Identification of Three Viruses Infecting Mulberry Varieties.

Viruses-mediated genome editing in plants is a powerful strategy to develop plant cultivars with important and novel agricultural traits. Mulberry alba is an important economic tree species that has been cultivated in China for more than 5000 years. So far, only a few viruses have been identified from mulberry trees, and their application potential is largely unknown. Therefore, mining more virus resources from the mulberry tree can pave the way for the establishment of useful engineering tools. In this study, eight old mulberry plants were gathered in seven geographic areas for virome analysis. Based on transcriptome analysis, we discovered three viruses associated with mulberries: Citrus leaf blotch virus isolate mulberry alba 2 (CLBV-ML2), Mulberry-associated virga-like virus (MaVLV), and Mulberry-associated narna-like virus (MaNLV). The genome of CLBV-ML2 was completely sequenced and exhibited high homology with Citriviruses, considered to be members of the genus Citrivirus, while the genomes of MaVLV and MaNLV were nearly completed lacking the 5' and 3' termini sequences. We tentatively consider MaVLV to be members of the family Virgaviridae and MaNLV to be members of the genus Narnavirus based on the results of phylogenetic trees. The infection experiments showed that CLBV-ML2 could be detected in the inoculated seedlings of both N. benthamiana and Morus alba, while MaVLV could only be detected in N. benthamiana. All of the infected seedlings did not show obvious symptoms.

Integrative Analyses of Transcriptomics and Metabolomics in Sex Differentiation of Mulberry Flowers.

Sex determination and sex differentiation of plants are important physiological processes of plant development. Mulberry (Morus indica L.) is an important economic tree being cultivated in sericulture countries, and mulberry leaf is commonly used for sericulture. The transcriptomic and metabolomic differences between the staminate flowers (SFs) and pistillate flowers (PFs) of mulberry were investigated by RNA sequencing and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Overall, we uncovered 4,230 genes and 209 metabolites are significantly differentially expressed between the SFs and PFs of mulberry. The combined transcriptomic and metabolomic analysis revealed these differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs) are involved in flavonoid biosynthesis, galactose metabolism, plant-pathogen interaction, and starch and sucrose metabolism, and these detected DEGs and DEMs may be associated with sex differentiation of mulberry through the regulation of the enrichment pathways, such as the MAPK pathway, flavonoid biosynthesis, galactose metabolism, plant-pathogen interaction, and starch and sucrose metabolism. This study will provide a rich source for the analysis of the molecular mechanism of mulberry sex differentiation processes.

Sex-Specific Differences in the Physiological and Biochemical Performance of Arbuscular Mycorrhizal Fungi-Inoculated Mulberry Clones Under Salinity Stress.

Arbuscular mycorrhizal fungi (AMF) are often considered bioameliorators. AMF can promote plant growth under various stressful conditions; however, differences between male and female clones in mycorrhizal strategies that protect plants from the detrimental effects of salinity are not well studied. In this study, we aimed to examine the interactive effects of salinity and AMF on the growth, photosynthetic traits, nutrient uptake, and biochemical responses of Morus alba males and females. In a factorial setup, male and female M. alba clones were subjected to three salinity regimes (0, 50, and 200 mM NaCl) and planted in soil with or without Funneliformis mosseae inoculation. The results showed that NaCl alone conferred negative effects on the growth, salinity tolerance, photosynthetic performance, and shoot and root ionic ratios (K+/Na+, Ca2+/Na+, and Mg2+/Na+) in both sexes; in contrast, mycorrhizal inoculation mitigated the detrimental effects of salinity. Furthermore, the mycorrhizal effects were closely correlated with Mn2+, proline, and N concentrations. Females benefited more from AMF inoculation as shown by the enhancements in their biomass accumulation, and N, proline, K+, Mg2+, Fe2+, Zn2+, and Mn2+ concentrations than males with mycorrhizal inoculation under saline conditions. In comparison, male plants inoculated with AMF showed improvements in biomass allocated to the roots, P, and peroxidase concentrations under saline conditions. These sex-specific differences suggest that male and female mulberry clones adopted different mycorrhizal strategies when growing under saline conditions. Overall, our results provide insight into the sex-specific difference in the performance of AMF-associated mulberry clones, suggesting that female mulberry could be more suitable for vegetation remediation than the male one, due to its higher salinity tolerance.

Protocatechualdehyde Induces S-Phase Arrest and Apoptosis by Stimulating the p27(KIP1)-Cyclin A/D1-CDK2 and Mitochondrial Apoptotic Pathways in HT-29 Cells.

Protocatechualdehyde (PCA) extracted from Phellinus gilvus exhibits anti-cancer activity in human colorectal carcinoma cells (HT-29). However, the underlying mechanisms remain poorly understood. We performed an in vitro study involving MTT, flow cytometry, RT-PCR, and western blot analyses to investigate the effects of PCA treatment on cell proliferation, cell cycle distribution, apoptosis, and expression of several cell cycle-related genes in HT-29 cells. The treatment enhanced S-phase cell cycle and apoptosis in HT-29 cells in a dose-dependent manner. Western blot results showed that PCA treatment decreased the expression levels of cyclin A, cyclin D1, and p27(KIP1) but increased those of cyclin-dependent kinase 2 (CDK2) in HT-29 cells. Furthermore, the expression levels of B-cell lymphoma/leukemia-2 (Bcl-2) and B-cell lymphoma/leukemia-xL (Bcl-xL) were down-regulated, whereas the levels of BH3-interacting domain death agonist (Bid), Bcl-2 homologous antagonist/killer (Bak), and cytosolic cytochrome c were significantly upregulated. Thus, the enzymes caspases-9, -3, -8, and -6 were found to be activated in HT-29 cells with PCA treatment. These results indicate that PCA-induced S-phase cell cycle arrest and apoptosis involve p27(KIP1)-mediated activation of the cyclin-A/D1-Cdk2 signaling pathway and the mitochondrial apoptotic pathway.

Hypoglycemic effect of deoxynojirimycin-polysaccharide on high fat diet and streptozotocin-induced diabetic mice via regulation of hepatic glucose metabolism.

Type 2 diabetes mellitus (T2DM) is currently considered a worldwide epidemic and finding effective therapeutic strategies against this disease is highly important. A deoxynojirimycin-polysaccharide mixture (DPM) has previously been shown to exert hypoglycemic effects on alloxan- or streptozotocin (STZ)-induced diabetic mice. The purpose of the present study was to evaluate the therapeutic effects and underlying mechanism(s) of DPM on T2DM induced by high fat diet following low-dose STZ treatment in mice. After daily oral treatment of diabetic mice with DPM (150 mg/kg b.w.) for 90 d, significant decline in blood glucose, pyruvate, triglyceride (TG), aspartate transaminase (AST), alanine transaminase (ALT), creatinine (Cr), lipid peroxide (LPO) and malondialdehyde (MDA) levels as well as evident increases in high density lipoprotein (HDL-c) and hepatic glycogen concentrations were observed. In the first stage, in which DPM was administered for 60 d, blood insulin levels did not undergo significant change but a significant decrease in the HOMA-IR index was detected. By contrast, the HOMA-IR index increased significantly in T2MD controls. In the second stage, in which DPM treatment was continued for another 30 d, insulin levels significantly increased in DPM-treated mice in comparison with T2DM controls. These results indicate that insulin resistance in the pre-diabetic period and the dysfunction of pancreatic ß-cells are ameliorated by DPM treatment. DPM also down-regulated protein levels of insulin receptor (IR) and gluconeogenic enzymes (pyruvate carboxylase, fructose-1, 6-bisphosphatase, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) in peripheral tissues (liver and/or muscle), but enhanced the expressions of insulin in pancreas, lipoprotein lipase (LPL) and glycolysis enzymes (glucokinase, phosphofructokinase, private kinase and pyruvate decarboxylase E1) in the liver. Furthermore, deoxynojirimycin (DNJ) and polysaccharide (P) were found to increase proliferation of hepatic LO-2 cells and scavenging of radicals in vitro. These results support the results of our biochemical analyses and underscore possible mechanisms underlying the protective effects of DPM on STZ-induced damage to the pancreas and the liver. Taken together, our findings suggest that DPM may be developed as an antihyperglycemic agent for the treatment of diabetes mellitus.

Expression of beclin 1 in non-small cell lung cancer: an immunohistochemical study.

BACKGROUND: Cigarette smoking causes a variety of adverse human health effects, including lung cancer. The molecular events associated with smoke-induced carcinogenesis are thought to be related in part to autophagy. Beclin 1 is an important autophagy-related protein involved in cell death and cell survival. AIM: The purpose of this investigation was to determine the beclin 1 protein and its association with cigarette smoke and the mutation of epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC). MATERIAL AND METHODOLOGY: Our study included 108 cases of non-small cell lung cancer who were admitted in our hospital. The beclin 1 protein was detected by immunohistochemistry and EGFR mutation by direct sequencing. RESULTS: Beclin 1 expression could be detected in 15 (13.9%) of 108 specimens. These studies investigated that beclin 1 expression was associated with heavy smoking, the gender and the histological type of NSCLC (P = 0.023, 0.035 and 0.039). No association of beclin 1 with EGFR mutation was found (P > 0.05). CONCLUSION: The results from these experiments indicate that heavy smoking may induce the beclin 1 protein in NSCLC.

Triptolide inhibits TGF-ß1 induced proliferation and migration of rat airway smooth muscle cells by suppressing NF-κB but not ERK1/2.

BACKGROUND: Airway remodeling contributes to increased mortality in asthma. We have reported that triptolide can inhibit airway remodeling in a mouse asthma model. In this study, we aimed to investigate the effect of triptolide on airway smooth muscle cells (ASMCs) proliferation, migration and the possible mechanism. METHODS: Rat airway smooth muscle cells were cultured and made synchronized, then pretreated with different concentrations of triptolide before stimulated by TGF-ß1. Cell proliferation was evaluated by cell counting and MTT assay. Flow cytometry was used to study the influence of triptolide on cell cycle. Migration was measured by Transwell analysis. Signal proteins (NF-κB p65 and ERK1/2) were detected by western blotting analysis. LDH releasing test and flow cytometry analysis of apoptosis were also performed to explore the potential cytotoxic or pro-apoptotic effects of triptolide. RESULTS: Triptolide significantly inhibited TGF-ß1 induced ASMC proliferation and migration (p<0.05). The cell cycle was blocked at G1/S-interphase by triptolide dose dependently. Western blotting analysis showed TGF-ß1 induced NF-κB p65 phosphorylation was inhibited by triptolide pretreatment, but ERK1/2 was not affected. No cytotoxic or pro-apoptotic effects were detected under the concentration of triptolide we used. CONCLUSIONS: Triptolide may function as an inhibitor of asthma airway remodeling by suppressing ASMCs proliferation and migration through inactivation of NF-κB pathway. This article is protected by copyright. All rights reserved.

Tumor-induced perturbations of cytokines and immune cell networks.

Until recently, the intrinsically high level of cross-talk between immune cells, the complexity of immune cell development, and the pleiotropic nature of cytokine signaling have hampered progress in understanding the mechanisms of immunosuppression by which tumor cells circumvent native and adaptive immune responses. One technology that has helped to shed light on this complex signaling network is the cytokine antibody array, which facilitates simultaneous screening of dozens to hundreds of secreted signal proteins in complex biological samples. The combined applications of traditional methods of molecular and cell biology with the high-content, high-throughput screening capabilities of cytokine antibody arrays and other multiplexed immunoassays have revealed a complex mechanism that involves multiple cytokine signals contributed not just by tumor cells but by stromal cells and a wide spectrum of immune cell types. This review will summarize the interactions among cancerous and immune cell types, as well as the key cytokine signals that are required for tumors to survive immunoediting in a dormant state or to grow and spread by escaping it. Additionally, it will present examples of how probing secreted cell-cell signal networks in the tumor microenvironment (TME) with cytokine screens have contributed to our current understanding of these processes and discuss the implications of this understanding to antitumor therapies.

Polysaccharide from Phellinus linteus induces S-phase arrest in HepG2 cells by decreasing calreticulin expression and activating the P27kip1-cyclin A/D1/E-CDK2 pathway.

ETHNOPHARMACOLOGY RELEVANCE: Our previous study showed that the proteoglycan P1 from Phellinus linteus (Mesima) exhibits significant anti-tumor activity against human hepatocellular carcinoma cells (HepG2); however, its molecular mechanism remains unknown. This study aims to provide insights into the mechanism of the anti-tumor activity of P1 against HepG2 cells. METHODS: We examined the effects of P1 on HepG2 cell proliferation in vitro and in vivo. Flow cytometry was used to analyze the cell cycle distribution and apoptosis. Proteomic analysis, real-time (RT)-PCR, and Western blot were carried out to observe the expression of several cell cycle control proteins in HepG2 cells. RESULTS: Both the volume and the weight of solid tumors were significantly decreased in P1-treated mice (200mg/kg) compared with the control. The HepG2 cells in the P1-treated tumors were significantly decreased, irregularly shaped, and smaller. P1 slightly increased the body weight of the tumor-bearing mice, which indicates that P1 is nontoxic to mammals at 200mg/kg. P1 also caused a significant dose-dependent increase in S phase arrest, but no apoptosis was observed in HepG2 cells. The results of the proteomic analysis, RT-PCR, and Western blot analysis showed that significantly downregulated expression of calreticulin, cyclin D1, cyclin E, and CDK2 and upregulated expression of P27 kip1 and cyclin A in the P1-treated HepG2 cells (200 µg/ml). CONCLUSION: These results suggest that calreticulin expression and the P27 kip1-cyclin A/D1/E-CDK2 pathway were involved in P1-induced S-phase cell cycle arrest in HepG2 cells.

Activation of P27kip1-cyclin D1/E-CDK2 pathway by polysaccharide from Phellinus linteus leads to S-phase arrest in HT-29 cells.

Our previous study showed that polysaccharide (P1) from Phellinus linteus exhibits a significant inhibitive activity on human colorectal carcinoma cells (HT-29). However its novel molecular mechanism remains unknown. To obtain insights into P1's mechanism of action, we examined its effects on cell proliferation in vitro and in vivo, cell cycle distribution, apoptosis, autophagy, and expression of several cell cycle interrelated proteins in HT-29 cells. Interestingly, we found that volume and weight of the solid tumor significantly decreased in P1 (200mg/kg)-treated mice compared with the control. However, slightly increased the body weight of the P1 treated tumor-bearing mice, with no significant increased ALT, AST levels in serum and LPO concentration in liver and kidney indicated that P1 has no toxicity to mammals at a dose of 200mg/kg. Furthermore, P1 caused a significantly dose-dependent increase in the S-phase cell cycle, but no apoptosis and autophagy in HT-29 cells. RT-PCR and Western blot results showed significantly down-regulated expressions of cyclin D1, cyclin E, and CDK2, as well as increased expressions of P27kip1 in P1 (100 µg/mL)-treated HT-29 cells. These results suggested that the activation of P27kip1-cyclin D1/E-CDK2 pathway is involved in P1-induced S-phase cell cycle arrest in HT-29 cells.

Antioxidant and anti-fatigue effects of anthocyanins of mulberry juice purification (MJP) and mulberry marc purification (MMP) from different varieties mulberry fruit in China.

Anthocyanins, copiously distributed in a variety of colored fruits and vegetables, are probably the most important group of visible plant pigments besides chlorophyll. And the mulberry fruit is one of the anthocyanins-rich fruits. Total flavonols, total phenolic acids and anthocyanins contents of ten varieties mulberry juice purification (MJP) and mulberry marc purification (MMP) were determined. The highest content was 965.63±4.90 mg RE/g, 690.83±7.38 mg GAE/g and 272.00±1.20 mg cyanidin-3-glucoside/g FW, respectively. Moreover, MJP and MMP exhibited high antioxidant activity, including total force reduction (TRP), Fe³âº reducing power (FRAP) and DPPH • radical scavenging capacity. In addition, the anti-fatigue activity of MJP and MMP was determined through mice-burden swimming experiments. Interestingly, the antioxidant and anti-fatigue capacities of MMP were much higher than those of MJP. The experimental results suggested that the generally discarded mulberry marc had greater value of development and utilization as food processing waste.

Cooperative anti-diabetic effects of deoxynojirimycin-polysaccharide by inhibiting glucose absorption and modulating glucose metabolism in streptozotocin-induced diabetic mice.

We had previously shown that deoxynojirimycin-polysaccharide mixture (DPM) not only decreased blood glucose but also reversed the damage to pancreatic ß-cells in diabetic mice, and that the anti-hyperglycemic efficacy of this combination was better than that of 1-deoxynojirimycin (DNJ) or polysachharide alone. However, the mechanisms behind these effects were not fully understood. The present study aimed to evaluate the therapeutic effects of DPM on streptozotocin (STZ)-induced diabetic symptoms and their potential mechanisms. Diabetic mice were treated with DPM (150 mg/kg body weight) for 90 days and continued to be fed without DPM for an additional 30 days. Strikingly, decrease of blood glucose levels was observed in all DPM treated diabetic mice, which persisted 30 days after cessation of DPM administration. Significant decrease of glycosylated hemoglobin and hepatic pyruvate concentrations, along with marked increase of serum insulin and hepatic glycogen levels were detected in DPM treated diabetic mice. Results of a labeled (13)C6-glucose uptake assay indicated that DPM can restrain glucose absorption. Additionally, DPM down-regulated the mRNA and protein expression of jejunal Na(+)/glucose cotransporter, Na(+)/K(+)-ATPase and glucose transporter 2, and enhanced the activities as well as mRNA and protein levels of hepatic glycolysis enzymes (glucokinase, phosphofructokinase, private kinase and pyruvate decarboxylas E1). Activity and expression of hepatic gluconeogenesis enzymes (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) were also found to be attenuated in diabetic mice treated with DPM. Purified enzyme activity assays verified that the increased activities of glucose glycolysis enzymes resulted not from their direct activation, but from the relative increase in protein expression. Importantly, our histopathological observations support the results of our biochemical analyses and validate the protective effects of DPM on STZ-induced damage to the pancreas. Thus, DPM has significant potential as a therapeutic agent against diabetes.

1-deoxynojirimycin inhibits glucose absorption and accelerates glucose metabolism in streptozotocin-induced diabetic mice.

We investigated the role of 1-deoxynojirimycin (DNJ) on glucose absorption and metabolism in normal and diabetic mice. Oral and intravenous glucose tolerance tests and labeled (13)C6-glucose uptake assays suggested that DNJ inhibited intestinal glucose absorption in intestine. We also showed that DNJ down-regulated intestinal SGLT1, Na(+)/K(+)-ATP and GLUT2 mRNA and protein expression. Pretreatment with DNJ (50 mg/kg) increased the activity, mRNA and protein levels of hepatic glycolysis enzymes (GK, PFK, PK, PDE1) and decreased the expression of gluconeogenesis enzymes (PEPCK, G-6-Pase). Assays of protein expression in hepatic cells and in vitro tests with purified enzymes indicated that the increased activity of glucose glycolysis enzymes was resulted from the relative increase in protein expression, rather than from direct enzyme activation. These results suggest that DNJ inhibits intestinal glucose absorption and accelerates hepatic glucose metabolism by directly regulating the expression of proteins involved in glucose transport systems, glycolysis and gluconeogenesis enzymes.

Evaluation of urban sprawl and urban landscape pattern in a rapidly developing region.

Urban sprawl is a worldwide phenomenon happening particularly in rapidly developing regions. A study on the spatiotemporal characteristics of urban sprawl and urban pattern is useful for the sustainable management of land management and urban land planning. The present research explores the spatiotemporal dynamics of urban sprawl in the context of a rapid urbanization process in a booming economic region of southern China from 1979 to 2005. Three urban sprawl types are distinguished by analyzing overlaid urban area maps of two adjacent study years which originated from the interpretation of remote sensed images and vector land use maps. Landscape metrics are used to analyze the spatiotemporal pattern of urban sprawl for each study period. Study results show that urban areas have expanded dramatically, and the spatiotemporal landscape pattern configured by the three sprawl types changed obviously. The different sprawl type patterns in five study periods have transformed significantly, with their proportions altered both in terms of quantity and of location. The present research proves that urban sprawl quantification and pattern analysis can provide a clear perspective of the urbanization process during a long time period. Particularly, the present study on urban sprawl and sprawl patterns can be used by land use and urban planners.

Hybrid of 1-deoxynojirimycin and polysaccharide from mulberry leaves treat diabetes mellitus by activating PDX-1/insulin-1 signaling pathway and regulating the expression of glucokinase, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in alloxan-induced diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: 1-Deoxynojirimycin (DNJ) discovered from mulberry trees has been reported to be a potent inhibitor of intestinal α-glycosidases (sucrase, maltase, glucoamylase), and many polysaccharides were useful in protecting against alloxan-induced pancreatic islets damage through their scavenging ability. This study was aimed to evaluate the therapeutic effect and potential mechanism(s) of the hybrid of DNJ and polysaccharide (HDP) from mulberry leaves on alloxan-induced diabetic mice. MATERIALS AND METHODS: Daily oral treatment with HDP (150 mg/kg body weight) to diabetic mice for 12 weeks, body weight and blood glucose were determined every week, oral glucose tolerance test was performed after 4 and 8 weeks, biochemical values were measured using assay kits and gene expressions were investigated by RT-PCR. RESULTS: A significant decline in blood glucose, glycosylated hemoglobin, triglyceride, aspartate transaminase and alanine transaminase levels and an evident increase in body weight, plasma insulin level and high density lipoprotein were observed in HDP treated diabetic mice. The polysaccharide (P1) showed a significant scavenging hydroxyl radicals and superoxide anion radical effects in vitro, which indicated that P1 could protect alloxan-induced pancreatic islets from damage by scavenging the free radicals and repaired the destroyed pancreatic ß-cells. Pharmacokinetics assay showed that DNJ could be absorbed from the gastrointestinal mucosa and diffused rapidly into the liver, resulted in postprandial blood glucose decrease and alleviated the toxicity caused by sustained supra-physiological glucose to pancreatic ß-cells. RT-PCR results indicated that HDP could modulate the hepatic glucose metabolism and gluconeogenesis by up/down-regulating the expression of rate-limiting enzymes (glucokinase, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) in liver and up-regulating the pancreatic and duodenal homeobox factor-1 (PDX-1), insulin-1 and insulin-2 expressions in pancreas. CONCLUSION: These findings suggested that HDP has complimentary potency to develop an antihyperglycemic agent for treatment of diabetes mellitus.

Expressions of connexin 32 and 26 and their correlation to prognosis of non-small cell lung cancer.

BACKGROUND AND OBJECTIVE: Gap junction intercellular communication (GJIC) plays an important role in regulating homeostasis and differentiation in many tissues. Connexins are gap junction proteins, whose expressions directly affect the function of GJIC. This study was to investigate expressions of connexin 32 and 26 proteins in non-small cell lung cancer (NSCLC), and their correlation to clinicopathological characters of NSCLC. METHODS: Immunohistochemistry was applied to detect expressions of connexin 32 and 26 in 77 NSCLC tissues. Correlations of connexin 32 and 26 expressions to smoking, tumor size, histological type, the degree of differentiation, lymph node metastasis and prognosis were analyzed. RESULTS: The positive rates of connexin 32 and 26 were 51.9% and 40.3% in the 77 samples, which were significantly higher than 20.3% and 30.5% in alveolar epithelium (p = 0.000, r = -0.322; p = 0.013, r = -0.215). Positive expression of connexion 32 was positively correlated with the differentiation degree of NSCLC tissues (p = 0.010, r = 0.345). The one- to five-year survival rates were higher in patients with positive connexion 32 expression than those without (p = 0.005). Moreover, the positive rate of connexin 26 was not correlated to smoking, tumor size, histological type, the degree of differentiation, lymph node metastasis and the postoperative survival time (p > 0.05). CONCLUSIONS: Expression of connexin 32 is closely correlated to the differentiation of NSCLC and affects the prognosis of NSCLC patients. Increasing the expression of connexin 32 may improve the prognosis of NSCLC.