Physiological response of chestnuts (Castanea mollissima Blume) infected by pathogenic fungi and their correlation with fruit decay.

The postharvest decay of chestnuts (Castanea mollissima Blume) limits their industrial utilization, and pathogenic fungi are the main cause for chestnut decay. In this study, the physiological changes and their correlation with the rotting degree of chestnuts were investigated during single or mixed infection by Fusarium proliferatum, Penicillium crustosum, and Alternaria alternata. During the infection period, the activities of cell wall degrading enzymes (CWDEs) and antioxidant enzymes firstly increased and then decreased, the contents of nutrients decreased, but the levels of physiological indexes increased. The mycelium and spores of pathogenic fungi colonized the intercellular space, and then the mycelium covered the entire tissue surface of chestnuts, leading to the disappearance of cell structure. Notably, the most prominent changes in physiological indexes and ultrastructure were observed in chestnuts infected with three pathogenic fungi. Furthermore, the rotting degree of chestnuts was positively correlated with the levels of CWDEs, antioxidant enzymes and malondialdehyde.

SSR Marker Acquisition and Application from Transcriptome of Captive Chinese Forest Musk Deer (Moschus berezovskii).

Forest musk deer (Moschus berezovskii) is one of the most endangered medicinally important wild animals in the world. Forest musk deer farming is the main way of production of musk. However, the single provenance and lack of genetic information lead to reduced genetic diversity of forest musk deer. Therefore, more SSR markers need to be developed to identify forest musk deer germplasm. In this study, bone marrow derived mesenchymal cells were used to construct cDNA library for transcriptome sequencing. The datasets were de novo assembled and annotated. 9 polymorphic simple sequence repeat (SSR) markers were finally identified and used to detect population genetic diversity. 6.07 Gb clean data were generated using Illumina sequencing technology, and de novo assembled into 138,591 transcripts and 81,553 unigenes. 5,777 simple sequence repeats (SSRs) were identified, in which there were 578 repeating motif types, with mono-nucleotide and tri-nucleotides comprising 55.88% and 25.60%, respectively. 100 primer pairs were designed to validate amplification and polymorphism using DNA from fecal samples. 9 polymorphic SSRs were developed and used to detect population genetic diversity of 122 forest musk deer in 2 farms. The average number of alleles per locus varied from 4 to 15 (average = 8.3). The observed heterozygosity (HO) per locus ranged from 0.102 to 0.941, while the expected heterozygosity (HE) per locus was from 0.111 to 0.651. All loci deviated significantly from the Hardy-Weinberg equilibrium (p < 0.001). The polymorphism information content (PIC) of these loci varied from 0.108 to 0.619. 9 polymorphic SSR markers were developed in this research. These sites can be used for breeding planning and conservation of germplasm resources.

Preliminary exploration on the ectomycorrhizal status of a wild edible Gomphus species from Southwest China.

A wild edible Gomphus species was discovered at local wild mushroom markets from May to November in Southwest China, where it was eaten for hundreds of years. However, litter information on the taxonomy is available. Whether Gomphus is a saprotrophic, parasitic, or ectomycorrhizal (ECM) fungus is unclear. In the present study, field investigation, fungi isolation, optimum medium, morphological description, molecular analyses, and preliminary exploration on mycorrhizal synthesis were carried out. The morphological and molecular analyses showed that the same species between Gomphus matijun and Gomphus sp. (zituoluo) might be the related species of Gomphus purpuraceus. Moreover, the root dry weight and first-lateral root number of inoculated seedlings were significantly enhanced by evaluating Pinus massoniana seedlings inoculated with G. matijun. Meanwhile, the levels of nine phytohormones, including five new phytohormones, in the roots of inoculated seedlings were upregulated. This study explored the mycorrhizal synthesis of the wild edible Gomphus species from Southwest China with P. massoniana Lamb. We concluded that G. matijun might be an ECM fungus. The mycorrhizal synthesis of G. matijun under pure culture conditions provided the basis for the next inoculation under controlled soil conditions, making the conservation and cultivation of G. matijun feasible in the future.

Whole-Genome Sequencing and Comparative Genomics Analysis of the Wild Edible Mushroom (Gomphus purpuraceus) Provide Insights into Its Potential Food Application and Artificial Domestication.

Gomphus purpuraceus (Iwade) Yokoyama is a species of wild fungi that grows in southwest China, considered an edible and medicinal fungus with potential commercial prospects. However, the detailed mechanisms related to the development of mycelium and the formation of the fruiting body are unclear. To obtain a comprehensive overview of genetic features, whole-genome and comparative genomics analyses of G. purpuraceus were performed. High-quality DNA was extracted from the mycelium, which was isolated from a fresh fruiting body of G. purpuraceus. The DNA sample was subjected to sequencing using Illumina and Oxford Nanopore sequencing platforms. A genome assembly totaling 40.15 Mb in 50 contigs with an N50 length of 2.06 Mb was generated, and 8705 putative predicted genes were found. Subsequently, phylogenetic analysis revealed a close evolutionary relationship between G. purpuraceus and Gomphus bonarii. Moreover, a total of 403 carbohydrate-active enzymes (CAZymes) were identified in G. purpuraceus, which included 147 glycoside hydrolases (GHs), 85 glycosyl transferases (GTs), 8 polysaccharide lyases (PLs), 76 carbohydrate esterases (CEs), 57 auxiliary activities (AAs) and 30 carbohydrate-binding modules (CBMs). Compared with the other 13 fungi (Laccaria bicolor, Russula virescens, Boletus edulis, etc.), the number and distribution of CAZymes in G. purpuraceus were similar to other mycorrhizal fungi. Furthermore, the optimization of culture medium for G. purpuraceus showed the efficient utilization of disaccharides such as sucrose and maltose. The genome of G. purpuraceus provides new insights into its niche, food applications and potential artificial domestication.

Isolation and Identification of Bone Marrow Mesenchymal Stem Cells from Forest Musk Deer.

The forest musk deer (Moschus berezovskii) is an endangered animal that produces musk that is utilized for medical applications worldwide, and this species primarily lives in China. Animal-derived musk can be employed as an important ingredient in Chinese medicine. To investigate the properties of bone marrow mesenchymal stem cells (MSCs) obtained from the bone marrow of forest deer for future application, MSCs were isolated and cultivated in vitro. The properties and differentiation of these cells were assessed at the cellular and gene levels. The results show that 81,533 expressed genes were detected by RNA sequencing, and marker genes of MSCs were expressed in the cells. Karyotype analysis of the cells determined the karyotype to be normal, and marker proteins of MSCs were observed to be expressed in the cell membranes. Cells were differentiated into osteoblasts, adipocytes, and chondroblasts. The expression of genes related to osteoblasts, adipocytes, and chondroblasts was observed to be increased. The results of this study demonstrate that the properties of the cells isolated from bone marrow were in keeping with the characteristics of MSCs, providing a possible basis for future research.

Demarcation of early esophageal squamous cell carcinoma during endoscopic submucosal dissection: A comparison study between Lugol's iodine staining and narrow-band imaging.

ABSTRACT: Lugol's iodine staining (LIS) and narrow-band imaging (NBI) are currently the most common methods applied in demarcating early esophageal squamous cell carcinoma (EESCCs) during endoscopic submucosal dissection. The purpose of the present study was to investigate the effects on clinical outcomes in comparison between LIS and NBI for the demarcation of EESCCs during endoscopic submucosal dissection.This was a single-center, retrospective, cohort study. A total of 172 patients were involved. 109 patients received demarcation of the lesion by LIS and 63 patients by NBI. Data on baseline characteristics, clinical outcomes and follow-up information were collected for analyses.The mean diameter of the lesions was 3.9 ±â€Š1.5 cm. R0 resection rate was 89.5%. The rate of total and in-hospital adverse events was 25.6% and 9.3%. The cumulative recurrence rate was 2.9% and 3-year disease-specific survival rate was 98.3%. Compared to patients of the LIS group, patients of the NBI group showed significantly shorter procedure time (44.8 ±â€Š32.2 v.s.57.0 ±â€Š40.6, P = .044), lower rate of using of scopolamine butylbromide (19.0% vs 35.8%, P = 0.021), reduced number of clips used (1.3 ±â€Š1.2  vs 1.8 ±â€Š1.5, P = .017) and alleviated discomfort evaluated by visual analog system score after operation (4.7 ±â€Š0.8 vs 5.5 ±â€Š1.0, P < .001). There was no significant difference of R0 resection rate, margin status, adverse events, cumulative recurrence rate and 3-year disease-specific survival rate between the two groups.Demarcation of EESCCs by NBI could achieve comparable accuracy and clinical outcomes with more convenience and safety compared with demarcation by LIS.

Phenolic composition and nutritional attributes of diaphragma juglandis fructus and shell of walnut (Juglans regia L.).

Phenolic composition and nutritional attributes of diaphragma juglandis fructus (Djf) and walnut shells (Ws) were investigated. Phenolic acids, hydroxybenzoic acid, isoflavone, and flavone were identified in the free phenolic fractions (FPFs) of both Djf and Ws. Bound phenolic fractions were less than FPFs both in content and diversity. The soluble dietary fiber contents of Djf and Ws were 25.56 g/100 g and 9.5 g/100 g, respectively. The contents of unsaturated fatty acids (1912.28 mg/kg and 9137.56 mg/kg, respectively) were significantly higher than that of saturated fatty acid both in Djf and Ws. The content of essential amino acids in Djf (9.67 mg/g) was significantly higher than that in Ws. More than eight types of monosaccharides were detected in Djf and Ws. The monosaccharide content of Djf (314.16 mg/g) was significantly higher than that of Ws (60.97 mg/g). Trehalose was the predominant component both in Djf (71.2%) and Ws (78.6%).

Upregulation of AUF1 is involved in the proliferation of esophageal squamous cell carcinoma through GCH1.

Esophageal squamous cell carcinoma (ESCC) has one of the highest mortality rates worldwide. AU-rich element RNA-binding factor 1 (AUF1) is an established RNA-binding protein. AUF1 influences the process of development, apoptosis and tumorigenesis via interacting with adenylate-uridylate rich elements (AREs) bearing mRNAs. However, the clinical relevance of AUF1 and its biological function in ESCC progression have not been reported. In the present study, we first investigated the expression of AUF1 in the ESCC tissue samles and normal samples. We found a significantly higher expression of AUF1 in ESCC tissues than that in normal tissues and tumor adjacent tissues. The expression of AUF1 correlated with ESCC stage (P=0.011) and marginally correlated with lymph node metastasis (P=0.055) of ESCC patients. Silencing of AUF1 by an siRNA inhibited the proliferation and enhanced the apoptosis of ESCC cells. mRNA profiling by microarray analysis revealed that AUF1 knockdown affected 285 genes (fold change ≥2) that function in multiple pathways. GTP cyclohydrolase I (GCH1), the rate limiting enzyme for BH4 synthesis, was found to be downregulated. One of the AU-rich elements in the 3'UTR of GCH1 was found to be responsive to AUF1 expression by luciferase assay. Knockdown of GCH1 suppressed cell proliferation and colony formation of ESCC cells. The expression of AUF1 significantly correlated with that of GCH1 in ESCC tissues. Taken together, we demonstrated the overexpression of AUF1 in esophageal carcinoma and identified GCH1 as AUF1's effector for the proliferation of ESCC cells.

[The expression and significance of hnRNPD in esophageal squamous cell carcinoma cells].

OBJECTIVE: To investigate the expression of heterogeneous nuclear ribonucleoprotein D (hnRNPD) in esophageal squamous cell carcinoma (ESCC) tissues and the relationship between hnRNPD expression and the clinicopathological features of ESCC, and to study the effect of down-regulated hnRNPD on the proliferation of ESCC cells and explore its potential mechanism. METHODS: The expression of hnRNPD protein in ESCC tissues and the normal paracancerous tissues were detected by immunohistochemistry. The siRNA-hnRNPD was transfected into ESCC cells and the silence effect was verified by Western blotting. MTT assay and clone formation assay were used to evaluate the proliferation of ESCC cells after down-regulation of hnRNPD genes. Cell apoptosis was examined by annexin V-phycoerythrin/7-aminoactinomycin D (annexin V-PE/7-AAD) staining and flow cytometry. RESULTS: The expression of hnRNPD protein in ESCC tissues was significantly higher than that of the normal paracancerous tissues, and the expression was closely related with neoplasm staging. Down-regulation of hnRNPD inhibited the proliferation and clonality of ESCC cells. Compared with the control group, siRNA targeting hnRNPD significantly promoted cell apoptosis. CONCLUSION: Down-regulation of hnRNPD inhibits the proliferation of ESCC cells by promoting cell apoptosis.

miR-34a inhibits the migration and invasion of esophageal squamous cell carcinoma by targeting Yin Yang-1.

Esophageal squamous cell carcinoma (ESCC), one of the most common gastrointestinal tumors, is known for its high mortality rate. microRNAs (miRNAs) have been reported to play important regulatory roles in cancer metastasis and progression. miR-34a has been demonstrated to be associated with the development of and metastasis in certain types of cancer via various target genes, but its function and targets in ESCC are unknown. The aim of this study was to examine whether the expression of miR-34a was significantly decreased in ESCC tissues, compared with normal esophageal tissues using RT-PCR and western blot analysis. The results showed that miR-34a overexpression increased apoptosis and decreased clonogenic formation, but inhibited invasion and migration in ESCC cells by suppressing MMP-2 and -9 expression. Yin Yang-1 (YY1), a widely distributed transcription factor that belongs to the GLI-Kruppel class of zinc finger proteins, was found to be a direct target of miR-34a in ESCC cell lines. Rescue experiments indicated that the suppressive effect of miR-34a on invasion and migration was mediated by activating YY1 expression. Results of the present study showed that miR-34a is associated with ESCC migration and provides a potential therapeutic and diagnostic target for ESCC.

The superoxide dismutase 1 3'UTR maintains high expression of the SOD1 gene in cancer cells: The involvement of the RNA-binding protein AUF-1.

Superoxide dismutase 1 (SOD1) is ubiquitously expressed and the predominant dismutase in the cytoplasm. Whereas transcriptional regulation of the SOD1 gene has been well characterized, posttranscriptional regulation of the gene remains largely unknown in eukaryotes. In this study, a full-length 3'UTR of the SOD1 transcript was cloned and characterized for its ability to regulate SOD1 gene expression in human cancer cells. Inclusion of the SOD1 3'UTR in the pGL3 reporter construct dramatically enhanced the reporter activity by 10- to 220-fold in various cell lines. RT-PCR analysis, however, indicated that the reporter gene mRNA levels were only modestly altered by the SOD1 3'UTR, suggesting that the SOD1 3'UTR enhances the reporter gene activity not simply by stabilizing the mRNA but primarily by promoting translation of the protein. Bioinformatics analysis showed multiple stem and loop structures of the SOD1 3'UTR, and alterations in this secondary structure led to remarkably reduced reporter gene activity. Importantly, introducing the SOD1 3'UTR into cancer cells attenuated endogenous SOD1 expression in a concentration-dependent manner, indicating the involvement of RNA trans-acting factors in this process. Using siRNA and RNA immunoprecipitation techniques, we identified AUF-1, an RNA-binding protein, as a positive regulator of SOD1 expression through its 3'UTR. Consequently, AUF-1 was found to regulate redox balance in our cell model systems. Furthermore, in human ovarian, esophageal, and pancreatic cancer tissues, the expression of SOD1 was significantly correlated with that of AUF-1, further supporting the importance of AUF-1 in regulating SOD1 gene expression.

Hypoxia-inducible factor 1α (HIF-1α) and reactive oxygen species (ROS) mediates radiation-induced invasiveness through the SDF-1α/CXCR4 pathway in non-small cell lung carcinoma cells.

Radiotherapy is an important procedure for the treatment of inoperable non-small cell lung cancer (NSCLC). However, recent evidence has shown that irradiation can promote the invasion and metastasis of several types of cancer, and the underlying mechanisms are not fully understood. This study aimed to investigate the molecular mechanism by which radiation enhances the invasiveness of NSCLC cells. We found that after irradiation, hypoxia-inducible factor 1α (HIF-1α) was increased and translocated into the nucleus, where it bound to the hypoxia response element (HRE) in the CXCR4 promoter and promoted the transcription of CXCR4. Furthermore, reactive oxygen species (ROS) also plays a role in the radiation-induced expression of CXCR4. Our results revealed that 2 Gy X-ray irradiation promoted the metastasis and invasiveness of H1299, A549 and H460 cells, which were significantly enhanced by SDF-1α treatment. Blocking the SDF-1α/CXCR4 interaction could suppress the radiation-induced invasiveness of NSCLC cells. The PI3K/pAkt and MAPK/pERK1/2 pathways were found to be involved in radiation-induced matrix metalloproteinase (MMP) expression. In vivo, irradiation promoted the colonization of H1299 cells in the liver and lung, which was mediated by CXCR4. Altogether, our findings have elucidated the underlying mechanisms of the irradiation-enhanced invasiveness of NSCLC cells.

Downregulation of ubiquitin inhibits the proliferation and radioresistance of non-small cell lung cancer cells in vitro and in vivo.

Radioresistance has been an important factor in restricting efficacy of radiotherapy for non-small cell lung cancer (NSCLC) patients and new approaches to inhibit cancer growth and sensitize irradiation were warranted. Despite the important role of ubiquitin/proteasome system (UPS) during cancer progression and treatment, the expression and biological role of ubiquitin (Ub) in human NSCLC has not been characterized. In this study, we found that ubiquitin was significantly overexpressed in 75 NSCLC tissues, compared to their respective benign tissues by immunohistochemistry (P < 0.0001). Knock-down of ubiquitin by mixed shRNAs targeting its coding genes ubiquitin B (UBB) and ubiquitin C (UBC) suppressed the growth and increased the radiosensitivity in NSCLC H1299 cells. Apoptosis and γ H2AX foci induced by X-ray irradiation were enhanced by knock-down of ubiquitin. Western blot and immunostaining showed that knock-down of ubiquitin decreased the expression and translocation of NF-κB to the nucleus by reduced phospho-IκBα after irradiation. Suppression of ubiquitin decreased the proliferation and radioresistance of H1299 transplanted xenografts in nude mice by promoting apoptosis. Taken together, our results demonstrate the critical role of ubiquitin in NSCLC proliferation and radiosensitivity. Targeting ubiquitin may serve as a potentially important and novel approach for NSCLC prevention and therapy.

MicroRNA profiling of atrial fibrillation in canines: miR-206 modulates intrinsic cardiac autonomic nerve remodeling by regulating SOD1.

BACKGROUND: A critical mechanism in atrial fibrillation (AF) is cardiac autonomic nerve remodeling (ANR). MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Numerous miRNAs are involved in diseases of the nervous and cardiovascular systems. OBJECTIVE: We aimed to assess the underlying role of miRNAs in regulating cardiac ANR in AF by right atrial tachypacing (A-TP) in canines. METHODS AND RESULTS: Following 4-week A-TP, the superior left ganglionated plexuses (SLGPs), which are embedded in the fat pads of the left atrium, were subjected to miRNA expression profiling to screen preferentially expressed miRNAs. Sixteen miRNAs showed significantly differential expression between the control and A-TP groups, including miR-206, miR-203, miR-224 and miR-137. In particular, we focused on miR-206, which was elevated ~10-fold in A-TP dogs. Forced expression of miR-206 through lentiviral infection based on A-TP in vivo significantly shortened the atrial effective refractory period (AERP) (81 ± 7 vs. 98 ± 7 ms, P < 0.05). Immunohistochemical analysis showed that the regeneration of nerves increased more than 2-fold by miR-206 overexpression (P < 0.01). The expression of superoxide dismutase 1 (SOD1) was repressed by miR-206 overexpression by Western blot and luciferase assay, indicative of SOD1 as a direct target of miR-206. Overexpression of miR-206 increased reactive oxygen species (ROS) levels in vitro and in vivo, whereas miR-206 silencing attenuated irradiation- or A-TP-induced ROS. Knockdown of SOD1 effectively abolished ROS reduction caused by miR-206 silencing. CONCLUSIONS: Our results found the differential expression of miRNAs in response to ANR in AF and elucidated the important role of miR-206 by targeting SOD1. The study illustrated the novel molecular mechanism of ANR and indicated a potential therapeutic target for AF.