The Rice Endophyte-Derived α-Mannosidase ShAM1 Degrades Host Cell Walls To Activate DAMP-Triggered Immunity against Disease.

Endophytes play an important role in shaping plant growth and immunity. However, the mechanisms for endophyte-induced disease resistance in host plants remain unclear. Here, we screened and isolated the immunity inducer ShAM1 from the endophyte Streptomyces hygroscopicus OsiSh-2, which strongly antagonizes the pathogen Magnaporthe oryzae. Recombinant ShAM1 can trigger rice immune responses and induce hypersensitive responses in various plant species. After infection with M. oryzae, blast resistance was dramatically improved in ShAM1-inoculated rice. In addition, the enhanced disease resistance by ShAM1 was found to occur through a priming strategy and was mainly regulated through the jasmonic acid-ethylene (JA/ET)-dependent signaling pathway. ShAM1 was identified as a novel α-mannosidase, and its induction of immunity is dependent on its enzyme activity. When we incubated ShAM1 with isolated rice cell walls, the release of oligosaccharides was observed. Notably, extracts from the ShAM1-digested cell wall can enhance the disease resistance of the host rice. These results indicated that ShAM1 triggered immune defense against pathogens by damage-associated molecular pattern (DAMP)-related mechanisms. Our work provides a representative example of endophyte-mediated modulation of disease resistance in host plants. The effects of ShAM1 indicate the promise of using active components from endophytes as plant defense elicitors for the management of plant disease. IMPORTANCE The specific biological niche inside host plants allows endophytes to regulate plant disease resistance effectively. However, there have been few reports on the role of active metabolites from endophytes in inducing host disease resistance. In this study, we demonstrated that an identified α-mannosidase protein, ShAM1, secreted by the endophyte S. hygroscopicus OsiSh-2 could activate typical plant immunity responses and induce a timely and cost-efficient priming defense against the pathogen M. oryzae in rice. Importantly, we revealed that ShAM1 enhanced plant disease resistance through its hydrolytic enzyme (HE) activity to digest the rice cell wall and release damage-associated molecular patterns. Taken together, these findings provide an example of the interaction mode of endophyte-plant symbionts and suggest that HEs derived from endophytes can be used as environmentally friendly and safe prevention agent for plant disease control.

Sequential targeting dual-responsive magnetic nanoparticle for improved therapy of lung metastatic breast cancer.

Lung metastatic breast cancer is a leading cause of cancer-related death in women and difficult to treat due to non-specific drug delivery. Herein a sequential targeting dual-responsive magnetic nanoparticle was fabricated, where Fe3O4 nanoparticle was used as magnetic core, then sequentially coated with tetraethyl orthosilicate, bis[3-(triethoxy-silyl)propyl] tetrasulfide, and 3-(trimethoxysilyl) propylmethacrylate to afford -C = C- on the surface for further polymerisation with acrylic acid, acryloyl-6-ethylenediamine-6-deoxy-ß-cyclodextrin using N, N-bisacryloylcy- stamine as cross-linker, obtaining pH/redox dual-responsive magnetic nanoparticle (MNPs-CD) to delivery doxorubicin (DOX) for suppressing lung metastatic breast cancer. Our results suggested DOX-loaded nanoparticle could target the lung metastases site by sequential targeting, in which they first be delivered to the lung and even the metastatic nodules through size-driven, electrical interaction, and magnetic field-guided mechanisms, then be effectively internalised into the cancer cells followed by intelligently triggering DOX release. MTT analysis demonstrated DOX-loaded nanoparticle exhibited high anti-tumour activity against 4T1 and A549 cells. 4T1 tumour-bearing mice were employed to confirm the higher specific accumulation in lung and improved anti-metastatic therapy efficiency of DOX by focussing an extracorporeal magnetic field on the biological target. Our findings suggested the as-proposed dual-responsive magnetic nanoparticle offered a prerequisite to inhibit lung metastasis of breast cancer tumours.

Development and characterization of an amorphous curcumin-Eudragit®E100 solid dispersions with improved solubility, stability, and pharmacokinetic properties.

The development of amorphous solid dispersions (ASD) is one way to overcome the bioavailability challenges of poorly water-soluble drug. Herein, Curcumin (CUR) was dispersed in the polymeric matrix of Eudragit®E100 by solvent evaporation, giving ASD, donated as CUR@Eudragit®E100. Solubility and stability of CUR were greatly enhanced. DSC and XRD analysis confirmed that the incorporated CUR was present in an amorphous state. The interaction between CUR and Eudragit®E100 was investigated through FTIR and molecular modelling studies which implied that -OH groups in CUR, and carboxyl and amino groups in Eudragit®E100 involved in the hydrogen bond formation. High resolution atomic force microscopy was employed to directly visualize the molecular morphology of Eudragit®E100 and CUR in CUR@Eudragit®E100 and the interaction between CUR and the polymer. pH influenced CUR release profile in which the sustained release pattern was revealed vs the physical mixtures. From the plasma concentration vs time profile graph, oral bioavailability of Cur@Eudragit®E100 was approximately 5-fold higher than that of native CUR. These results confirmed the potential of designing ASD to enhance the solubility and bioavailability of CUR, simultaneously deliver CUR through this alternative administration route.

Prediction of prognosis, immune infiltration and immunotherapy response with N6-methyladenosine-related lncRNA clustering patterns in cervical cancer.

LncRNAs and tumor microenvironment (TME) exert an important effect in antitumor immunity. Nonetheless, the role of m6A-related lncRNA clustering patterns in prognosis, TME and immunotherapy of cervical cancer (CC) remains unknown. Here, based on 7 m6A-related prognostic lncRNAs obtained from TCGA-CC dataset, two m6AlncRNA clustering patterns were determined. m6AlncRNA clusterA was characterized by immune cell infiltrates and immune activation. m6AlncRNA clusterB was characterized by enrichment of immune evasion and tumorigenic activation pathways as well as survival and clinical stage disadvantage. Then, principal component analysis algorithms were used to construct m6AlncRNAscore based on prognostic differentially expressed genes between two m6AlncRNA clusters to quantify m6AlncRNA clustering patterns. m6AlncRNAscore was an independent prognostic protective factor. Higher Th2 and Treg cells and enrichment of immunosuppressive pathways were observed in the low-m6AlncRNAscore group, with poorer survival. High-m6AlncRNAscore was characterized by increased infiltration of activated CD8 T cell, enrichment of immune activation pathways, lower IL-10 and TGF-beta1 levels, and higher immunophenscore values, indicating inflamed TME and better anti-tumor immunotherapy efficacy. Quantitative Real-Time Polymerase Chain Reaction was used for detection of m6A-related prognostic lncRNAs. Collectively, we identified two m6AlncRNA clustering patterns which play a nonnegligible role in the prognosis, TME heterogeneity and immunotherapy of CC patients.

m6A-Related lncRNAs Are Potential Prognostic Biomarkers of Cervical Cancer and Affect Immune Infiltration.

The correlation of m6A-related lncRNAs with the prognosis and immune microenvironment of cervical cancer is not yet clear. In this study, we identified 7 m6A-related prognostic lncRNAs by Pearson correlation and univariate Cox regression analyses based on TCGA-cervical cancer dataset. Then, patients were divided into two clusters by consensus clustering based on the 7 m6A-related prognostic lncRNA expression. Cluster 1 was characterized by survival and stage disadvantage, enrichment of immunosuppressive and carcinogenic activation pathways. Besides, cluster 1 had higher immunosuppressive factor TGFbeta and lower immune cell infiltration compared with cluster 2. According to the expression of 7 m6A-related lncRNA, a 6-m6A-related lncRNA risk score model was established in the training set by LASSO regression analysis. The high-risk group had worse overall survival than the low-risk group. No matter in the training or validation sets, the m6A-related lncRNA risk score was an independent prognostic factor for overall survival. Meanwhile, we validated the independent prognostic value of risk score in the disease-specific survival and progression-free survival by multivariate Cox analysis. The high-risk group was characterized by higher TGFbeta and regulatory T cell and was rich in malignant pathways. Additionally, we also detected and compared the expression levels of four m6A-related prognostic lncRNA in 9 tumor samples and 9 normal tissues using quantitative real-time polymerase chain reaction assay. In conclusion, the novel m6A-related lncRNA risk score is a potential prognostic predictor of cervical cancer patients. These 6 m6A-related lncRNAs might serve as key mediators of the immune microenvironment and represent promising therapeutic targets for improving cervical cancer prognosis.

ctc-[Pt(NH3)2(cinnamate)(valproate)Cl2] is a highly potent and low-toxic triple action anticancer prodrug.

Pt(iv) prodrugs have gained tremendous attention due to their indisputable advantages compared to cisplatin. Herein, new Pt(iv) derivatives with cinnamic acid at the first axial position, and inhibitor of matrix metalloproteinases-2 and -9, histone deacetylase, cyclooxygenase or pyruvate dehydrogenase at the second axial position are constructed to develop multi-action prodrugs. We demonstrate that Pt(iv) prodrugs are reducible and have superior antiproliferative activity with IC50 values at submicromolar concentrations. Notably, Pt(iv) prodrugs exhibit highly potent anti-tumour activity in an in vivo breast cancer model. Our results support the view that a triple-action Pt(iv) prodrug acts via a synergistic mechanism, which involves the effects of CDDP and the effects of axial moieties, thus jointly leading to the death of tumour cells. These findings provide a practical strategy for the rational design of more effective Pt(iv) prodrugs to efficiently kill tumour cells by enhancing their cellular accumulation and tuning their canonical mechanism.

Detection of α-globin gene deletions using denaturing high-performance liquid chromatography and multiplex ligation-dependent probe amplification.

BACKGROUND: Multiplex ligation-dependent probe amplification (MLPA) has been used to detect deletions and mutations of the α-globin gene for diagnosis of α-thalassemia. MLPA reaction products are usually separated and analyzed by high-voltage capillary gel electrophoresis (CGE). The goal of this study was to find and use a cost-effective method to separate and analyze MLPA products. METHODS: Blood samples were collected from China. DNA was extracted and amplified by PCR using fluorescently labeled primers. In this study, denaturing high-performance liquid chromatography (DHPLC) was used to separate and analyze the reaction products. And the optimal separation conditions were determined using nondenaturing columntemperature. RESULTS: The DHPLC conditions were optimized and have been applied to separate MLPA products and 27 of the MLPA products from 50 to 320 bp were well separated. DHPLC was able to separate up to 37 reaction products that differed by 4-12 base pairs and detected target gene deletions by differences in peak size. Compared with CGE, both the specificity and sensitivity of DHPLC for the 107 DNA samples were 100%. CONCLUSIONS: DHPLC could be used to test routinely for α-globin gene mutations and deletions. Combined with MLPA, DHPLC is a low-cost, simple to use, accurate technique with practical value.

An assay of gene copy number and its application based on heteroduplex products.

The aim of this study was to set up a simple and efficient method for detecting gene copy number, based on heteroduplex products from single-tube PCR/DHPLC. Single-nucleotide polymorphisms (SNPs) on the α-globin gene and chromosome 21 were used as examples. And the formula for quantitative calculation of gene copy number was deduced-based on the peak heights of homoduplexes and heteroduplexes on the DHPLC pattern. 27 samples (14 normal DNA and 13 cases of trisomy-21) were assessed with this method, and 160 samples (48 normal DNA and 112 α-thalassemia samples) were assessed with this method combined with a duplex PCR/DHPLC. Results for 184 of 187 cases were concordant with the known genotypes; three cases of trisomy-21 could not be detected because the target SNPs were homozygous. In conclusion, quantitative assessment of heteroduplex products from single-tube PCR/DHPLC is simple and rapid, and can be used to detect α-thalassemia gene deletions (α(-3.7), α(-4.2)) and trisomy-21.