Seven new species of Alternaria (Pleosporales, Pleosporaceae) associated with Chinese fir, based on morphological and molecular evidence.

Chinese fir (Cunninghamialanceolata) is a special fast-growing commercial tree species in China and has significant ecological and economic value. However, it experienced damage from leaf blight caused by pathogenic fungi of the genus Alternaria. To determine the diversity of Alternaria species associated with leaf blight of Chinese fir in China, infected leaves were collected from five major cultivation provinces (Fujian, Henan, Hunan, Jiangsu and Shandong provinces). A total of 48 fungal strains of Alternaria were obtained. Comparison of morphology and phylogenetic analyses, based on nine loci (ITS, SSU, LSU, GAPDH, RPB2, TEF1, Alt a1, endoPG and OPA10-2) of the representative isolates as well as the pairwise homoplasy index tests, revealed that the fungal strains belonged to seven undescribed taxa of Alternaria, which are described here and named as Alternariacunninghamiicolasp. nov., A.dongshanqiaoensissp. nov., A.hunanensissp. nov., A.kunyuensissp. nov., А. longqiaoensissp. nov., A.shandongensissp. nov. and A.xinyangensissp. nov. In order to prove Koch's postulates, pathogenicity tests on detached Chinese fir leaves revealed significant pathogenicity amongst these species, of which A.hunanensis is the most pathogenic to Chinese fir. This study represents the first report of A.cunninghamiicola, A.dongshanqiaoensis, A.hunanensis, A.kunyuensis, A.longqiaoensis, A.shandongensis and A.xinyangensis causing leaf blight on Chinese fir. Knowledge obtained in this study enhanced our understanding of Alternaria species causing leaf blight on Chinese fir and was crucial for the disease management and the further studies in the future.

Morphological and phylogenetic analyses reveal three new species of Fusarium (Hypocreales, Nectriaceae) associated with leaf blight on Cunninghamialanceolata in China.

Chinese fir (Cunninghamialanceolata) is a special fast-growing commercial tree species in China with high economic value. In recent years, leaf blight disease on C.lanceolata has been observed frequently. The diversity of Fusarium species associated with leaf blight on C.lanceolata in China (Fujian, Guangxi, Guizhou, and Hunan provinces) was evaluated using morphological study and molecular multi-locus analyses based on RNA polymerase second largest subunit (RPB2), translation elongation factor 1-alpha (TEF-1α), and RNA polymerase largest subunit (RPB1) genes/region as well as the pairwise homoplasy index tests. A total of five Fusarium species belonging to four Fusarium species complexes were recognized in this study. Two known species including Fusariumconcentricum and F.fujikuroi belonged to the F.fujikuroi species complex, and three new Fusarium species were described, i.e., F.fujianense belonged to the F.lateritium species complex, F.guizhouense belonged to the F.sambucinum species complex, and F.hunanense belonged to the F.solani species complex. To prove Koch's postulates, pathogenicity tests on C.lanceolata revealed a wide variation in pathogenicity and aggressiveness among the species, of which F.hunanense HN33-8-2 caused the most severe symptoms and F.fujianense LC14 led to the least severe symptoms. To our knowledge, this study also represented the first report of F.concentricum, F.fujianense, F.fujikuroi, F.guizhouense, and F.hunanense causing leaf blight on C.lanceolata in China.

Preventive effect of LCZ696 on hypoxic pulmonary hypertension in rats via regulating the PI3K/AKT signaling pathway.

Hypoxic pulmonary hypertension (HPH) is a devastating disease worldwide; however, effective therapeutic drugs are lacking. This study investigated the effects and underlying mechanisms of LCZ696 treatment on hypoxia-induced pulmonary hypertension. Male Sprague-Dawley (SD) rats were kept in a hypobaric chamber with an oxygen concentration of 5% for 4 weeks. Rats were treated with either LCZ696 (18 mg/kg, 36 mg/kg, and 72 mg/kg) or sildenafil. The mean pulmonary artery pressure (mPAP), right ventricle hypertrophy index (RVHI), and lung system index were measured. Hematoxylin-eosin (HE) staining, Masson staining, and immunofluorescence staining were used for histological analysis. Enzyme linked immunosorbent assay (ELISA) kits were used to determine the concentrations of inflammatory and hypoxia-related factors. Western blotting was used to examine the expression of apoptotic and PI3K/AKT signaling pathway proteins in rat lung tissue. Hypoxia increased mPAP, RVHI, and lung system index and induced pulmonary vascular remodeling, pulmonary arteriomyosis, and pulmonary artery fibrosis. LCZ696 treatment reduced the increase in mPAP, RVHI, and the lung system index and ameliorated the induced pathological changes. Hypoxia upregulated expression of NF-kB, TNF-α, IL-6, HIF-1α, and Vascular endothelial growth factor (VEGF), decreased the ratio of Bax/Bcl-2, and activated the PI3K/AKT signaling pathway in lung tissue, and these effects were partially reversed by treatment with LCZ696. These results demonstrated that LCZ696 can ameliorate hypoxia-induced HPH by suppressing apoptosis, inhibiting the inflammatory response, and inhibiting the PI3K/AKT signaling pathway. It provides a reference for clinical rational drug use and lays a foundation for the study of HPH therapeutic drugs.

Screening and identification of differentially expressed microRNAs in diffuse large B-cell lymphoma based on microRNA microarray.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of B-cell non-Hodgkin lymphoma in adults and the pathogenesis of DLBCL is multifactorial and complex. Understanding the molecular mechanisms involved in DLBCL is important to identify new therapeutic targets. The present study aimed to screen and identify differentially expressed microRNAs (miRNAs/miRs) between diffuse large B-cell lymphoma (DLBCL) and control [lymph node reactive hyperplasia (LRH)] groups, and to investigate whether miRNAs associated with DLBCL could serve as potential therapeutic targets. In total, 5 DLBCL experimental samples and 5 control samples were obtained from fresh patient tissues. Firstly, the fresh samples were analyzed using miRNA microarray to identify differentially expressed miRNAs. Next, three databases (TargetScan, microRNA.org and PITA) were used to predict by intersection the potential target genes of the 204 differential miRNAs identified, and a Venn diagram of the results was performed. Subsequently, the target genes of differential miRNAs were analyzed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Finally, to validate the miRNA microarray data, reverse transcription-quantitative PCR (RT-qPCR) was performed for 8 differentially expressed miRNAs (miR-193a-3p, miR-19a-3p, miR-19b-3p, miR-370-3p, miR-1275, miR-490-5p, miR-630 and miR-665) using DLBCL and LRH fresh samples. In total, 204 miRNAs exhibited differential expression, including 105 downregulated and 54 upregulated miRNAs. The cut-off criteria were set as P≤0.05 and fold-change ≥2. A total of 7,522 potential target genes for the 204 miRNAs were predicted. Potential target genes were enriched in the following pathways: 'Cancer', 'MAPK signaling pathway', 'regulation of actin cytoskeleton', 'focal adhesion', 'endocytosis', 'Wnt signaling pathway', 'axon guidance', 'calcium signaling pathway' and 'PI3K/AKT signaling pathway'. A total of 8 miRNAs were validated by RT-qPCR, and 4 miRNAs (miR-19b-3p, miR-193a-3p, miR-370-3p and miR-490-5p) exhibited low expression levels in DLBCL (P<0.05), while miR-630 was highly expressed in DLBCL (P<0.05). Overall, the present study screened 204 differentially expressed miRNAs and analyzed the expression levels of 8 differentially expressed miRNAs in DLBCL. These differentially expressed miRNAs may serve as therapeutic targets for improvement of therapeutic efficacy in DLBCL in the future.

Proteomics reveals protein phosphatase 1γ as a biomarker associated with Hippo signal pathway in glioma.

Hub proteins related with Hippo signal pathway in glioma were investigated using proteomics methods (Tandem Mass Tag, TMT) to determine the differentially expressed proteins in glioblastoma (GBM). Ingenuity Pathway Analysis (IPA) was performed to complement proteomic findings by identifying the top canonical pathways as well as to suggest novel proteins for the targeted therapy of glioma. A total of 222 formalin-fixed paraffin-embedded (FFPE) glioma tissue samples were used to verify the expression of protein phosphatase 1γ (PP1γ), Yes-associated protein 1 (YAP1), and SOX2 via immunohistochemistry. Bioinformatics analysis revealed these proteins as crucial in the Hippo signaling pathway in GBM. Spearman correlation was performed to analyze the relationship of these three proteins, and survival analysis was conducted to investigate their effects on prognosis. Among the 5808 proteins identified by TMT with the standard of P-value < 0.05 and fold change (FC) of>1.2 or <0.83, 1398 upregulated and 1060 downregulated differentially expressed proteins were found. IPA revealed that the Hippo signaling was activated in the top 10 canonical pathways, and PP1γ was activated in the Hippo signaling. Immunohistochemistry analysis indicated that PP1γ, YAP1, and SOX2 were highly and positively expressed in glioma. PP1γ expression was related to WHO grade (p = 0.003) and ki-67 expression (p = 0.012). Low PP1γ expression was associated with IDH1-mut in low-grade glioma (LGG; WHO grades II and III) (p = 0.037). PP1γ was positively correlated with YAP1 (p < 0.001; r = 0.259) and SOX2 (p = 0.009; r = 0.175). In survival analysis, age, WHO grade, ki-67 expression, and PP1γ expression independently predicted a short OS in total cohort (p < 0.05). Therefore, PP1γ is a hub protein associated with Hippo signal pathway in glioma, and its expression indicates poor prognosis in patients with glioma. Therefore, PP1γ may be a promising prognostic biomarker and a therapeutic target in glioma.

Expression of Foxp3 and interleukin-7 receptor and clinicopathological characteristics of patients with diffuse large B-cell lymphoma.

The most specific biomarker on the surface of regulatory T cells (Tregs) is the forkhead/wingeded-helix protein 3 (Foxp3). In contrast, the expression of interleukin-7 receptor (IL-7R) is low or negative in Tregs. The present study aimed to investigate the expression of Foxp3 and IL-7R in diffuse large B-cell lymphoma (DLBCL), and to analyse the clinicopathological characteristics of patients with DLBCL and their association with overall survival (OS). Immunohistochemistry was performed to detect the expression of Foxp3 and IL-7R on routinely processed formalin-fixed and paraffin-embedded specimens. The χ2 test was used to analyse the association between the expression of Foxp3 and IL-7R and the clinicopathological characteristics of patients with DLBCL. Survival curves were used to investigate the effect of Foxp3 and IL-7R on patient prognosis. The results demonstrated that high Foxp3 expression in tissue was associated with non- germinal centre B-cell (GCB)-type disease (P=0.012), International Prognostic Index score >0 (P=0.012), stage 3 or 4 tumour (P=0.045) and disease progression and stabilization period (P=0.032). In addition, IL-7R expression was associated with non-GCB-type disease (P=0.001) and extranodal lymphoma (P=0.008). Furthermore, expression of Foxp3 and IL-7R was not associated with OS (P=0.447 and P=0.201, respectively). Foxp3 and IL-7R expression in non-GCB-type lymphoma was significantly higher compared with that in GCB lymphoma. The expression of Foxp3 and IL-7R may therefore help the development of individualized treatment, prognostic prediction and therapy stratification.

TCL1 as a hub protein associated with the PI3K/AKT signaling pathway in diffuse large B-cell lymphoma based on proteomics methods.

This study aimed to investigate the hub protein related to the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in diffuse large B-cell lymphoma (DLBCL). We used proteomics methods (iTRAQ) to explore the differentially expressed proteins in the non-germinal center B-cell -like (non-GCB) DLBCL in our previous study. In this study, a total of 137 formalin-fixed paraffin-embedded DLBCL tissue samples were analyzed via immunohistochemistry to verify the expression of TCL1, AKT1 + 2+3, IKKß and to determine the differentially expressed proteins associated with the PI3K/AKT signaling pathway. Spearman correlation was used to analyze the relationship between these proteins, and survival analysis was used to investigate their effects on prognosis. Immunohistochemistry analysis indicated that TCL1, AKT1 + 2+3, and IKKß were highly positively expressed in DLBCL. Results showed that the expression of TCL1 was related to ethnicity (p = 0.022), primary site (p = 0.045), Ann Arbor stage (p = 0.037), the International Prognostic Index (p = 0.005), ß2-microglobulin (p = 0.030), BCL2 expression (p < 0.001), and Ki-67 expression (p = 0.008). A positive correlation was found between TCL1 and AKT1 + 2+3 (p < 0.001; r = 0.475). A positive correlation was also found between AKT1 + 2+3 and IKKß (p < 0.001; r = 0.342). In survival analysis, anemia, non-treatment with R­CHOP, positive TCL1 expression, and Ki-67 expression≥50% independently predicted short progression-free survival and overall survival in the total cohort (p < 0.05). Thus, TCL1 as a hub protein is associated with the PI3K/AKT signaling pathway in DLBCL. TCL1 expression indicated a poor prognosis in patients with DLBCL. With further studies, TCL1 may be established as a reliable prognostic biomarker and potential immunotherapeutic target for improving therapeutic efficacy for DLBCL in the future.

Identification of core differentially methylated genes in glioma.

Differentially methylated genes (DMGs) serve a crucial role in the pathogenesis of glioma via the regulation of the cell cycle, proliferation, apoptosis, migration, infiltration, DNA repair and signaling pathways. This study aimed to identify aberrant DMGs and pathways by comprehensive bioinformatics analysis. The gene expression profile of GSE28094 was downloaded from the Gene Expression Omnibus (GEO) database, and the GEO2R online tool was used to find DMGs. Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of the DMGs were performed by using the Database for Annotation Visualization and Integrated Discovery. A protein-protein interaction (PPI) network was constructed with Search Tool for the Retrieval of Interacting Genes. Analysis of modules in the PPI networks was performed by Molecular Complex Detection in Cytoscape software, and four modules were performed. The hub genes with a high degree of connectivity were verified by The Cancer Genome Atlas database. A total of 349 DMGs, including 167 hypermethylation genes, were enriched in biological processes of negative and positive regulation of cell proliferation and positive regulation of transcription from RNA polymerase II promoter. Pathway analysis enrichment revealed that cancer regulated the pluripotency of stem cells and the PI3K-AKT signaling pathway, whereas 182 hypomethylated genes were enriched in biological processes of immune response, cellular response to lipopolysaccharide and peptidyl-tyrosine phosphorylation. Pathway enrichment analysis revealed cytokine-cytokine receptor interaction, type I diabetes mellitus and TNF signaling pathway. A total of 20 hub genes were identified, of which eight genes were associated with survival, including notch receptor 1 (NOTCH1), SRC proto-oncogene (also known as non-receptor tyrosine kinase, SRC), interleukin 6 (IL6), matrix metallopeptidase 9 (MMP9), interleukin 10 (IL10), caspase 3 (CASP3), erb-b2 receptor tyrosine kinase 2 (ERBB2) and epidermal growth factor (EGF). Therefore, bioinformatics analysis identified a series of core DMGs and pathways in glioma. The results of the present study may facilitate the assessment of the tumorigenicity and progression of glioma. Furthermore, the significant DMGs may provide potential methylation-based biomarkers for the precise diagnosis and targeted treatment of glioma.

Quantitative proteomics analysis of differentially expressed proteins in activated B-cell-like diffuse large B-cell lymphoma using quantitative proteomics.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with unclear pathogenesis. DLBCL accounts for 30%-35% of all non-Hodgkin lymphomas (NHLs) and is an aggressive subtype of mature B-cell neoplasm. At present, half of DLBCL cases can be cured, although one-third of patients experience recurrence after treatment and enter advanced tumor stage. This study aimed to investigate the differentially expressed proteins in activated B-cell-like-DLBCL (ABC-DLBCL) through quantitative proteomics (iTRAQ). Seven ABC-DLBCL experimental samples and eight control samples (reactive hyperplasia of the lymph node) were obtained from fresh tissues. The exclusion criteria were expressed as follows: (1) patients with other lymphoid diseases; and (2) patients undergoing chemical treatment. A total of 5974 proteins were identified. P value < 0.05 and multiple expressions were more than 1.2-fold. A total of 131 upregulated and 204 downregulated differentially expressed proteins were identified. Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analysis were performed. Protein-protein interaction (PPI) network analysis was conducted. The expression levels of HSP90AB1, GNA13, LAMB2, LAMA5, YWHAZ, and IKBKB were evaluated through PRM and TCGA to validate the accuracy of iTRAQ and liquid chromatography-tandem mass spectrometry results. Results of differential multiple and t-test showed differences in the expression levels of six target proteins between the control and experimental groups. To the best of our knowledge, the present study is the first to identify proteins associated with ABC-DLBCL using iTRAQ technology. Our results provide new insights into the pathogenesis of ABC-DLBCL. The combination of ABC-DLBCL-associated signaling pathway proteins and targeted therapy to reverse drug resistance is of great significance in improving the comprehensive treatment of lymphoma and reducing mortality of affected individuals. The feasibility of the present study is limited due to the number of samples, and future studies are required to determine the function of proteins in ABC-DLBCL development.

Association between clinicopathological features and survival in patients with primary and paired metastatic colorectal cancer and KRAS mutation.

The KRAS gene mutation is involved in several types of tumors. However, the potential role of the KRAS mutation in human primary and paired metastatic colorectal cancer (CRC) among different nationalities is poorly understood. In the present study, we assessed the relationship between KRAS mutation status and overall survival (OS) and disease-free survival (DFS) in 230 patients with primary and paired metastatic CRC. The KRAS mutation rate in primary CRC tissue was 43.0% (99/230), which was higher than in paired metastatic CRC, which was 31.9% (23/72; P<0.001). Clinicopathologically, the KRAS gene mutation rate was higher in tumors that had infiltrated more deeply (T3, T4) and in lymph node (LN) metastases (N1/N2) (P=0.029 and P=0.010, respectively). The KRAS gene status did not differ between the Han and Uyghur nationalities in both primary and metastatic CRC. In 72 paired cases, the KRAS mutation rate in primary CRC was significantly higher than in metastatic CRC (P<0.001) and in metastatic CRC that had infiltrated more deeply (T3, T4) (P=0.034). In the metastatic cases, the KRAS gene mutation rate was higher in patients aged over 65 years (P=0.035). Specifically, KRAS mutation was correlated with a poorer OS and DFS (P=0.004 and P=0.029, respectively). In our study, 35 patients with wild-type KRAS who received cetuximab targeted therapy had a better DFS than patients with mutant KRAS (P=0.029). The results of the current study demonstrate that the KRAS status is significantly associated with infiltrating LN metastases and the TNM stage in primary CRC. In addition, the results show that the KRAS mutation is significantly more common in primary tumors than in paired metastatic CRC, and the KRAS mutation is correlated with a shorter OS and DFS, as patients with wild-type KRAS who received cetuximab experienced a longer DFS.

Loss of B-cell receptor expression defines a subset of diffuse large B-cell lymphoma characterized by silent BCR/PI3K/AKT signaling and a germinal center phenotype displaying low-risk clinicopathologic features.

B-cell receptor (BCR) signaling is crucial for the survival of normal and neoplastic B cells, and inhibitors targeting BCR signaling pathways have shown promising therapeutic outcomes for patients with B-cell lymphomas. In the current study, we analyzed de novo diffuse large B-cell lymphoma without BCR expression (DLBCL, BCR) in 25 cases to determine the BCR/phosphatidylinositol-3-kinase/AKT (BCR/PI3K/AKT) signaling status, clinicopathologic features, and underlying causes leading to the loss of BCR. On the basis of clinical features, 15 (60%) DLBCL, BCR patients were classified into the low-risk group, and 18 (86%) experienced complete remission. Morphologically and immunophenotypically, DLBCL, BCR demonstrated centroblastic cytology (21/25, 84%) and germinal center B-cell-like cell origin (18/25, 72%). Other components in BCR complexity remained intact, on the basis of immunohistochemical findings. Epstein-Barr virus infection, deficiency in B-lineage transcription factors (PAX5, Oct-2, and Bob.1), and oncogene rearrangement did not seem to be associated with BCR loss. The activated form of signaling proteins (pSYK and pAKT) involved in the BCR/PI3K/AKT pathway were expressed at low levels in DLBCL, BCR tissue. In vitro validation revealed that in DLBCL, BCR cell lines, the BCR/PI3K/AKT pathway did not respond to BCR stimulation or inhibition. Our findings suggest that DLBCL, BCR was characterized by a silent BCR/PI3K/AKT pathway, germinal center phenotype, and low risk and may not be a candidate for BCR-targeted therapies.

[Correlation of amplification of chromosome 1 with histologic typing of thymic epithelial tumors].

OBJECTIVE: To study the correlation between amplification of chromosome 1 and histological typing and clinical staging of thymic epithelial tumors according to the WHO classification. METHODS: Amplification of chromosome 1 was detected by interphase fluorescence in-situ hybridization (FISH) in 60 cases of thymic epithelial tumors, including type A thymoma (2 cases), type AB (19 cases), B1 (4 cases), B2 (14 cases), B3 (11 cases), metaplastic thymoma (2 cases), and thymic carcinoma (8 cases) and 11 samples of normal thymus. RESULTS: Gain on chromosome 1 was found in 19 cases (31.7%) of thymic epithelial tumors, and none was detected in normal thymic tissues (P < 0.05). The positive rates of gain on chromosome 1 were statistically different among various histological subtypes of thymic epithelial tumors (P < 0.05), in which the highest rate of detection was in thymic carcinoma (6/8), the second, type B3 (6/11), followed by type A (1/2), type AB (4/19), type B2 (2/14) and type B1 (0). The positive rate of gain on chromosome 1 in type B3 had no statistical difference from thymic carcinoma (P > 0.05), but significantly higher than that in other types of thymoma (P < 0.05). In addition, the polysomy rate of chromosome 1 was significantly different among the thymic epithelial tumors at different clinical stages (P = 0.023), and that at stages III and IV was statistically higher than that in stages I and II (P = 0.003) but there was no significant difference between stage I and stage II tumors (P = 0.750). CONCLUSIONS: Gain on chromosome 1 is more common in thymic carcinoma and type B3 thymoma than that in other subtypes of thymic epithelial tumors. Thymoma of type B3 may have different genetic features from other subtypes. Detection of gain on chromosome 1 by FISH is helpful in the differential diagnosis and prediction of prognosis in patients with thymic epithelium tumors.