Ultrasonic microbubbles promote mesenchymal stem cell homing to the fibrotic liver via upregulation of CXCR4 expression.

OBJECTIVE: To investigate the mechanism of ultrasound microbubbles (UTMB) promoting stem cells homing to fibrotic liver. METHODS: Bone marrow derived mesenchymal stem cells (BMSCs) were divided into 5 groups with or without ultrasound microbubbles and continuously irradiated with ultrasound conditions of frequency 1 MHZ and output power 0.6 W/cm2 for different times, and then injected into a mouse model of liver fibrosis through the tail vein with or without ultrasound microbubbles, with sound intensity. The effect of ultrasound microbubbles on MSC expression of CXC chemokine receptor 4 (CXCR4) and homing fibrotic liver was evaluated by flow cytometry (FCM), western blot (WB) and immunohistochemistry (IHC) analysis. RESULTS: The level of CXCR4 expression was significantly higher in the ultrasound microbubble group than in the non-intervention group (P < 0.05), and the number of MSC and the rate of CXCR4 receptor positivity in the ultrasound microbubble-treated liver tissues were significantly higher than in the non-intervention group (P < 0.01). CONCLUSION: Ultrasonic microbubbles can promote the expression of CXCR4 on the surface of MSCs, thus improving the homing rate of MSCs in fibrotic liver.

Protective effect of L-pipecolic acid on constipation in C57BL/6 mice based on gut microbiome and serum metabolomic.

BACKGROUND: Functional constipation (FC) in children affects their growth, development and quality of life. L-pipecolic acid (L-PA) was decreased in FC children based on gut microbiome and serum metabolomic. In this study, loperamide-induced constipation in mice was used to evaluate the effects of L-PA on constipated mice. METHOD: 26 FC and 28 healthy children were recruited. Stool samples and serum samples were subjected to 16S rDNA sequencing and ultra-performance liquid chromatography/quadrupole time of flight (UPLC-Q/TOF-MS) approach, respectively. A loperamide-induced mouse constipation model was developed, and all mice were randomly divided into control (Con), loperamide (Lop) and L-PA (Lop + L-PA) treatment groups (6 mice per group). The mice in the Lop + L-PA group were given L-PA (250 mg/kg, once a day) and loperamide; the Lop group was given loperamide for 1 week, and the Con group was given saline. The fecal parameters and intestinal motility of mice in each group were detected. serum 5-HT levels and colon 5-HT expression were detected by ELISA and immunohistochemistry, respectively; qRT-PCR was used to detect the expression of AQP3 and 5-HT4R mRNA in each group. RESULTS: 45 differential metabolites and 18 significantly different microbiota were found in FC children. The α and ß diversity of gut microbiota in FC children was significantly reduced. Importantly, serum L-PA was significantly reduced in FC children. The KEGG pathway enrichment were mainly enriched in fatty acid biosynthesis, lysine degradation, and choline metabolism. L-PA was negatively associated with Ochrobactrum, and N6, N6, N6-trimethyl-l-lysine was positively associated with Phascolarcrobacterium. In addition, L-PA improved the fecal water content, intestinal transit rate, and increased the serum 5-HT levels in constipated mice. Moreover, L-PA increased the expression of 5-HT4R, reduced AQP3, and regulated constipation-associated genes. CONCLUSIONS: Gut microbiota and serum metabolites were significantly altered in children with FC. The abundance of Phascolarctobacterium and Ochrobactrum and serum L-PA content were decreased in FC children. L-PA was found to alleviate the fecal water content, increase intestinal transit rate and the first black stool defecation time. L-PA improved constipation by increasing 5-HT and 5-HT4R expression while down-regulating AQP3 expression.

Meta-analysis of the impact of future climate change on the area of woody plant habitats in China.

Climate change poses a very serious threat to woody plants, and it is important to study its impact on the distribution dynamics of woody plants in China. However, there are no comprehensive quantitative studies on which factors influence the changes in the area of woody plant habitats in China under climate change. In this meta-analysis, we investigated the future suitable habitat area changes of 114 woody plant species in 85 studies based on MaxEnt model predictions to summarize the future climate change impacts on woody plant habitat area changes in China. It was found that climate change will result in a 3.66% increase in the overall woody plant suitable areas and a 31.33% decrease in the highly suitable areas in China. The mean temperature of the coldest quarter is the most important climatic factor, and greenhouse gas concentrations were inversely related to the area of future woody plant suitable areas. Meanwhile, shrubs are more climate-responsive than trees, drought-tolerant plants (e.g., Dalbergia, Cupressus, and Xanthoceras) and plants that can adapt quickly (e.g., Camellia, Cassia, and Fokienia) and their appearance will increase in the future. Old World temperate, Trop. Asia and Trop. Amer. disjuncted, and the Sino-Himalaya Floristic region are more vulnerable. Quantitative analysis of the possible risks to future climate change in areas suitable for woody plants in China is important for global woody plant diversity conservation.

CsMYC2 is involved in the regulation of phenylpropanoid biosynthesis induced by trypsin in cucumber (Cucumis sativus) during storage.

Trypsin has a new activity of scavenging superoxide anion and generating hydrogen peroxide. Trypsin can significantly improve the storage quality of C. sativus. To illustrate the mechanism of trypsin-induced resistance in fruits and vegetables, an integrated analysis of widely targeted metabolomics and transcriptomics was carried out. Transcriptomic results showed that 1068 genes highly related to phenylpropanoid biosynthesis gathered in the brown module were obtained by WGCNA. In KEGG analysis, differentially expressed genes (DEGs) were also highly enriched in EIP (Environmental Information Processing) pathways "Plant hormone signal transduction (map04075)" and "MAPK signaling pathway-plant (map04016)". Next, 87 genes were identified as the leading edge by GSEA analysis. So far, CsMYC2 was highlighted as a key transcription factor that regulates phenylpropanoid biosynthesis identified by GSEA and WGCNA. Furthermore, the major route of biosynthesis of phenylpropanoid compounds including coumarins, lignins, chlorogenic acid, flavonoids, and derivatives regulated by trypsin was also illustrated by both transcriptomic and metabolomic data. Results of O2PLS showed that CsMYC2 was positively correlated with Rosmarinic acid-3-O-glucoside, Epigallocatechin, Quercetin-3-O-sophoroside (Baimaside), and so on. Correlation between CsMYC2, phenylpropanoid related genes, and metabolites in C. sativus was illustrated by co-expression networks. Roles of CsMYC2 were further checked in C. sativus by VIGS. The results of this study might give new insight into the exploration of the postharvest resistance mechanism of C. sativus induced by trypsin and provide useful information for the subsequent mining of resistance genes in C. sativus.

IL-2/GM-CSF enhances CXCR3 expression in CAR-T cells via the PI3K/AKT and ERK1/2 pathways.

OBJECTIVE: To investigate the effects of cytokines IL-2 and GM-CSF on CXCR3 expression and chemotaxis of CAR-T cells. BACKGROUND: High lymphocyte infiltration within the tumor is a basic requirement for good results in tumor immunotherapy; C-X-C motif chemokine receptor 3 (CXCR3) is an important factor for the chemotaxis of lymphocytes to tumor tissues. The tumor microenvironment can exhibit diverse cytokine suppression or promote antitumor immunity. Both interleukin (IL)-2 and granulocyte macrophage colony-stimulating factor (GM-CSF) contribute to the regulation of immunosuppression in the tumor microenvironment. However, the effects of IL-2 and GM-CSF on CXCR3 expression on the T cell surface and its mechanisms are not well understood. Here, we explored the effects of polycytokines on CXCR3 expression in chimeric antigen receptor T cells (CAR-T cells) and on HuH-7 in situ hepatocellular carcinoma. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated, followed by purifying using CD3 immunomagnetic beads. Cells were divided into three groups. After 24h of activation using CD3/CD28 antibody, T cells were transfected using lentiviral vector, pGC-SV40-EGFP-GPC3-CAR. Three culture methods were used to amplify the transfected T cells. Method 'A' was to incubate T cells with CD3/CD28 antibody; method 'B' was with CD3/CD28 antibody and IL-2 at a final concentration of 1000 U/ml; method 'C' was with method B in addition of GM-CSF at a final concentration of 1000 U/ml. The phosphorylation of MAPK and PI3K/AKT was determined by western blot. The chemotaxis effect of CAR-T cells on Huh-7 HCCIA in situ was assayed by immunofluorescence and immunohistochemistry. RESULTS: The CD3/CD28/IL-2/GM-CSF combination is the most potent for stimulating activated CAR-T cell proliferation and CXCR3 expression in vitro; CD3/CD28/IL-2 induces CAR-T cell expression of CXCR3 through the activation of the PI3K/APK pathway and GM-CSF induces CXCR3 expression in CAR-T cells through the activation of ERK1/2 rather than the p38 MAPK signaling pathway. CAR-GPC3-T cells with high CXCR3 expression showed increased chemotaxis ability to HuH in situ hepatocellular carcinoma, and considerably inhibited the growth of in situ tumors in nude mouse livers. CONCLUSION: A multi-factorial amplification protocol can effectively improve CXCR3 expression on the surface of activated CAR-T cells in vitro, as well as enhance the chemotaxis ability of CAR-T cells in vivo, which significantly inhibit the growth of liver cancer.

Application of alkaline phosphatase­to­platelet ratio as a novel noninvasive index predicts liver fibrosis in patients with chronic hepatitis B.

Assessment of the extent of liver fibrosis is a crucial requirement for the design of antiviral treatments for patients with chronic hepatitis B (CHB). Several non-invasive predictive indices have been developed as potential alternatives to liver biopsy for fibrosis assessment. The present study aimed to establish a novel non-invasive method for predicting liver fibrosis in patients with CHB. A total of 382 patients with CHB who underwent liver biopsy and pathological examination at The Second Hospital of Anhui Medical University (Hefei, China) were enrolled into the present study. Liver fibrosis was assessed according to the meta-analysis of histological data in viral hepatitis scoring system. Logistic regression analyses were performed to explore possibly significant characteristics associated with liver fibrosis. In addition, potential correlations between the alkaline phosphatase (AKP)-to-platelet count (PLT) ratio (APPR) and the aspartate transaminase-to-platelet ratio index (APRI), fibrosis index based on four factors (FIB-4) and γ-glutamyl transpeptidase-to-platelet ratio (GPR) were assessed using Spearman's correlation analysis. Subsequently, the performance of APPR was compared with APRI, FIB-4 and GPR using receiver operating characteristic (ROC) analysis. Logistic regression analysis identified AKP and PLT to be significant independent predictors of fibrosis. Therefore, an index was then constructed for predicting the degree of fibrosis, which was expressed using the formula APPR=AKP (IU/ml)/PLT (1x109/l). APPR was found to be positively associated with the fibrotic stage of the liver in addition to being positively correlated with APRI, FIB-4 and GPR. The area under the ROC curve (AUROC) values of APPR were also significantly higher compared with those of APRI and FIB-4 in predicting significant fibrosis but were equal to those of GPR. However, for advanced fibrosis and cirrhosis, the AUROC value of APPR was shown to be higher compared with that of APRI, FIB-4 and GPR. In conclusion, these observations suggest that APPR is a viable marker that can be used to assess liver fibrosis in patients with CHB.

[The expression of interferon-stimulating gene (STING/TMEM173) in liver tissue and its correlation with liver inflammation in patients with chronic hepatitis B].

Objective To investigate the relationship between the expression and distribution of interferon-stimulating gene/transmembrane protein 173(STING/TMEM173) in liver tissue and the grade of liver inflammation in patients with chronic hepatitis B, and to explore the underlying mechanisms in vitro. Methods The expression of STING/TMEM173 protein in liver tissue of 62 naive patients with chronic hepatitis B was detected by immunohistochemistry. Rank sum test and spearman correlation coefficient were used to analyze the correlation between hepatic STING/TMEM173 expression and liver inflammation grades as well as serum ALT levels. After transient or stable transfection by HBV whole genome plasmid, the expression of STING/TMEM173 in HepG2 cells was determined by Western blot analysis. The peripheral blood mononuclear cells (PBMCs) were stimulated by supernatant of HepG2.2.15 cells containing intact HBV virions, and the expression STING/TMEM173 gene was detected by real-time PCR. Results The results of immunohistochemical showed that STING/TMEM173 protein was higher in liver tissues of CHB patients and mainly expressed in inflammatory cells of liver tissue, and the expression of STING/TMEM173 protein was positively correlated with liver inflammation grade as well as serum ALT level. After transient and stable transfection by HBV whole genome plasmid, the STING/TMEM173 protein decreased significantly in HepG2 cells. In addition, HepG2.2.15 cell supernatant containing intact HBV virions promoted the expression of STING/TMEM173 in PBMC in a dose-dependent manner at RNA level. Conclusion HBV can up-regulate the expression of STING/TMEM173 protein in inflammatory cells of liver tissue, and the number of liver inflammatory cells expressing STING/TMEM173 may reflect the severity of liver inflammation.

Serum sCD14 as a Biomarker for Significant Liver Inflammation in Chronic Hepatitis B Patients with Normal or Mildly Elevated ALT.

BACKGROUND: CD14 is a pattern recognition receptor constitutively expressed in different types of immune cells, either in a membrane-anchored (mCD14) or in a soluble (sCD14) form. This study investigated whether hepatic CD14 expression levels were correlated with the grades of liver inflammation as well as the potential usefulness of serum sCD14 as a biomarker for predicting liver inflammation in chronic hepatitis B (CHB) patients with normal or mildly elevated ALT. METHODS: A total of 216 treatment-naive CHB patients with normal or mildly elevated ALT who underwent liver biopsy were recruited. Hepatic expression level of CD14 was measured using immunohistochemistry and real-time PCR. Serum sCD14 concentrations were determined with an enzyme-linked immunosorbent assay. Correlations between hepatic CD14, serum sCD14, and liver inflammation grade were analyzed. Univariate and multivariate analysis were performed to identify significant liver inflammation-associated factors. The receiver operating characteristic curve was used to assess the discriminating power of serum sCD14 to significant liver inflammation in CHB patients with normal or mildly elevated ALT. RESULTS: Both hepatic expression levels of CD14 and serum sCD14 concentrations significantly increased with the aggravation of liver inflammation. Moreover, hepatic expression levels of CD14, serum sCD14 concentrations, and liver inflammation grades were positively correlated with each other. Three parameters including alkaline phosphatase (ALP), neutrophil, and sCD14 were identified as independent predictors of significant liver inflammation. Subsequently, a diagnostic equation named model-sCD14 was developed incorporating sCD14 and other variables (ALP and neutrophils) with p < 0.05 in multivariate logistic analysis. The area under receiver operating characteristic curve (AUC) of sCD14 for predicting significant liver inflammation was 0.788 and the optimal cutoff was 27.14 ng/mL, with a sensitivity of 66.67%, a specificity of 81.70%, positive predictive value of 60.01%, and negative predictive value of 85.62%. When sCD14 was replaced by model-sCD14, the AUC value increased from 0.788 to 0.843 (z = 2.311, p = 0.021), with sensitivity of 77.78%, specificity of 77.12%, positive predictive value of 58.33%, and negative predictive value of 89.39%. CONCLUSIONS: Serum sCD4 has the potential to discriminate significant liver inflammation from CHB patients with normal or mildly increased ALT levels.

Erratum: Ceramide synthase-4 orchestrates the cell proliferation and tumor growth of liver cancer in vitro and in vivo through the nuclear factor-κB signaling pathway.

[This corrects the article DOI: 10.3892/ol.2017.6365.].

Antifungal Effects of Fusion Puroindoline B on the Surface and Intracellular Environment of Aspergillus flavus.

Aspergillus flavus infection is a major issue for safe food storage. In this study, we constructed an efficient prokaryotic expression system for puroindoline B (PINB) protein to detect its antifungal activity. The Puroindoline b gene was cloned into pET-28a (+) vector and expressed in Escherichia coli. Treatment with fusion PINB revealed that it inhibits mycelial growth of A. flavus, a common grain mold. Moreover, fusion PINB-treated A. flavus mycelium withered and exhibited a sunken spore head. As fusion PINB concentration increased, electrical conductivity in mycelium also increased, indicative of cell membrane damage. Furthermore, intracellular malate dehydrogenase and succinate dehydrogenase activity decreased, revealing a disruption in the tricarboxylic acid cycle. Moreover, the dampened activity of the ion pump Na+K+-ATPase negatively affected the intracellular regulation of both ions. Catalase and superoxide dismutase activity decreased, thus reducing antioxidant capacity, a result confirmed with an increase in malondialdehyde content. Changes to the GSH/GSSG ratio indicated a shift to an intracellular oxidative state. At the same time, laser scanning confocal microscopy assay showed the accumulation of reactive oxygen species and nuclear damage. Therefore, the PINB fusion protein may have the potential to control A. flavus in grain storage and food preservation.

[Spectral Characteristics and Source Analysis of WSOC of PM2.5 in Winter of Xi'an].

The spectral characteristics and sources of water-soluble organic compounds (WSOC) in PM2.5 in winter were studied by using UV-vis absorption spectroscopy, three-dimensional fluorescence spectroscopy, parallel factor analysis, and backward trajectory model. The results showed that the concentration of WSOC in PM2.5 was 4.66-14.75 µg ·m-3. The values of E2/E3, E3/E4, S275-295, SUVA254, AAE, and MAE365 of WSOC were, respectively, in the range of 2.85-4.32, 2.21-3.56, 0.0099-0.0127 nm-1, 2.35-3.89 m2 ·g-1, 2.66-4.60, and 1.51-2.60 m2 ·g-1. The E2/E3, E3/E4, S275-295, and AAE values of WSOC at the sampling site in the southern suburb of Xi'an, China (Xi'an University of Architecture and Technology) were higher than those at the sampling site in the northern suburb (sports park), while the values of SUVA254 and MAE365 were lower. There were four fluorescent components in WSOC identified by the EEMs-PARAFAC model: C1 and C2 were fulvic acid-like and protein-like, respectively, and C3 and C4 were humus-like components. The fluorescence intensities and the sum of the fluorescent components were positively correlated with the concentrations of PM2.5, OC, WSOC, and A254 value (P<0.01). The fluorescence index (FI), biological source index (BIX), and humic index (HIX) values of WSOC were 1.75-2.12, 1.14-1.46, and 1.18-2.06, respectively. During the monitoring period, the air mass transmission trajectory was dominated by the local southwest of short-distance transmission, and its trajectory accounted for more than 50%. The pollutant emissions from Xinjiang, Inner Mongolia, and Gansu also made significant contributions to the air pollution levels in Xi'an in winter. There was a small difference in the carbon component content of PM2.5 in the northern and southern suburbs of Xi'an. The molecular weight, humification degree, and light absorption capacity of WSOC at the southern suburb sampling site were lower than those in the northern suburb where the wavelength dependence of light absorption intensity was relatively stronger. The WSOC mainly originated from biological sources or both from biological and terrestrial sources. Local transmission had the most significant contribution to PM2.5 and WSOC in winter.

[Effect of salvianolic acid B on high-glucose induced renal tubular epithelial-mesenchymal transition in rats and its mechanism].

The aim of this paper was to observe the effect of salvianolic acid B(Sal B) on high-glucose induced renal tubular epithelial-mesenchymal transition(EMT) in rats, and to explore its possible mechanisms of prevention and treatment of diabetic nephropathy. The rat renal tubular epithelial NRK-52 E cells were cultured in vitro. The cells were divided into control group, high glucose group, high glucose+10 µmol·L~(-1)Sal B group(Sal B), the above 3 groups were set at 6, 12, 24 and 48 h for dynamic observation; high glucose+Sal B different concentration(1, 5, 10 µmol·L~(-1)) groups, high glucose+5.0 µmol·L~(-1) pioglitazone group, high glucose+10 µmol·L~(-1)Sal B+5 µmol·L~(-1)GW9662 group. The protein expression levels of PPARγ, PTEN, α-SMA, E-cadherin and PI3 K/Akt signaling molecules were determined by Western blot. The mRNA expression of PPARγ and PTEN were detected by Real-time PCR. The viabi-lity of NRK52 E cells was determined by MTT assay. The results showed that as compared with control group, the mRNA and protein expression levels of PPARγ and PTEN in high glucose group gradually reduced, the protein expression levels of α-SMA and p-Akt~((Thr308))gradually increased, and the protein expression of E-cadherin gradually reduced(P<0.05). As compared with high glucose group, when increases in Sal B doses, the mRNA and protein expression levels of PPARγ, PTEN in high glucose + different concentrations of Sal B groups gradually increased, the protein expression levels of α-SMA and p-Akt~((Thr308)) gradually reduced, and the protein expression of E-cadherin gradually increased(P<0.05), however, the effect of 1 µmol·L~(-1)concentration of Sal B on the expression of PPARγ mRNA and protein and PTEN mRNA was not significantly different. As compared with high glucose group, the mRNA and protein expression levels of PPARγ mRNA(except 6 h) and protein(except 6 h), PTEN mRNA(except 6 h) and protein(except 6, 12 h) kept increasing, the protein expression levels of α-SMA and p-Akt~((Thr308))(except 6 h) continued to reduce, the protein expression of E-cadherin kept increasing in high glucose+10 µmol·L~(-1) Sal B dynamic observation group(P<0.05). As compared with high glucose group, Sal B and the pioglitazone(PIO) can greatly enhance the expression of PPARγ, PTEN at mRNA and protein levels, enhance the expression of E-cadherin at protein levels, and reduce the expression of α-SMA, p-Akt~((Thr308))protein level(P<0.05), there was no significant difference between the two groups. However, the expression levels of PPARγ and PTEN mRNA and protein, E-cadherin, α-SMA and p-Akt(Thr308) protein in the Sal B+GW9662 control group were not statistically significant compared with the high glucose group. The effect of Sal B was blocked by the PPARγ antagonist GW9662. It can be concluded that Sal B can suppress the NRK52 E cells induced by high-glucose EMT. The mechanism may be related to the activation of PPARγ with Sal B, and the up-regulation of PTEN expression, and thereby inhibiting the fibrosis effect of PI3 K/Akt signaling pathway.

MicroRNA-27a targets Sfrp1 to induce renal fibrosis in diabetic nephropathy by activating Wnt/ß-Catenin signalling.

Diabetic nephropathy (DN) commonly causes end-stage renal disease (ESRD). Increasing evidence indicates that abnormal miRNA expression is tightly associated with chronic kidney disease (CKD). This work aimed to investigate whether miR-27a can promote the occurrence of renal fibrosis in DN by suppressing the expression of secreted frizzled-related protein 1 (Sfrp1) to activate Wnt/ß-catenin signalling. Therefore, we assessed the expression levels of miR-27a, Sfrp1, Wnt signalling components, and extracellular matrix (ECM)-related molecules in vitro and in vivo. Sfrp1 was significantly down-regulated in a high-glucose environment, while miR-27a levels were markedly increased. A luciferase reporter assay confirmed that miR-27a down-regulated Sfrp1 by binding to the 3' untranslated region directly. Further, NRK-52E cells under high-glucose conditions underwent transfection with miR-27a mimic or the corresponding negative control, miR-27a inhibitor or the corresponding negative control, si-Sfrp1, or combined miR-27a inhibitor and si-Sfrp1. Immunoblotting and immunofluorescence were performed to assess the relative expression levels of Wnt/ß-catenin signalling and ECM components. The mRNA levels of Sfrp1, miR-27a, and ECM-related molecules were also detected by quantitative real-time PCR (qPCR). We found that miR-27a inhibitor inactivated Wnt/ß-catenin signalling and reduced ECM deposition. Conversely, Wnt/ß-catenin signalling was activated, while ECM deposition was increased after transfection with si-Sfrp1. Interestingly, miR-27a inhibitor attenuated the effects of si-Sfrp1. We concluded that miR-27a down-regulated Sfrp1 and activated Wnt/ß-catenin signalling to promote renal fibrosis.

Effects of freezing treatments on the quality of frozen cooked noodles.

Freezing process is one of the key steps in making frozen cooked noodles. Ice crystal formed in freezing process affects the quality of frozen cooked noodles. In this paper, we studied the effect of freezing treatment on frozen cooked noodles. Frozen cooked noodles were evaluated for microstructure and texture properties explored with a scanning electron microscope and texture analyzer at - 20 °C, - 30 °C and - 40 °C respectively. The results indicated that the microstructure and texture properties of frozen cooked noodles were significantly (P < 0.05) improved by a lower freezing temperature than a higher temperature. This present study also showed that the freezing rate is not the only parameter responsible for microstructure and texture properties that occur during freezing; the difference of flours also can be a factor. These findings, if generally applicable to frozen cooked noodle products, could have important economic implications for the convenience of the food industry.

Expression and purification of recombinant puroindoline A protein in Escherichia coli and its antifungal effect against Aspergillus flavus.

Aspergillus flavus is the main cause of postharvest agricultural commodity loss. In this study, puroindoline A (PINA) protein was expressed in Escherichia coli, purified, and its antifungal properties against A. flavus were characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the molecular weight of the recombinant PINA protein was approximately 44 kDa. PINA exerted a powerful antifungal effect against A. flavus at 42.42 µg/mL on potato dextrose agar culture medium. Flow cytometry and scanning electron microscopy revealed that the spore morphology was damaged by PINA exposure; spores were depressed and broken, suggesting that the cell wall was impaired. Transmission electron microscopy and propidium iodide staining illustrated significant changes in intracellular spore structure, indicating cell membrane damage. 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide staining indicated decreased mitochondrial membrane potential. Large nuclear condensation and DNA fragmentation were detected by 4',6-diamidino-2-phenylindole staining. The expression of genes related to the cell wall, cell membrane, and spore germination significantly changed in PINA-treated cells; this illustrated the probable mode of PINA action on A. flavus through cell wall destruction and triggered cell membrane, mitochondrial, and DNA damage leading to cell death. The antifungal mechanism of wheat PINA protein on A. flavus has been demonstrated in this study, and has potential application in preventing postharvest loss in the agricultural industry.

Dynamic proteomic changes in soft wheat seeds during accelerated ageing.

Previous research demonstrated that soft wheat cultivars have better post-harvest storage tolerance than harder cultivars during accelerated ageing. To better understand this phenomenon, a tandem mass tag-based quantitative proteomic analysis of soft wheat seeds was performed at different storage times during accelerated ageing (germination ratios of 97%, 45%, 28%, and 6%). A total of 1,010 proteins were differentially regulated, of which 519 and 491 were up- and downregulated, respectively. Most of the differentially expressed proteins were predicted to be involved in nutrient reservoir, enzyme activity and regulation, energy and metabolism, and response to stimulus functions, consistent with processes occurring in hard wheat during artificial ageing. Notably, defense-associated proteins including wheatwin-2, pathogenesis-related proteins protecting against fungal invasion, and glutathione S-transferase and glutathione synthetase participating in reactive oxygen species (ROS) detoxification, were upregulated compared to levels in hard wheat during accelerated ageing. These upregulated proteins might be responsible for the superior post-harvest storage-tolerance of soft wheat cultivars during accelerated ageing compared with hard wheat. Although accelerated ageing could not fully mimic natural ageing, our findings provided novel dynamic proteomic insight into soft wheat seeds during seed deterioration.

Ceramide synthase-4 orchestrates the cell proliferation and tumor growth of liver cancer in vitro and in vivo through the nuclear factor-κB signaling pathway.

Liver cancer is one of the leading causes of cancer-associated mortalities worldwide, partly due to the absence of effective therapeutic targets and diagnostic biomarkers. Therefore, novel molecular targets are critical to develop new therapeutic approaches for liver cancer. In the present study, ceramide synthase-4 (CERS4) was investigated as a novel molecular target for liver cancer. High expression of CERS4 in liver cancer tissues was detected by reverse transcription polymerase chain reaction and western blot analysis. Subsequently, CERS4 was silenced by lentivirus-mediated RNA interfere, and the proliferation rates of liver cancer cells were significantly suppressed (P<0.001). In addition, the weight and volume of the tumors were reduced subsequent to silencing of CERS4 in liver cancer cells, revealed by an in vivo study using Balb/c nude mice. In addition, the nuclear factor (NF)-κB signaling pathway was affected following knockdown of CERS4 in liver cancer cells. The present results proposed that CERS4 is an important regulator of liver cancer cell proliferation and indicated that CERS4 may be a potential anticancer therapeutic target and a promising diagnostic biomarker for human liver cancer.

Upconverted Photosensitization of Tb Visible Emission by NIR Yb Excitation in Discrete Supramolecular Heteropolynuclear Complexes.

Addition of Tb3+ salts to a solution of a (YbLD) complex in D2O resulted in the formation of [(YbLD)2Tbx] (x = 1 to 3) complexes that, upon NIR excitation at 980 nm, showed an unprecedented Yb to Tb upconversion sensitization phenomenon resulting in the observation of the typical green emission of Tb.

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers.

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r=0.78) was better than that of the RAPD marker system (r=0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.

Comparative metabolomics reveals the mechanism of avermectin production enhancement by S-adenosylmethionine.

It was found that S-adenosylmethionine (SAM) could effectively improve avermectin titer with 30-60 µg/mL addition to FH medium. To clearly elucidate the mechanism of SAM on intracellular metabolites of Streptomyces avermitilis, a GC-MS-based comparative metabolomics approach was carried out. First, 230 intracellular metabolites were identified and 14 of them remarkably influenced avermectin biosynthesis were discriminative biomarkers between non-SAM groups and SAM-treated groups by principal components analysis (PCA) and partial least squares (PLS). Based on further key metabolic pathway analyses, these biomarkers, such as glucose, oxaloacetic acid, fatty acids (in soybean oil), threonine, valine, and leucine, were identified as potentially beneficial precursors and added in medium. Compared with single-precursor feeding, the combined feeding of the precursors and SAM markedly increased the avermectin titer. The co-feeding approach not only directly verified our hypothesis on the mechanism of SAM by comparative metabolomics, but also provided a novel strategy to increase avermectin production.