Low let-7d exosomes from pulmonary vascular endothelial cells drive lung pericyte fibrosis through the TGFßRI/FoxM1/Smad/ß-catenin pathway.

The pathogenesis of pulmonary fibrosis (PF) was mediated by the progressive deposition of excessive extracellular matrix, but little is known about the regulatory mechanisms of fibrogenesis by lung pericytes. The mouse PF model was established by treatment with bleomycin, followed by isolation of exosomes from mouse broncho-alveolar lavage fluids by the centrifuge method. Relative mRNA/microRNA levels and protein expression were assessed by qRT-PCR and Western blotting, respectively. The binding of let-7d with gene promoter was validated by dual-luciferase reporter assay. Protein interactions were verified via GST pull-down and co-immunoprecipitation. Nuclear retention of Smad3 was analysed by extraction of cytoplasmic and nuclear fraction of pericytes followed by Western blotting. Association of FoxM1 with gene promoter was detected by EMSA and ChIP-PCR methods. FoxM1 expression is significantly elevated in human lung fibroblasts of PF patients and mouse PF model. The expression of let-7d is repressed in exosomes derived from broncho-alveolar lavage fluids of PF mice. Let-7d or FoxM1 knockdown suppressed the expression of FoxM1, Smad3, ß-catenin, Col1A and α-SMA expression in mouse lung pericytes under TGF-ß1 treatment. FoxM1 overexpression elevated above gene expression in mouse lung pericytes under TGF-ß1 treatment. Let-7d directly targets TGFßRI to regulate FoxM1 and downstream gene expression in mouse lung pericytes. FoxM1 directly interacts with Smad3 proteins to promote Smad3 nuclear retention and binds with ß-catenin promoter sequence to promote fibrogenesis. Exosomes with low let-7d from pulmonary vascular endothelial cells drive lung pericyte fibrosis through activating the TGFßRI/FoxM1/Smad/ß-catenin signalling pathway.

Yoga for asthma.

BACKGROUND: Asthma is a common chronic inflammatory disorder affecting about 300 million people worldwide. As a holistic therapy, yoga has the potential to relieve both the physical and psychological suffering of people with asthma, and its popularity has expanded globally. A number of clinical trials have been carried out to evaluate the effects of yoga practice, with inconsistent results. OBJECTIVES: To assess the effects of yoga in people with asthma. SEARCH METHODS: We systematically searched the Cochrane Airways Group Register of Trials, which is derived from systematic searches of bibliographic databases including the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, CINAHL, AMED, and PsycINFO, and handsearching of respiratory journals and meeting abstracts. We also searched PEDro. We searched ClinicalTrials.gov and the WHO ICTRP search portal. We searched all databases from their inception to 22 July 2015, and used no restriction on language of publication. We checked the reference lists of eligible studies and relevant review articles for additional studies. We attempted to contact investigators of eligible studies and experts in the field to learn of other published and unpublished studies. SELECTION CRITERIA: We included randomised controlled trials (RCTs) that compared yoga with usual care (or no intervention) or sham intervention in people with asthma and reported at least one of the following outcomes: quality of life, asthma symptom score, asthma control, lung function measures, asthma medication usage, and adverse events. DATA COLLECTION AND ANALYSIS: We extracted bibliographic information, characteristics of participants, characteristics of interventions and controls, characteristics of methodology, and results for the outcomes of our interest from eligible studies. For continuous outcomes, we used mean difference (MD) with 95% confidence interval (CI) to denote the treatment effects, if the outcomes were measured by the same scale across studies. Alternatively, if the outcomes were measured by different scales across studies, we used standardised mean difference (SMD) with 95% CI. For dichotomous outcomes, we used risk ratio (RR) with 95% CI to measure the treatment effects. We performed meta-analysis with Review Manager 5.3. We used the fixed-effect model to pool the data, unless there was substantial heterogeneity among studies, in which case we used the random-effects model instead. For outcomes inappropriate or impossible to pool quantitatively, we conducted a descriptive analysis and summarised the findings narratively. MAIN RESULTS: We included 15 RCTs with a total of 1048 participants. Most of the trials were conducted in India, followed by Europe and the United States. The majority of participants were adults of both sexes with mild to moderate asthma for six months to more than 23 years. Five studies included yoga breathing alone, while the other studies assessed yoga interventions that included breathing, posture, and meditation. Interventions lasted from two weeks to 54 months, for no more than six months in the majority of studies. The risk of bias was low across all domains in one study and unclear or high in at least one domain for the remainder.There was some evidence that yoga may improve quality of life (MD in Asthma Quality of Life Questionnaire (AQLQ) score per item 0.57 units on a 7-point scale, 95% CI 0.37 to 0.77; 5 studies; 375 participants), improve symptoms (SMD 0.37, 95% CI 0.09 to 0.65; 3 studies; 243 participants), and reduce medication usage (RR 5.35, 95% CI 1.29 to 22.11; 2 studies) in people with asthma. The MD for AQLQ score exceeded the minimal clinically important difference (MCID) of 0.5, but whether the mean changes exceeded the MCID for asthma symptoms is uncertain due to the lack of an established MCID in the severity scores used in the included studies. The effects of yoga on change from baseline forced expiratory volume in one second (MD 0.04 litres, 95% CI -0.10 to 0.19; 7 studies; 340 participants; I(2) = 68%) were not statistically significant. Two studies indicated improved asthma control, but due to very significant heterogeneity (I(2) = 98%) we did not pool data. No serious adverse events associated with yoga were reported, but the data on this outcome was limited. AUTHORS' CONCLUSIONS: We found moderate-quality evidence that yoga probably leads to small improvements in quality of life and symptoms in people with asthma. There is more uncertainty about potential adverse effects of yoga and its impact on lung function and medication usage. RCTs with a large sample size and high methodological and reporting quality are needed to confirm the effects of yoga for asthma.

NLS-RARα promotes proliferation and inhibits differentiation in HL-60 cells.

A unique mRNA produced in leukemic cells from a t(15;17) acute promyelocytic leukemia (APL) patient encodes a fusion protein between the retinoic acid receptor α (RARα) and a myeloid gene product called PML. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RARα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. The resultant NLS-RARα fusion protein mainly localizes to, and functions within, the cell nucleus. It is speculated that NLS-RARα may act in different ways from the wild-type RARα, but its biological characteristics have not been reported. This study takes two approaches. Firstly, the NLS-RARα was silenced with pNLS-RARα-shRNA. The mRNA and protein expression of NLS-RARα were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect the differentiation of cells. Secondly, the NLS-RARα was over-expressed by preparation of recombinant adenovirus HL-60/pAd-NLS-RARα. The assays of mRNA and protein expression of NLS-RARα, and cell proliferation, were as above. By contrast, cell differentiation was stimulated by all trans retinoic acid (ATRA) (2.5µmol/L) at 24h after virus infection of pAd-NLS-RARα, and then detected by CD11b labeling two days later. The transcription and translation of C-MYC was detected in HL-60/pAd-NLS-RARα cells which treated by ATRA. Our results showed that compared to the control groups, the expression of NLS-RARα was significantly reduced in the HL-60/pNLS-RARα-shRNA cells, and increased dramatically in the HL-60/pAd-NLS-RARα cells. The proliferation was remarkably inhibited in the HL-60/pNLS-RARα-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-NLS-RARα cells. FCM outcome revealed the differentiation increased in HL-60/pNLS-RARα-shRNA cells, and decreased in the HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. The expression of C-MYC increased strikingly in HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. Down-regulation of NLS-RARα expression inhibited the proliferation and induced the differentiation of HL-60 cells. On the contrary, over-expression of NLS-RARα promoted proliferation and reduced the ATRA-induced differentiation of HL-60 cells.

Effect of GINS2 on proliferation and apoptosis in leukemic cell line.

Although previous researches have demonstrated that GINS2 express abundantly and abnormally in many malignant solid tumors, such as breast cancer, melanoma and hepatic carcinoma. However, the role and precise molecular mechanism in acute promyelocytic leukemia (APL) are rarely reported. In this current study, we investigated the possible effect and particular mechanism of GINS2 in occurrence and development of APL. We synthesized interference plasmid targeted GINS2 successfully in vitro and also constructed recombinant adenovirus vector carrying GINS2 gene in order to down-regulate or up-regulate GINS2 expression from two aspects of positive and negative in APL. After siRNA were transfected into HL60 cells, both GINS2 expression level of mRNA and protein in interfering group were down-regulated when compared with control groups. Together, MTT and flow cytometry technology showed that cell growth was significantly inhibited. Moreover, the expression lever of Bax was distinctly increased whereas Bcl2 was dramatically decreased in transfected group. Further experiments revealed that down-regulation of GINS2 expression inhibited DNA replication and had a G2/M phase block in HL60 cells. What's more, ATM, CHK2, and P53 gene could involve in the pathogenic signaling pathways of HL60 cells when GINS2 gene was down-regulated. On the contrary, after HL60 cells were infected by recombinant adenovirus vector which contained GINS2 gene, we observed that over-expression of GINS2 could promote HL-60 cell proliferation. What's more, GINS2 might implicate a potential target for leukemia gene therapy.

PML(NLS(-)) inhibits cell apoptosis and promotes proliferation in HL-60 cells.

Promyelocytic leukemia (PML) is a cell-growth suppressor, and PML-retinoic acid receptor α (PML-RARα) is known as a fusion gene of acute promyelocytic leukemia (APL). Studies have reported that neutrophil elastase(NE) cleaved bcr-1-derived PML-RARα in early myeloid cells leading to the removal of nuclear localization signal (NLS) from PML. The resultant PML without NLS named PML(NLS(-)). PML(NLS(-)) mainly locates and functions in the cytoplasm. PML(NLS(-)) may act in different ways from PML, but its biological characteristics have not been reported. In this study, the PML (NLS(-)) was silenced with shRNA [HL-60/pPML(NLS(-))-shRNA] and over-expressed by preparation of recombinant adenovirus [HL-60/pAd-PML(NLS(-))]. The mRNA and protein expression of PML(NLS(-)) were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect apoptotic cells. The transcription of BCL-2, BAX and C-MYC was detected in HL-60/pAd-PML(NLS(-)) cells. Our results showed that compared to the control group, the expression of PML(NLS(-)) was significantly reduced in the HL-60/pPML(NLS(-))-shRNA cells, and increased significantly in the HL-60/pAd-PML(NLS(-)) cells. The proliferation was significantly inhibited in the HL-60/pPML(NLS(-))-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-PML(NLS(-)) cells treated with 60 µmol/L emodin. FCM revealed the apoptosis increased in HL-60/pPML(NLS(-))-shRNA cells, and decreased in the HL-60/pAd-PML(NLS(-)) cells. The expression of BAX decreased significantly, while that of BCL-2 and C-MYC increased significantly in HL-60/ pAd-PML(NLS(-)) cells. Down-regulation of PML(NLS(-)) expression inhibits the proliferation and induces the apoptosis of HL-60 cells. On the contrary, over-expression of PML(NLS(-)) promotes the proliferation and reduce the emodin-induced apoptosis of HL-60 cells.

[Effect of PML(NLS-) gene mediated by recobinant adenovirus vector on emodin-induced apoptosis of HL-60 cells].

OBJECTIVE: To determine the effect and mechanism of action of PML(NLS-) gene on emodin-induced apoptosis of human HL-60 cells. METHODS: HL-60 cells were infected with recombinant adenovirus Ad-PML (NLS-) and Ad-KZ, respectively. The PML(NLS-) gene was detected by Real-time PCR(RT-PCR) and Western blot. The proliferation level of the HL-60 cells was determined by MTT method. The HL-60 cells were treated with 60 micromol/L emodin for 72 h and then analyzed by flow cytometry for their cell cycle and apoptosis rate. The transcription levels of apoptosis-related BCL-2, BAX and C-MYC genes were determined by RT-PCR. The translation levels of those genes were determined by Western blot. RESULTS: Compared with normal controls and the HL-60 cells infected with Ad-KZ, the mRNA and protein expression levels of PML(NLS-) gene increased significantly in the HL-60 cells infected with Ad-PML( NLS-). Increased proliferation levels of the Ad-PML (NLS-) infected HL-60 cells were observed in those treated with 60 pmol/L emodin, which showed decreased percentage of cells at Gx phase, increased percentage of cells at S phase, and decreased emodin-induced apoptosis. The levels of mRNA transcription and protein expression of BAX gene decreased, while those of BCL-2 and C-MYC genes increased significantly. CONCLUSION: The over-expression of PML(NLS-) gene might promote the proliferation and arrest the apoptosis of HL-60 cells by up-regulating the expressions of BCL-2 and C-MYC genes and down-regulating the expression of BAX gene.

[Effect of recombinant adenovirus carrying NLS-RARalpha gene on the proliferation of HL-60 cell and the differentiation of HL-60 cells induced by ATRA and relevant mechanism].

OBJECTIVE: To explore the effect and mechanism of recombined adenovirus carrying NLS-RARalpha gene on proliferation of HL-60 cells and the differentiation of HL-60 cells induced by ATRA. METHODS: HL-60 cells was infected with Ad-NLS-RARalpha and control virus Ad-KZ. The efficiency of infection was detected by FCM. The mRNA and protein levels of NLS-RARalpha were assessed by Real-time PCR (RT-PCR) and Western blot, respectively. MTT assay were applied to determine proliferation of HL-60 cells. Cell surface differentiation antigen CD11b of infected HL-60 cell induced by ATRA was examined by FCM. The mRNA and protein levels of C-MYC of infected HL-60 cell induced by ATRA were determined by Real-time PCR (RT-PCR) and Western blot assay. RESULTS: The efficiency of infection of Ad-NLS-RARalpha and Ad-KZ on HL-60 cell was 70%-80%. The mRNA and protein levels of NLS-RARalpha gene of HL-60 cells which infected with Ad-NLS-RARalpha were both obviously higher than that of the cells which infected with Ad-KZ and non-infected (P < 0.05). The proliferation ability of HL-60 cell infected with Ad-NLS-RARalpha was significantly increased (P < 0.05). The level of CD11b of HL-60 cell infected with Ad-NLS-RARalpha and induced by ATRA was clearly decreased than control groups (P < 0.05). The mRNA and protein levels of C-MYC gene of HL-60 cells infected with Ad-NLS-RARalpha and induced by ATRA were both obviously higher than that of the cells which infected with Ad-KZ and non-infected (P < 0.05). CONCLUSION: The recombined adenovirus Ad-NLS-RARalpha can increase the proliferation ability of HL-60 cell, and inhibit the differentiation of HL-60 cell through reduce the expression level of C-MYC gene.

Identification of differentially expressed genes and signaling pathways with Candida infection by bioinformatics analysis.

BACKGROUND: Opportunistic Candida species causes severe infections when the human immune system is weakened, leading to high mortality. METHODS: In our study, bioinformatics analysis was used to study the high-throughput sequencing data of samples infected with four kinds of Candida species. And the hub genes were obtained by statistical analysis. RESULTS: A total of 547, 422, 415 and 405 differentially expressed genes (DEGs) of Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis groups were obtained, respectively. A total of 216 DEGs were obtained after taking intersections of DEGs from the four groups. A protein-protein interaction (PPI) network was established using these 216 genes. The top 10 hub genes (FOSB, EGR1, JUNB, ATF3, EGR2, NR4A1, NR4A2, DUSP1, BTG2, and EGR3) were acquired through calculation by the cytoHubba plug-in in Cytoscape software. Validated by the sequencing data of peripheral blood, JUNB, ATF3 and EGR2 genes were  significant statistical significance. CONCLUSIONS: In conclusion, our study demonstrated the potential pathogenic genes in Candida species and their underlying mechanisms by bioinformatic analysis methods. Further, after statistical validation, JUNB, ATF3 and EGR2 genes were attained, which may be used as potential biomarkers with Candida species infection.

Observation of composite gap solitons in optically induced nonlinear lattices in LiNbO3:Fe crystal.

We observe experimentally, the first time to our knowledge, two types of composite gap solitons in optically induced one-dimensional nonlinear lattice in LiNbO(3) crystal. We observe the staggered bright composite gap soliton when a single Gauss probe beam is incident at Bragg angle as well as a dipole probe beam is incident at normal incidence. When a single Gauss beam is at normal incidence, the in-phase bright composite gap solitons are observed.

Yoga for asthma.

BACKGROUND: Asthma is a common chronic inflammatory disorder affecting about 300 million people worldwide. As a holistic therapy, yoga has the potential to relieve both the physical and psychological suffering of people with asthma, and its popularity has expanded globally. A number of clinical trials have been carried out to evaluate the effects of yoga practice, with inconsistent results. OBJECTIVES: To assess the effects of yoga in people with asthma. SEARCH METHODS: We systematically searched the Cochrane Airways Group Register of Trials, which is derived from systematic searches of bibliographic databases including the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, CINAHL, AMED, and PsycINFO, and handsearching of respiratory journals and meeting abstracts. We also searched PEDro. We searched ClinicalTrials.gov and the WHO ICTRP search portal. We searched all databases from their inception to 22 July 2015, and used no restriction on language of publication. We checked the reference lists of eligible studies and relevant review articles for additional studies. We attempted to contact investigators of eligible studies and experts in the field to learn of other published and unpublished studies. SELECTION CRITERIA: We included randomized controlled trials (RCTs) that compared yoga with usual care (or no intervention) or sham intervention in people with asthma and reported at least one of the following outcomes: quality of life, asthma symptom score, asthma control, lung function measures, asthma medication usage, and adverse events. DATA COLLECTION AND ANALYSIS: We extracted bibliographic information, characteristics of participants, characteristics of interventions and controls, characteristics of methodology, and results for the outcomes of our interest from eligible studies. For continuous outcomes, we used mean difference (MD) with 95% confidence interval (CI) to denote the treatment effects, if the outcomes were measured by the same scale across studies. Alternatively, if the outcomes were measured by different scales across studies, we used standardized mean difference (SMD) with 95% CI. For dichotomous outcomes, we used risk ratio (RR) with 95% CI to measure the treatment effects. We performed meta-analysis with Review Manager 5.3. We used the fixed-effect model to pool the data, unless there was substantial heterogeneity among studies, in which case we used the random-effects model instead. For outcomes inappropriate or impossible to pool quantitatively, we conducted a descriptive analysis and summarized the findings narratively. MAIN RESULTS: We included 15 RCTs with a total of 1048 participants. Most of the trials were conducted in India, followed by Europe and the United States. The majority of participants were adults of both sexes with mild to moderate asthma for six months to more than 23 years. Five studies included yoga breathing alone, while the other studies assessed yoga interventions that included breathing, posture, and meditation. Interventions lasted from two weeks to 54 months, for no more than six months in the majority of studies. The risk of bias was low across all domains in one study and unclear or high in at least one domain for the remainder.There was some evidence that yoga may improve quality of life (MD in Asthma Quality of Life Questionnaire (AQLQ) score per item 0.57 units on a 7-point scale, 95% CI 0.37 to 0.77; 5 studies; 375 participants), improve symptoms (SMD 0.37, 95% CI 0.09 to 0.65; 3 studies; 243 participants), and reduce medication usage (RR 5.35, 95% CI 1.29 to 22.11; 2 studies) in people with asthma. The MD for AQLQ score exceeded the minimal clinically important difference (MCID) of 0.5, but whether the mean changes exceeded the MCID for asthma symptoms is uncertain due to the lack of an established MCID in the severity scores used in the included studies. The effects of yoga on change from baseline forced expiratory volume in one second (MD 0.04 liters, 95% CI -0.10 to 0.19; 7 studies; 340 participants; I2 = 68%) were not statistically significant. Two studies indicated improved asthma control, but due to very significant heterogeneity (I2 = 98%) we did not pool data. No serious adverse events associated with yoga were reported, but the data on this outcome was limited. AUTHORS CONCLUSIONS: We found moderate-quality evidence that yoga probably leads to small improvements in quality of life and symptoms in people with asthma. There is more uncertainty about potential adverse effects of yoga and its impact on lung function and medication usage. RCTs with a large sample size and high methodological and reporting quality are needed to confirm the effects of yoga for asthma.

[Neutrophil elastase inhibitor on proliferation and apoptosis of U937 cells].

OBJECTIVE: To study and compare the effect of neutrophil elastase inhibitors (GW311616A and sivelestat) on the proliferation and apoptosis of U937 cells. METHODS: Inhibitory effects of GW311616A and sivelestat on the proliferation of U937 cells were assayed by MTT assay. The morphologic changes of U937 cells were detected by transmission electron microscope, and apoptosis was observed by AnnexinV-FITC/PI staining. The changes of cell cycle and apoptosis were detected by flow cytometry. The expression of NE in U937 cells was observed by indirect immunofluorescence, the variations of content and activity of NE in U937 cells were measured through ELISA assay and colorimetric method. RESULTS: MTT showed that both NE inhibitors could inhibit the proliferation of U937 cells in a dose dependent manner. The IC50 of GW311616A and sivelestat were 150 and 214 µmol/L respectively. The inhibition effect of GW311616A was significantly higher than of sivelestat (P<0.01). Typical apoptosis morphological changes of U937 cells was observed through electron microscope. AnnexinV-FITC/PI staining showed that U937 cells could be induced to undergo apoptosis by the two inhibitors, the apoptosis ratio of 150µmol/L GW311616A group (13.60%) was significantly higher than that of 150µmol/L sivelestat group (3.69%)(P<0.01). The result of flow cytometry indicated that the apoptosis ratio of 150 µmol/L GW311616A group was 14.61%, U937 cell cycle was mainly blocked in G2/M phase; meanwhile 150 µmol/L sivelestat group as 4.25% with cell cycle in S phase. The fluorescence intensity of GW311616A group obviously decreased than of sivelestat group. And the two inhibitors could reduce the content and activity of NE in U937 cells, but the effect of GW311616A was significantly higher than of sivelestat (P<0.01). CONCLUSION: GW311616A and sivelestat could inhibit the proliferation and cause apoptosis of U937 cells. Furthermore, GW311616A was more effective and harmful to cells than sivelestat.