Transcriptome analysis reveals the impact of NETs activation on airway epithelial cell EMT and inflammation in bronchiolitis obliterans.

Bronchiolitis obliterans (BO) is a chronic airway disease that was often indicated by the pathological presentation of narrowed and irreversible airways. However, the molecular mechanisms of BO pathogenesis remain unknown. Although neutrophil extracellular traps (NETs) can contribute to inflammatory disorders, their involvement in BO is unclear. This study aims to identify potential signaling pathways in BO by exploring the correlations between NETs and BO. GSE52761 and GSE137169 datasets were downloaded from gene expression omnibus (GEO) database. A series of bioinformatics analyses such as differential expression analysis, gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and gene set enrichment analysis (GSEA) were performed on GSE52761 and GSE137169 datasets to identify BO potential signaling pathways. Two different types of BO mouse models were constructed to verify NETs involvements in BO. Additional experiments and bioinformatics analysis using human small airway epithelial cells (SAECs) were also performed to further elucidate differential genes enrichment with their respective signaling pathways in BO. Our study identified 115 differentially expressed genes (DEGs) that were found up-regulated in BO. Pathway enrichment analysis revealed that these genes were primarily involved in inflammatory signaling processes. Besides, we found that neutrophil extracellular traps (NETs) were formed and activated during BO. Our western blot analysis on lung tissue from BO mice further confirmed NETs activation in BO, where neutrophil elastase (NE) and myeloperoxidase (MPO) expression were found significantly elevated. Transcriptomic and bioinformatics analysis of NETs treated-SAECs also revealed that NETs-DEGs were primarily associated through inflammatory and epithelial-to-mesenchymal transition (EMT) -related pathways. Our study provides novel clues towards the understanding of BO pathogenesis, in which NETs contribute to BO pathogenesis through the activation of inflammatory and EMT associated pathways. The completion of our study will provide the basis for potential novel therapeutic targets in BO treatment.

Andrographolide inhibits murine embryonic neuronal development through PFKFB3-mediated glycolytic pathway.

Dysregulation of neuronal development may cause neurodevelopmental disorders. However, how to regulate embryonic neuronal development and whether this regulation can be medical interrupted are largely unknown. This study aimed to investigate whether and how andrographolide (ANP) regulates embryonic neuronal development. The pregnant mice at embryonic day 10.5 (E10.5) were administrated with ANP, and the embryonic brains were harvested at E17.5 or E18.5. Immunofluorescence (IF), Immunohistochemistry (IHC) performed to determine whether ANP is critical in regulating neuronal development. Real-time quantitative PCR, western blotting, cell counting kit-8 assay, Flow Cytometry assay, Boyden Chamber Migration assay carried out to evaluate whether ANP regulates neuronal proliferation and migration. Protein-protein interaction, CO-immunoprecipitation and IF staining carried out to evaluate whether ANP regulates the interaction between PFKFB3, NeuN and TBR1. Knockdown or overexpression of PFKFB3 by adenovirus infection were used to determine whether ANP inhibits neuronal development through PFKFB3 mediated glycolytic pathway. Our data indicated that ANP inhibited the maturation of embryonic neurons characterized by suppressing neuronal proliferation and migration. ANP regulated the interaction between PFKFB3, NeuN, and TBR1. Knockdown of PFKFB3 aggravated ANP mediated inhibition of neuronal proliferation and migration, while overexpression of PFKFB3 attenuated ANP mediated neuronal developmental suppression. In summary, ANP suppressed the expression of PFKFB3, and interrupted the interaction between TRB1 and NeuN, resulting in suppressing neuronal proliferation, migration and maturation and eventually inhibiting murine embryonic neuronal development.

Effect of F11R Gene Knockdown on Malignant Biological Behaviors of Pancreatic Cancer Cells.

F11R receptor (F11R/junctional adhesion molecule-A/F11R-A) is preferentially concentrated at tight junctions and influences epithelial cell morphology and migration. Numerous studies have shown that the aberrant expression of F11R contributes to tumor progression including pancreatic cancer. However, the significance of F11R in various tumors is controversial, and the role of F11R in regulating the malignant behaviors of human pancreatic cancer is unknown. To investigate the role of F11R in the carcinogenesis of pancreatic cancer and the potential targets of F11R as a therapeutic target for pancreatic cancer, we knocked down F11R in the pancreatic cancer cell line PANC-1 using lentiviral approaches. We found that F11R silencing led to decreased cell proliferation, a loss of cell invasiveness, cell cycle arrest in the G1 phase, and enhanced cell apoptosis. The present results suggest that F11R may be a promising therapeutic target for pancreatic cancer.

IL10-modified Human Mesenchymal Stem Cells inhibit Pancreatic Cancer growth through Angiogenesis Inhibition.

In the present study, we constructed the recombinant plasmid IL10-PEGFP-C1 and successfully transfected into human mesenchymal stem cells. After culturing for 72 h, the levels of IL6 and TNF-α in the supernatant of the MSCs-IL10 group were significantly lower than the vector group and the control group (17.6 ± 0.68vs73.8 ± 0.8 and 74.4 ± 1.5) µg/L and (65.05 ± 3.8 vs 203.2 ± 2.4 and 201.3 ± 3.7) µg/L, respectively (p < 0.001) .The animal experiments showed that the volume of subcutaneous tumors in the MSCs-IL10 group in vivo was a significantly less level compared to that in MSC control and the blank control groups (76.84 ± 20.11) mm3 vs (518. 344 ± 48.66) mm3, (576.99± 49.88) mm3, (P < 0. 05) and they have a longer life time. Further we found the mass concentrations of IL6 and TNF-α in the blood serum of MSC-IL10 group were lower than the vector group and the control group (64.42 ± 10.9 vs120.83 ± 15.52 and 122.65 ± 13.71) and (40.05 ± 5.63 vs 126.78 ±1.89 and 105.83 ± 2.16) µg/L respectively (p < 0.001). CD31 immunohistochemistry and alginate encapsulation experiments showed tumor angiogenesis were inhibited in MSCs-IL10 group in comparison to the control and vector group (P < 0.001), FITC-labeled dextran intake was also lower than the other groups (P < 0.01). Collectively, this study suggested IL10 could inhibit the growth of the transplanted tumor in vivo and prolong survival of mice, and the primary mechanism may be the indirect inhibition of pro-inflammatory cytokines IL6 and TNF-α secretion and tumor angiogenesis formation.

Racial disparities in survival and age-related outcome in postsurgery breast cancer patients in a new york city community hospital.

Breast cancer survival has significantly improved over the past two decades. However, the diagnosis of breast cancer is lower and the mortality rate remains higher, in African American women (AA) compared to Caucasian-American women. The purpose of this investigation is to analyze postoperative events that may affect breast cancer survival. This is a retrospective analysis of prospectively collected data from The Brooklyn Hospital Center cancer registry from 1997 to 2010. Of the 1538 patients in the registry, 1226 are AA and 269 are Caucasian. The study was divided into two time periods, 1997-2004 (period A) and 2005-2010 (period B), in order to assess the effect of treatment outcomes on survival. During period A, 5-year survival probabilities of 75.37%, 74.53%, and 78.70% were seen among all patients, AA women and Caucasian women, respectively. These probabilities increased to 87.62%, 87.15% and 89.99% in period B. Improved survival in AA women may be attributed to the use of adjuvant chemotherapy, radiation, and hormonal therapy. Improved survival in Caucasian patients was attributed to the use of radiation therapy, as well as earlier detection resulting in more favorable tumor grades and pathological stages.

Cotranslational assembly of the yeast SET1C histone methyltransferase complex.

While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus. Importantly, the protein-RNA interaction was sensitive to EDTA, to the translation elongation inhibitor puromycin and to the inhibition of translation initiation in prt1-1 mutants. Taken together, our results support the idea that SET1 mRNA binding was dependent on translation and that SET1RC assembled on nascent Set1 in a cotranslational manner. Moreover, we show that cellular accumulation of Set1 is limited by the availability of certain SET1C components, such as Swd1 and Swd3, and suggest that cotranslational protein interactions may exert an effect in the protection of nascent Set1 from degradation.

Elimination of rheumatoid synovium in situ using a Fas ligand 'gene scalpel'.

Surgical synovectomy to remove the inflammatory synovium can temporarily ameliorate rheumatoid inflammation and delay the progress of joint destruction. An efficient medically induced programmed cell death (apoptosis) in the rheumatoid synovium might play a role similar to synovectomy but without surgical tissue damage. Gene transfer of Fas ligand (FasL) has increased the frequency of apoptotic cells in mouse and rabbit arthritic synovium. In this study, we investigated whether repeated FasL gene transfer could remove human inflammatory synovial tissue in situ and function as a molecular synovectomy. Briefly, specimens of human synovium from joint replacement surgeries and synovectomies of rheumatoid arthritis (RA) patients were grafted subcutaneously into male C.B-17 severe combined immunodeficiency (SCID) mice. Injections of a recombinant FasL adenovirus (Ad-FasL) into the grafted synovial tissue at the dosage of 10(11) particles per mouse were performed every two weeks. Three days after the fifth virus injection, the mice were euthanized by CO2 inhalation and the human synovial tissues were collected, weighed and further examined. Compared to the control adenovirus-LacZ (Ad-LacZ) and phosphate buffered saline (PBS) injected RA synovium, the Ad-FasL injected RA synovium was dramatically reduced in size and weight (P < 0.005). The number of both synoviocytes & mononuclear cells was significantly reduced. Interestingly, an approximate 15-fold increased frequency of apoptotic cells was observed in RA synovium three days after Ad-FasL injection, compared with control tissues. In summary, our in vivo investigation of gene transfer to human synovium in SCID mice suggests that repeated intra-articular gene transfer of an apoptosis inducer, such as FasL, may function as a 'gene scalpel' for molecular synovectomy to arrest inflammatory synovium at an early stage of RA.

Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP.

Dicer is a multi-domain RNase III-related endonuclease responsible for processing double-stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the purification and properties of a recombinant human Dicer. The protein cleaves dsRNAs into approximately 22 nucleotide siRNAs. Accumulation of processing intermediates of discrete sizes, and experiments performed with substrates containing modified ends, indicate that Dicer preferentially cleaves dsRNAs at their termini. Binding of the enzyme to the substrate can be uncoupled from the cleavage step by omitting Mg(2+) or performing the reaction at 4 degrees C. Activity of the recombinant Dicer, and of the endogenous protein present in mammalian cell extracts, is stimulated by limited proteolysis, and the proteolysed enzyme becomes active at 4 degrees C. Cleavage of dsRNA by purifed Dicer and the endogenous enzyme is ATP independent. Additional experiments suggest that if ATP participates in the Dicer reaction in mammalian cells, it might be involved in product release needed for the multiple turnover of the enzyme.