset and delaying the progression of myocardial dysfunction. Herein, a novel Au-Se nanoprobe with strong anti-interference capability was developed for simultaneous real-time in situ monitoring the expression of Lon protease (Lon) and Caspase-3 with high-fidelity in living cardiomyocytes. As Lon upregulation plays a major role in the initiation of hypoxia-induced apoptosis and Caspase-3 is a marker protein for apoptosis, the nanoprobe has been successfully applied for imaging the activation of Lon-Caspase-3 apoptotic signalling pathway and assessing the state of cardiomyocytes under hypoxic conditions. Furthermore, combining with mitochondrial H2O2 probe-MitoPY1, the nanoprobe was also used to confirm the synergistic effect of Lon and ROS on hypoxia-induced apoptosis of cardiomyocytes and evaluate the function of ROS scavenger on attenuating such apoptosis. This work proposed a promising strategy for early diagnosis, prevention and treatment of hypoxic-ischemic myocardial dysfunction.

Tricolor imaging of MMPs to investigate the promoting roles of inflammation on invasion and migration of tumor cells.

The expression levels of matrix metalloproteinases (MMPs) are closely related to the degree of inflammation which facilitates tumor cells' invasion and migration. A tricolor fluorescence nanoprobe based on high-fidelity gold-selenium (Au-Se) nanoplatform was designed and constructed for simultaneously imaging matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-7 (MMP-7) and matrix metalloproteinase-9 (MMP-9) to thoroughly investigate the tumor cells' invasion and migration behaviors under inflammation environment. The nanoprobe was assembled by attaching Au NPs with three different peptide substrates respectively labeled with fluorescein isothiocyanate (FITC), 5-carboxytetramethylrhodamine (5-TAMRA) and cyanine 5 (Cy5) via the Au-Se bond. The nanoprobe can specifically respond to MMP-2/7/9, thereby triggering the fluorophores' fluorescence that quenched previously by fluorescence resonance energy transfer (FRET) to realize the MMP-2/7/9's visualization in biological systems. Moreover, as the inflammation stimulated by different concentrations lipopolysaccharide (LPS), the expression of MMP-2/7/9 in SMMC-7721 cells was observed to be significantly enhanced by confocal laser scanning microscope (CLSM) imaging, and inflammation was further proved to intensify SMMC-7721 cells' invasion and migration by transwell invasion and migration experiments. Therefore, the nanoprobe can be used to monitor biomarkers to provide a visual system for the degree of invasion and migration of tumor cells in an inflammatory environment, and also offer a new strategy for the study of the correlation between various active biomacromolecules and specific intracellular pathways in cells.

Reconstruction of nano-flares based on Au-Se bonds for high-fidelity detection of RNA in living cells.

We have, for the first time, developed a Au-Se-DNA nanoprobe by upgrading the conventional Au-S bonds of nano-flares to more stable Au-Se bonds for high-fidelity imaging of target RNAs in living cells. The design concept is potentially introduced into various Au-DNA nanosensors that offer wide application prospects in research and clinical practice.

An Au-Se nanoprobe for the evaluation of the invasive potential of breast cancer cells via imaging the sequential activation of uPA and MMP-2.

Urokinase-type plasminogen activator (uPA) has been shown to activate matrix metalloproteinase-2 (MMP-2) that leads to the migration and invasion of breast cancer cells. Overexpressed uPA and MMP-2 are regarded as signs of malignant tumors in clinical practice. Therefore, real-time monitoring of the sequential activation of these two signal molecules may have important implications for the evaluation of the invasive potential and tumor progression of breast cancer. However, due to the complicated intracellular environment, visualizing the dynamic changes of protein expression levels in living cells with a noninvasive method is still a great challenge. Here, a novel gold-selenium (Au-Se) fluorescent nanoprobe with excellent selectivity and strong anti-interference capability was designed for the simultaneous in situ imaging of uPA and MMP-2 and real-time monitoring of their changes in living cells. The imaging results demonstrated that the nanoprobe achieved a better prevention of glutathione interference compared to the conventional Au-S nanoprobe, thus it could be applied to actually reflect the expression level of uPA and MMP-2 in different breast cancer cells. Furthermore, the Au-Se nanoprobe could visually present the activation process of the two signal molecules, which play a dual role of insuring the invasiveness evaluation of breast cancer cells. Overall, our work offers a visual biomarker detection method for the judgment of the degree of breast cancer malignancy, and also provides an effective strategy to investigate the relationships among signal molecules of other signaling pathways in the future.

An Aptamer-Based Near-Infrared Fluorescence Nanoprobe for Detecting and Imaging of Phospholamban Micropeptide in Cardiomyocytes.

A growing body of evidence indicates that micropeptides encoded by long noncoding RNAs (lncRNAs) act independently or as regulators of larger proteins in fundamental biological processes, especially in the maintenance of cellular homeostasis. However, due to their small size and low intracellular expression, visual monitoring of micropeptides in living cells is still a challenge. In this work, we have designed and synthesized an aptamer-based near-infrared fluorescence nanoprobe for fluorescence imaging of phospholamban (PLN), which is an intracellular micropeptide that affects calcium homeostasis, and is closely associated with human heart failure in the clinic. The nanoprobe could respond specifically to PLN with excellent selectivity, high sensitivity, good nuclease stability, and biocompatibility, and it was successfully applied for imaging of changes in PLN levels in cardiomyocytes and in frozen sections of heart tissues. Further combined with clinical myocardial biopsy, we believe that the developed nanoprobe should be of great significance in later molecular pathology study of heart failure, which may help with diagnosis of early heart failure in the future. More importantly, for the first time nanoprobes were applied to visually monitor the changes of micropeptides in living cells and in frozen tissue sections, and the design concept of the aptamer-based nanoprobe can be extended to fluorescence detection of other micropeptides.

Identification of WDR12 as a novel oncogene involved in hepatocellular carcinoma propagation.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant cancer worldwide. Importantly, the precise mechanisms causing HCC pathogenicity are still unknown. The identification of potential oncogenes plays significant roles in finding novel therapeutic targets for human HCC. PURPOSE: WDR12 (WD repeat protein 12), a member of WD repeats family, plays crucial roles in the ribosome biogenesis pathway. However, Whether WDR12 contributes to HCC development remains unknown. The objective of this study was to elucidate the role of WDR12 in HCC development. METHODS: The expression level of WDR12 in HCC tissues and adjacent non-tumor tissues were detected form Gene Expression Omnibus (GEO) database. The expression level of WDR12 in HCC cell lines were examined by RT-PCR and western blot. Kaplan-Meier analysis were used to analyze the effect of WDR12 level on overall and disease-free survival of HCC patients. To examine whether WDR12 supports development of HCC, we inhibited expression of WDR12 by using an shRNA-encoding lentivirus system. Effects of WDR12 knockdown were evaluated on cell-growth, cell-proliferation and cell-migration. The mechanisms involved in HCC cells growth, proliferation and migration were analyzed by western blot assay. RESULTS: In silico analysis of HCC data sets showed that elevated expression of WDR12 correlated with high serum AFP level, high vascular invasion, high histologic grade and high TNM stage in HCC patients. Furthermore, up-regulated expression of WDR12 significantly correlated with the short overall survival and recurrence time of HCC patients. The shRNA-mediated knockdown of WDR12 expression resulted in reduced proliferation and migration of HepG2 and Huh-7 cells. Notably, inhibition of WDR12 resulted in decreased phosphorylation of AKT, mTOR and S6K1. CONCLUSION: Our study indicates that WDR12 contributes to HCC propagation, and indicates that suppression of WDR12 may be a potential strategy for human HCC treatment.

SPG6 supports development of acute myeloid leukemia by regulating BMPR2-Smad-Bcl-2/Bcl-xl signaling.

Acute myeloid leukemia (AML) is the most common acute leukemia affecting adults. To effectively treat AML, new molecular targets and therapeutic approaches must be identified. In silico analysis of several available databases of AML patients showed that the expression of Spastic Paraplegia 6 Protein (SPG6) significantly inversely correlates with the overall survival of AML patients. To determine whether SPG6 supports AML development, we employed an shRNA-encoding lentivirus system to inhibit SPG6 expression in human AML cells including NB4 and MV4-11 cells. Knockdown expression of SPG6 resulted in decreased cell growth and elevated apoptosis of these leukemia cells. Notably, the SPG6 deficiency resulted in higher BMPR2 expression indicating that BMPR2 signaling contributes to AML pathogenesis. Furthermore, SPG6 deficiency promoted phosphorylation of Smad1/5/9 and decreased transcription of Bcl-2 and Bcl-xl. Our study suggests that SPG6 contributes to AML pathogenesis, and suggests that inhibition of SPG6 may be novel strategy for treating human AML.

MiR-541-5p regulates lung fibrosis by targeting cyclic nucleotide phosphodiesterase 1A.

AIM OF THE STUDY: Idiopathic pulmonary fibrosis (IPF) is a lethal human disease with short survival time and few treatment options. In this study, we aim to demonstrate that cyclic nucleotide phosphodiesterase 1A (PDE1A), a Ca2+/calmodulin-stimulating PDE family member, plays a critical role in the induction of fibrosis and angiogenesis in the lung. MATERIALS AND METHODS: To induce pulmonary damage, adult male SD rats were treated with bleomycin in a dose of 6 mg/kg body weight by a single intratracheal instillation. For in vivo silencing of PDE1A in rat lung, a nonspecific control siRNA or PDE1A-specific siRNA was used to treat rat through nasal instillation. Human normal pulmonary fibroblasts MRC-5 and hFL1 and rat lung fibroblasts were used as in vitro model. Immunohistochemistry and immunoflurescence staining were performed to detect PDE1A and α-SMA expression. Reverse transcription-qPCR was performed to detect microRNA and mRNA expression. In vitro wound healing assay was performed to detect pulmonary fibroblasts'mortality ability. RESULTS: In vitro studies showed that PDE1A can stimulate lung fibroblasts to undergo myofibroblastic changes. This led to the identification of miR-541-5p as one of the miRNA candidates associated with bleomycin response. We found that miR-541-5p expression is downregulated in TGF-ß-treated lung fibroblasts and the rat pulmonary fibrosis model. Overexpression of miR-541-5p in lung fibroblasts inhibited mortality of human lung fibroblasts. CONCLUSIONS: MiR-541-5p is a key effector in lung fibroblastsby by regulating PDE1A expression at protein translation level and its overexpression is protective against bleomycin-induced lung fibrosis.

TLR9 Regulates the NF-κB-NLRP3-IL-1ß Pathway Negatively in Salmonella-Induced NKG2D-Mediated Intestinal Inflammation.

TLRs are key sensors for conserved bacterial molecules and play a critical role in host defense against invading pathogens. Although the roles of TLRs in defense against pathogen infection and in maintaining gut immune homeostasis have been studied, the precise functions of different TLRs in response to pathogen infection in the gut remain elusive. The present study investigated the role of TLR signaling in defense against the Gram-negative bacterial pathogen Salmonella typhimurium The results indicated that TLR9-deficient mice were more susceptible to S. typhimurium infection compared with wild-type and TLR2- or TLR4-deficient mice, as indicated by more severe intestinal damage and the highest bacterial load. TLR9 deficiency in intestinal epithelial cells (IECs) augmented the activation of NF-κB and NLRP3 inflammasomes significantly, resulting in increased secretion of IL-1ß. IL-1ß increased the expression of NKG2D on intestinal intraepithelial lymphocytes and NKG2D ligands on IECs, resulting in higher susceptibility of IECs to cytotoxicity of intestinal intraepithelial lymphocytes and damage to the epithelial barrier. We proposed that TLR9 regulates the NF-κB-NLRP3-IL-1ß pathway negatively in Salmonella-induced NKG2D-mediated intestinal inflammation and plays a critical role in defense against S. typhimurium infection and in the protection of intestinal integrity.

Cell surface GRP78 facilitates hepatoma cells proliferation and migration by activating IGF-IR.

The 78kDa glucose regulated protein (GRP78) is a multifunctional chaperone that is involved in a variety of cellular processes. Insulin like growth factor I receptor (IGF-IR) often aberrant expresses in many types of tumor cells. The IGF-IR signaling plays key roles in carcinogenesis and maintenance of the malignant phenotype. The crosstalk between GRP78 and IGF-IR molecules has not well been illuminated. Here, we demonstrated a reciprocal regulation of GRP78 expression and IGF-IR pathway activation. IGF-I induced GRP78 expression in hepatoma cells. IGF-IR knockdown or IGF-IR inhibitor repressed GRP78 expression. Both phosphatidylinositol 3-kianase (PI3K) and mitogen-activated protein kinase (MAPK) pathways involved in IGF-I induction of GRP78 expression. Interestingly, treatment of hepatoma cells with IGF-I re-distributes GRP78 from endoplasmic reticulum (ER) to cell surface and promotes its physical interaction with IGF-IR. Also, GRP78 promotes IGF-IR phosphorylation and activation. Blocked of GRP78 by small interfering RNA or inhibition of GRP78 function by (-)-epigallocatechin gallate (EGCG) blocks IGF-I induced IGF-IR phosphorylation and its downstream signaling. Further, blocked cell surface GRP78 with antibody inhibits IGF-I stimulated cellular proliferation and migration. These data reveal an essential role for the molecular chaperone GRP78 in IGF-IR signaling and implicate the use of GRP78 inhibitors in blocking IGF-IR signaling in hepatoma cells.

ADCY7 supports development of acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common adult acute leukemia. Despite treatment, the majority of the AML patients relapse within 5 years. In silico analysis of several available databases of AML patients showed that the expression of adenylate cyclase 7 (ADCY7) significantly inversely correlates with the overall survival of AML patients. To determine whether ADCY7 supports AML development, we employed an shRNA-encoding lentivirus system to inhibit adcy7 expression in human AML cells including U937, MV4-11, and THP-1 cells. The ADCY7 deficiency resulted in decreased cell growth, elevated apoptosis, and lower c-Myc expression of these leukemia cells. This indicates that G protein-coupled receptor signaling contributes to AML pathogenesis. Our study suggests that inhibition of ADCY7 may be novel strategy for treating leukemia.

Toll-Like receptor 2 (TLR2) and TLR9 play opposing roles in host innate immunity against Salmonella enterica serovar Typhimurium infection.

Toll-like receptors (TLRs) are evolutionarily conserved host proteins that are essential for effective host defense against pathogens. However, recent studies suggest that some TLRs can negatively regulate immune responses. We observed here that TLR2 and TLR9 played opposite roles in regulating innate immunity against oral infection of Salmonella enterica serovar Typhimurium in mice. While TLR9-/- mice exhibited shortened survival, an increased cytokine storm, and more severe Salmonella hepatitis than wild-type (WT) mice, TLR2-/- mice exhibited the opposite phenomenon. Further studies demonstrated that TLR2 deficiency and TLR9 deficiency in macrophages both disrupted NK cell cytotoxicity against S. Typhimurium-infected macrophages by downregulating NK cell degranulation and gamma interferon (IFN-γ) production through decreased macrophage expression of the RAE-1 NKG2D ligand. But more importantly, we found that S. Typhimurium-infected TLR2-/- macrophages upregulated inducible nitric oxide synthase (iNOS) expression, resulting in a lower bacterial load than that in WT macrophages in vitro and livers in vivo as well as low proinflammatory cytokine levels. In contrast, TLR9-/- macrophages showed decreased reactive oxygen species (ROS) expression concomitant with a high bacterial load in the macrophages and in livers of TLR9-/- mice. TLR9-/- macrophages were also more susceptible than WT macrophages to S. Typhimurium-induced necroptosis in vitro, likely contributing to bacterial spread and transmission in vivo. Collectively, these findings indicate that TLR2 negatively regulates anti-S. Typhimurium immunity, whereas TLR9 is vital to host defense and survival against S. Typhimurium invasion. TLR2 antagonists or TLR9 agonists may thus serve as potential anti-S. Typhimurium therapeutic agents.