Network-Based Analysis of Software Change Propagation

The object-oriented software systems frequently evolve to meet new change requirements. Understanding the characteristics of changes aids testers and system designers to improve the quality of softwares. Identifying important modules becomes a key issue in the process of evolution. In this context, a novel network-based approach is proposed to comprehensively investigate change distributions and the correlation between centrality measures and the scope of change propagation. First, software dependency networks are constructed at class level. And then, the number of times of cochanges among classes is minded from software repositories. According to the dependency relationships and the number of times of cochanges among classes, the scope of change propagation is calculated. Using Spearman rank correlation analyzes the correlation between centrality measures and the scope of change propagation. Three case studies on java open source software projects Findbugs, Hibernate, and Spring are conducted to research the characteristics of change propagation. Experimental results show that (i) change distribution is very uneven; (ii) PageRank, Degree, and CIRank are significantly correlated to the scope of change propagation. Particularly, CIRank shows higher correlation coefficient, which suggests it can be a more useful indicator for measuring the scope of change propagation of classes in object-oriented software system

Gut REG3γ-Associated Lactobacillus Induces Anti-inflammatory Macrophages to Maintain Adipose Tissue Homeostasis

Gut microbiota may not only affect composition of local immune cells but also affect systemic immune cells. However, it is not completely clear how gut microbiota modulate these immune systems. Here, we found that there exist expanded macrophage pools in huREG3γtgIEC mice. REG3γ-associated Lactobacillus, which is homology to Lactobacillus Taiwanese, could enlarge macrophage pools not only in the small intestinal lamina propria but also in the spleen and adipose tissues. STAT3-mediated signal(s) was a critical factor in the Lactobacillus-mediated anti-inflammatory macrophages. We also offered evidence for critical cellular links among REG3γ-associated Lactobacillus, tissue macrophages, and obesity diseases. Anti-inflammatory macrophages in the lamina propria, which are induced by REG3γ-associated Lactobacillus, may migrate into adipose tissues and are involved in resistance against high-fat diet-mediated obesity. Thus, REG3γ-associated Lactobacillus-induced anti-inflammatory macrophages in gut tissues may play a role in adipose tissue homeostasis

MicroRNA-200c Promotes Suppressive Potential of Myeloid-Derived Suppressor Cells by Modulating PTEN and FOG2 Expression.

Myeloid-derived suppressor cells (MDSCs) constitute one of the major populations that potently suppress anti-tumor immune responses and favor tumor growth in tumor microenvironment. However, the mechanism(s) regulating the differentiation and suppressive function of tumor-associated MDSCs remain(s) unclear. Here, we identified a microRNA-200c (miR-200c), whose expression was dramatically induced by tumor-derived factors. Meanwhile, we also demonstrated that GM-CSF was a main inducer of miR-200c in tumor environment, and miR-200c in turn promoted the expansion and immune suppressive activity of MDSCs via targeting phosphatase and tensin homolog (PTEN) and friend of Gata 2 (FOG2), which can lead to STAT3 and PI3K/Akt activation. Finally, we examined in vivo suppressive function of miR-200c transfected MDSCs and found that miR-200c could remarkably promote tumor growth via modifying MDSCs. Thus, GM-CSF induced miR-200c in tumor environment plays a critical role in governing the expansion and functions of tumor-associated MDSCs and serves as a potential target in immunotherapy against tumor

The Gut Epithelial Receptor LRRC19 Promotes the Recruitment of Immune Cells and Gut Inflammation

Commensal microbes are necessary for a healthy gut immune system. However, the mechanism involving these microbes that establish and maintain gut immune responses is largely unknown. Here, we have found that the gut immune receptor leucine-rich repeat (LRR) C19 is involved in host-microbiota interactions. LRRC19 deficiency not only impairs the gut immune system but also reduces inflammatory responses in gut tissues. We demonstrate that the LRRC19-associated chemokines CCL6, CCL9, CXCL9, and CXCL10 play a critical role in immune cell recruitment and intestinal inflammation. The expression of these chemokines is associated with regenerating islet-derived (REG) protein-mediated microbiotas. We also found that the expression of REGs may be regulated by gut Lactobacillus through LRRC19-mediated activation of NF-κB. Therefore, our study establishes a regulatory axis of LRRC19, REGs, altered microbiotas, and chemokines for the recruitment of immune cells and the regulation of intestinal inflammation

MiR-200c is upregulated by tumor associated GM-CSF.

<p>(A) Generation of BM cells derived CD11b⁺Gr1⁺ cells. BM cells derived CD11b⁺Gr1⁺ cells were generated by culturing with GM-CSF (40 ng/ml) or GM-CSF (40 ng/ml) with IL-6 (40 ng/ml), TNFα (40 ng/ml), or IL-4 (40 ng/ml) plus PGE2 (1.0 μg/ml). The proportion of Gr-1⁺CD11b⁺ cells was analyzed by FAScan. (B) Expression of miR-200c in differently derived MDSCs. BM cells derived CD11b⁺Gr1⁺ cells were generated by culturing with GM-CSF or GM-CSF with IL-6, TNFα, or IL-4 plus PGE2. Total RNAs were isolated from either fresh (fresh) or MDSCs (days 2 or 4 after exposed to the simulators) and miR-200c content was detected by qRT-PCR. Data are normalized to miRNA level in fresh BM cells. (C) Expression of miR-200c in different cytokines treated BM cells. BM cells were treated with different tumor-derived factors GM-CSF, IL-6, TNFα, PGE2 or TGFβ. Ctr., no tumor-derived factor. MiR-200c expression was examined after 24 h. (D) Expression of miR-200c in different doses of GM-CSF treated MDSC. BM cells were cultured with different concentrations of GM-CSF and then expression of miR-200c was analyzed. (E) GM-CSF levels in the supernatants of different tumor cell lines. The tumor supernatants were collected and analyzed by ELISA. (F) Effects of anti-GM-CSF on the expression of miR-200c. BM cells were cultured with 4T1, CT-26 or ID8 TCCM (25% v/v), without or with 10 μg/ml anti-GM-CSF Ab for 36 h. MiR-200c transcriptional levels were detected by qRT-PCR. Ctr., only medium; TCCM, medium with 25% tumor supernatants; TCCM+mAb, medium with 25% tumor supernatants plus anti-murine GM-CSF antibodies. Data are a representative of three independent experiments. *, p<0.05; **, P<0.01.</p

Silencing PTEN and FOG2 enhances immune suppressive activity of MDSCs.

<p>(A, B, C and D) Production of ROS (A), H₂O₂ (B), NO- (C) and arginase activity (D) in differently transfected cells. BM cells were transfected with FOG2 siRNA (siFOG2), PTEN siRNA (siPTEN) or both siFOG2 and siPTEN, and then cultured in the presence of GM-CSF and IL-6. The production of ROS, H₂O₂, NO- and arginase activity were measured. Ctr.oligoes,, control oligoes. The figures shown here are representative results from three independent experiments. *, p<0.05; **, P<0.01.</p

MiR-200c is highly expressed in tumor-associated MDSCs.

<p>(A and B) Expression of mature (A) and primary (B) miR-200c in CD11b⁺Gr-1⁺ cells isolated from the bone marrow of mice with different tumors. Ctrl-C57, B16 and Lewis were respectively CD11b⁺Gr1⁺ cells from tumor-free C57BL/6 mice, C57BL/6 mice bearing B16 melanoma and C57BL/6 bearing Lewis lung cancer; Ctrl-Balb andCT-26 were respectively CD11b⁺Gr1⁺ cells from tumor-free Balb/c mice and Balb/c mice with CT-26 tumor. (C and D) Expression of mature (C) and primary (D) miR-200c in tumor cell conditioned medium (TCCM) treated BM cells. BM cells were cultured with TCCM from B16 melanoma (B16), CT-26 colon cancer (CT-26), ID8 ovarian cancer (1D8) or 4T1 breast cancer (4T1, 25% v/v) for 36 h. (E) Expression of miR-200c in BM cells treated by different doses of TCCM. BM cells were treated with serial dilution of 4T1 TCCM for 36 h. 10, 20 and 50% indicated the concentrations of TCCM in medium. Each experiment was independently performed three times. *, p<0.05; **, P<0.01.</p

MiR-200c enhances the expression of immune suppression associated genes in MDSCs.

<p>(A) Phosphorylation of STAT3, Akt and p65 in differently transfected cells. BM cells were transfected with miR-200c mimics (miR-200cM), miR-200c inhibitor (miR-200c) or control oligoes (Ctr. oligoes) and then cultured in the presence of GM-CSF and IL-6. After further culturing for 3 days, phospho-STAT3,-Akt and-p65 were analyzed by Western blot. (B) Expression of immuno-suppression associated genes in CD11b⁺Gr-1⁺ cells isolated from spleens of mice with Lewis or B16 tumors. Ctr., CD11b⁺Gr-1⁺ cells isolated from spleens of tumor-free mice. (C and D) Expression of immune suppression associated genes in differently transfected BM cells. BM cells were transfected with miR-200c mimics (miR-200cM), miR-200c inhibitors (miR-200cI), or control oligoes (Ctr. oligoes) and then cultured with GM-CSF and IL-6. After further culturing for 3 days, immunosuppressive genes were detected by qRT-PCR (C) and Western blot (D). Each experiment was independently performed three times and for Western blotexperiments, the representative results are shown here. *, p<0.05; **, P<0.01</p

MiR-200c regulates the expression of FOG2 and PTEN.

<p>(A) Effect of miR-200c on the luciferase activity. 3’-UTR conserved site of PTEN and its mutated version for miR-200c pairing were exhibited in upper part. 293T cells were cotransfected with the luciferase reporter vectors and miR-200c mimics (miR-200cM) or control oligoes (Ctr.oligoes). The luciferase activity was measured after transfection for 36 hrs according to the protocol described in Materials and Methods (lower). Vector, pSiCHECK-2 vector; WT 3’-UTR, wild type PTEN 3’UTR; Mut-3’-UTR, PTEN 3’UTR with mutation in the putative miR-200c binding sites. (B) Effects of MiR-200c on the expression of PTEN and FOG2. MDSCs were transfected with miR-200c mimics (miR-200cM), miR-200c inhibitors (miR-200cI) or control oligoes (Ctr. oligoes),and then protein of PTEN and FOG2were detected by Western blot. The right histogram represents the relative densitometric value corresponding bands in the Western blot. (C) Effects of TCCM on the expression of miR-200c, PTEN and FOG2. BM cells were exposed to TCCM and then the expression of miR-200c (upper) and PTEN (middle) and FOG2 (lower) was detected at the indicated time points. BMC, bone marrow cells which were not exposed to TCCM. (D) Effect of anti-miR-200c on the expression of PTEN and FOG2 in TCCM treated BM cells. BM cells were transfected by miR-200c inhibitors (miR-200cI) or control oligoes (Ctr. oligoes), and then exposed to TCCM. Transcriptional levels of PTEN (upper) and FOG2 (lower) were detected by qRT-PCR at the indicated time. (E) Protein levels of PTEN and FOG2 in BM cells after exposed to TCCM. Protein levels of PTEN and FOG2 in BM cells after exposed to TCCM were detected by Western blot at the indicated time. Each experiment was independently performed three times. *, p<0.05; **, P<0.01.</p