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.

Identification of Hub Genes in the Pathogenesis of Bronchiolitis Obliterans via Bioinformatic Analysis and Experimental Verification.

Background: Bronchiolitis obliterans (BO) is a chronic disease that can arise as a complication of severe childhood pneumonia and can also impact the long-term survival of patients after lung transplantation. However, the precise molecular mechanism underlying BO remains unclear. We aimed to identify BO-associated hub genes and their molecular mechanisms. Methods: BO-associated transcriptome datasets (GSE52761, GSE137169, and GSE94557) were downloaded from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs). Additional bioinformatics analyses, such as Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Protein-Protein Interaction (PPI) analyses, were performed to determine functional roles and DEG-associated regulatory networks. Prediction of hub genes using the 12 algorithms available in the Cytohubba plugin of Cytoscape software was also performed. Verification was performed using the BO mouse model. Results: Our results revealed 57 DEGs associated with BO, of which 18 were down-regulated and 39 were up-regulated. The Cytohubba plugin data further narrowed down the 57 DEGs into 9 prominent hub genes (CCR2, CD1D, GM2A, TFEC, MPEG1, CTSS, GPNMB, BIRC2, and CTSZ). Genes such as CCR2, TFEC, MPEG1, CTSS, and CTSZ were dysregulated in 2,3-butanedione-induced BO mice, whereas TFEC, CTSS, and CTSZ were dysregulated in nitric acid-induced BO mouse models. Conclusion: Our study identified and validated four novel BO biomarkers, which may allow further investigation into the development of distinct BO diagnostic markers and novel therapeutic avenues.

Shoutai pills for threatened abortion: A protocol for systematic review and meta-analysis.

BACKGROUND: Threatened abortions are a serious health risk for women. Deferiprone tablets are commonly used in the treatment of clinical delivery. Traditional Chinese medicine, a characteristic medical system inherited for thousands of years, often applies Shoutai pills in the treatment of Threatened abortion and has achieved good results. This systematic review and meta-analysis aimed to evaluate the efficacy and safety of Shoutai pills combined with dedrogesterone tablets for the treatment of early preterm abortion. METHODS: Electronic searches of clinical randomized controlled trials in PubMed, Web of Science, MEDLINE, EMBASE, China National Knowledge Infrastructure, Wanfang database, and China Scientific Journal Database (VIP) were conducted. References to the included literature, gray literature in Open Grey, and other relevant literature such as clinical studies registered in ClinicalTrials.gov, were also manually searched. Relevant data were extracted, and a meta-analysis was performed using Reviewer Manager 5.4. RESULTS: The results of this study will be submitted to peer-reviewed journals. CONCLUSION: This study provides high-quality evidence on the efficacy and safety of Shoutai pills in combination with dedrogesterone tablets for the treatment of preterm abortion.

Andrographolide suppresses hypoxia-induced embryonic hyaloid vascular system development through HIF-1a/VEGFR2 signaling pathway.

Introduction: Ocular abnormalities and the development of retinal vasculature may cause postnatal retinopathy. In the past decade, tremendous progress has been made in identifying the mechanisms that regulate retina vasculature. However, the means of regulating embryonic hyaloid vasculature development is largely unknown. This study aims to determine whether and how andrographolide regulates embryonic hyaloid vasculature development. Methods: Murine embryonic retinas were used in this study. Whole mount isolectin B4 (IB4) staining, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and immunofluorescence staining (IF) were performed to determine whether andrographolide is critical for embryonic hyaloid vasculature development. BrdU incorporation assay, Boyden chamber migration assay, spheroid sprouting assay, and Matrigel-based tube formation assay were performed to evaluate whether andrographolide regulates the proliferation and migration of vascular endothelial cells. Molecular docking simulation and Co-immunoprecipitation assay were used to observe protein interaction. Results: Hypoxia conditions exist in murine embryonic retinas. Hypoxia induces HIF-1a expression; high-expressed HIF-1a interacts with VEGFR2, resulting in the activation of the VEGF signaling pathway. Andrographolide suppresses hypoxia-induced HIF-1a expression and, at least in part, interrupts the interaction between HIF-1a and VEGFR2, causing inhibiting endothelial proliferation and migration, eventually inhibiting embryonic hyaloid vasculature development. Conclusion: Our data demonstrated that andrographolide plays a critical role in regulating embryonic hyaloid vasculature development.

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.

The Relationship between Overweight/Obesity and Executive Control in College Students: The Mediating Effect of BDNF and 5-HT.

The main aim of this study was to explore the association between overweight/obesity and executive control (EC) in young adults, and to further analyze the mediating effect of brain-derived neurotrophic factor (BDNF) and serotonin (5-hydroxytryptamine (5-HT)) on the relationship between overweight/obesity and EC. A total of 449 college students aged between 18 and 20 years were recruited for the study between March and December 2019. Their height and weight were then measured professionally. Subsequently, body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). The EC of the participants was then estimated using the Flanker task, while their serum BDNF levels and 5-HT levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit. Finally, the multiple intermediary models in SPSS were used to analyze the mediating effect of 5-HT and BDNF between overweight/obesity and EC. The result show that the overweight/obesity of college students was positively correlated with the response of EC (p ≤ 0.005). However, it was negatively correlated with BDNF (p ≤ 0.05) and 5-HT (p ≤ 0.05). Moreover, BDNF (p ≤ 0.001) and 5-HT (p ≤ 0.001) were negatively correlated with the response of EC. The BDNF level played a partial mediating role between overweight/obesity and EC that accounted for 7.30% of the total effect value. Similarly, the 5-HT of college students played a partial mediating role between overweight/obesity and EC that accounted for 8.76% of the total effect value. Gender and age had no regulatory effect on the relationship between overweight/obesity, BDNF, 5-HT, and EC. This study provides the evidence that 5-HT and BDNF mediated the association between overweight/obesity and executive control. It is indicated that 5-HT and BDNF might be the biological pathways underpinning the link between overweight/obesity and executive control.

Andrographolide Promotes Interaction Between Endothelin-Dependent EDNRA/EDNRB and Myocardin-SRF to Regulate Pathological Vascular Remodeling.

INTRODUCTION: Pathological vascular remodeling is a hallmark of various vascular diseases. Smooth muscle cell (SMC) phenotypic switching plays a pivotal role during pathological vascular remodeling. The mechanism of how to regulate SMC phenotypic switching still needs to be defined. This study aims to investigate the effect of Andrographolide, a key principle isolated from Andrographis paniculate, on pathological vascular remodeling and its underlying mechanism. METHODS: A C57/BL6 mouse left carotid artery complete ligation model and rat SMCs were used to determine whether Andrographolide is critical in regulating SMC phenotypic switching. Quantitative real-time PCR, a CCK8 cell proliferation assay, BRDU incorporation assay, Boyden chamber migration assay, and spheroid sprouting assay were performed to evaluate whether Andrographolide suppresses SMC proliferation and migration. Immunohistochemistry staining, immunofluorescence staining, and protein co-immunoprecipitation were used to observe the interaction between EDNRA, EDNRB, and Myocardin-SRF. RESULTS: Andrographolide inhibits neointimal hyperplasia in the left carotid artery complete ligation model. Andrographolide regulates SMC phenotypic switching characterized by suppressing proliferation and migration. Andrographolide activates the endothelin signaling pathway exhibited by dramatically inducing EDNRA and EDNRB expression. The interaction between EDNRA/EDNRB and Myocardin-SRF resulted in promoting SMC differentiation marker gene expression. CONCLUSION: Andrographolide plays a critical role in regulating pathological vascular remodeling.

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.

TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing.

Dicer is a key enzyme involved in RNA interference (RNAi) and microRNA (miRNA) pathways. It is required for biogenesis of miRNAs and small interfering RNAs (siRNAs), and also has a role in the effector steps of RNA silencing. Apart from Argonautes, no proteins are known to associate with Dicer in mammalian cells. In this work, we describe the identification of TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) as a protein partner of human Dicer. We show that TRBP is required for optimal RNA silencing mediated by siRNAs and endogenous miRNAs, and that it facilitates cleavage of pre-miRNA in vitro. TRBP had previously been assigned several functions, including inhibition of the interferon-induced double-stranded RNA-regulated protein kinase PKR and modulation of HIV-1 gene expression by association with TAR. The TRBP-Dicer interaction shown raises interesting questions about the potential interplay between RNAi and interferon-PKR pathways.

Human dicer: purification, properties, and interaction with PAZ PIWI domain proteins.

Dicer is a multidomain ribonuclease that processes double-stranded RNAs (dsRNAs) to 21-nt small interfering RNAs (siRNAs) during RNA interference and excises microRNAs (miRNAs) from precursor hairpins. PAZ and PIWI domain (PPD) proteins, also involved in RNAi and miRNA function, are the best-characterized proteins known to interact with Dicer. PPD proteins are the core constituents of effector complexes, RISCs and miRNPs, mediating siRNA and miRNA function. In this chapter we describe overexpression and purification of recombinant human Dicer, its biochemical properties, and mapping of domains responsible for Dicer-PPD protein interactions.

Single processing center models for human Dicer and bacterial RNase III.

Dicer is a multidomain ribonuclease that processes double-stranded RNAs (dsRNAs) to 21 nt small interfering RNAs (siRNAs) during RNA interference, and excises microRNAs from precursor hairpins. Dicer contains two domains related to the bacterial dsRNA-specific endonuclease, RNase III, which is known to function as a homodimer. Based on an X-ray structure of the Aquifex aeolicus RNase III, models of the enzyme interaction with dsRNA, and its cleavage at two composite catalytic centers, have been proposed. We have generated mutations in human Dicer and Escherichia coli RNase III residues implicated in the catalysis, and studied their effect on RNA processing. Our results indicate that both enzymes have only one processing center, containing two RNA cleavage sites and generating products with 2 nt 3' overhangs. Based on these and other data, we propose that Dicer functions through intramolecular dimerization of its two RNase III domains, assisted by the flanking RNA binding domains, PAZ and dsRBD.

Characterization of the interactions between mammalian PAZ PIWI domain proteins and Dicer.

PAZ PIWI domain (PPD) proteins, together with the RNA cleavage products of Dicer, form ribonucleoprotein complexes called RNA-induced silencing complexes (RISCs). RISCs mediate gene silencing through targeted messenger RNA cleavage and translational suppression. The PAZ domains of PPD and Dicer proteins were originally thought to mediate binding between PPD proteins and Dicer, although no evidence exists to support this theory. Here we show that PAZ domains are not required for PPD protein-Dicer interactions. Rather, a subregion of the PIWI domain in PPD proteins, the PIWI-box, binds directly to the Dicer RNase III domain. Stable binding between PPD proteins and Dicer was dependent on the activity of Hsp90. Unexpectedly, binding of PPD proteins to Dicer inhibits the RNase activity of this enzyme in vitro. Lastly, we show that PPD proteins and Dicer are present in soluble and membrane-associated fractions, indicating that interactions between these two types of proteins may occur in multiple compartments.

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.