Transition of tumor-associated macrophages from MHC class IIhi to MHC class IIlow mediates tumor progression in mice

<p>Abstract</p> <p>Background</p> <p>Tumor-associated macrophages (TAMs) are the most abundant immune cells within the tumor stroma and play a crucial role in tumor development. Although clinical investigations indicate that high levels of macrophage (MΦ) infiltration into tumors are associated with a poor prognosis, the exact role played by TAMs during tumor development remains unclear. The present study aimed to investigate dynamic changes in TAM major histocompatibility complex (MHC) class II expression levels and to assess the effects of these changes on tumor progression.</p> <p>Results</p> <p>Significant inhibition of tumor growth in the murine hepatocellular carcinoma Hepa1-6 model was closely associated with partial TAM depletion. Strikingly, two distinct TAM subsets were found to coexist within the tumor microenvironment during Hepa1-6 tumor development. An MHC class II^hiTAM population appeared during the early phase of tumor development and was associated with tumor suppression; however, an MHC class II^lowTAM population became increasingly predominant as the tumor progressed.</p> <p>Conclusions</p> <p>Tumor progression was positively correlated with increasing infiltration of the tumor tissues by MHC class II^lowTAMs. Thus, targeting the transition of MΦ may be a novel strategy for drug development and immunotherapy.</p

TRIM32 Regulates Skeletal Muscle Stem Cell Differentiation and Is Necessary for Normal Adult Muscle Regeneration

Limb girdle muscular dystrophy type 2H (LGMD2H) is an inherited autosomal recessive disease of skeletal muscle caused by a mutation in the TRIM32 gene. Currently its pathogenesis is entirely unclear. Typically the regeneration process of adult skeletal muscle during growth or following injury is controlled by a tissue specific stem cell population termed satellite cells. Given that TRIM32 regulates the fate of mammalian neural progenitor cells through controlling their differentiation, we asked whether TRIM32 could also be essential for the regulation of myogenic stem cells. Here we demonstrate for the first time that TRIM32 is expressed in the skeletal muscle stem cell lineage of adult mice, and that in the absence of TRIM32, myogenic differentiation is disrupted. Moreover, we show that the ubiquitin ligase TRIM32 controls this process through the regulation of c-Myc, a similar mechanism to that previously observed in neural progenitors. Importantly we show that loss of TRIM32 function induces a LGMD2H-like phenotype and strongly affects muscle regeneration in vivo. Our studies implicate that the loss of TRIM32 results in dysfunctional muscle stem cells which could contribute to the development of LGMD2H

An essential role for the Id1/PI3K/Akt/NFkB/survivin signalling pathway in promoting the proliferation of endothelial progenitor cells in vitro

The enhancement of re-endothelialisation is a critical therapeutic option for repairing injured blood vessels. Endothelial progenitor cells (EPCs) are the major source of cells that participate in endothelium repair and contribute to re-endothelialisation by reducing neointima formation after vascular injury. The over-expression of the inhibitor of differentiation or DNA binding 1 (Id1) significantly improved EPC proliferation. This study aimed to investigate the effects of Id1 on the phosphatidylinositol-3-kinase (PI3K)/Akt/nuclear factor kappa B (NFκB)/survivin signalling pathway and its significance in promoting EPC proliferation in vitro. Spleen-derived EPCs were cultured as previously described. Id1 was presented at low levels in EPCs, and was rapidly up-regulated by stimulation with vascular endothelial growth factor. We demonstrated that transient transfection of Id1 into EPCs activated the PI3K/Akt/NFκB/survivin signalling pathway and promoted EPC proliferation. The proliferation of EPCs was extensively inhibited by silencing of endogenous Id1, and knockdown of Id1 expression led to suppression of PI3K/Akt/NFκB/survivin signalling pathway in EPCs. In addition, blockade by the PI3K-specific inhibitor LY294002, Akt inhibitor, the NFκB inhibitor BAY 11-7082, the survivin inhibitor Curcumin, or the survivin inhibitor YM155 reduced the effects of Id1 transfection. These results suggest that the Id1/PI3K/Akt/NFκB/survivin signalling pathway plays a critical role in EPC proliferation. The Id1/PI3K/Akt/NFκB/survivin signalling pathway may represent a novel therapeutic target in the prevention of restenosis after vascular injury

Differential relationships of family drinking with alcohol expectancy among urban school children

<p>Abstract</p> <p>Background</p> <p>Positive alcohol outcome expectancy has consistently been linked with problematic drinking, but there is little population-based evidence on its role on early stages of drinking in childhood. The present study seeks to understand the extent to which drinking of family members is differentially associated with the endorsement of alcohol expectancy in late childhood.</p> <p>Methods</p> <p>A representative sample of 4th and 6th graders (N = 2455) drawn from 28 public schools in an urban region of Taiwan completed a self-administered paper-and-pencil questionnaire. Each student provided information on alcohol expectancy, drinking experiences, and individual and family attributes. Complex survey analyses were performed to evaluate the relationship, with stratification by children's alcohol drinking history.</p> <p>Results</p> <p>An estimated 29% of the 4^thgraders and 43% of the 6^thgraders had initiated alcohol consumption (over 40% of them had drank on three or more occasions). Alcohol drinking-related differences appear in both the endorsement and the correlates of alcohol expectancy. Positive alcohol expectancy was strongly associated with family drinking, particularly the dimension of "enhanced social behaviors"; negative alcohol expectancy was inversely associated with drinking frequency. Among alcohol naïve children, significant connections appear between paternal drinking and three dimensions of positive alcohol expectancy (i.e., enhanced social behaviors:β_wt= 0.15, promoting relaxation or tension reduction:β_wt= 0.18, and global positive transformation:β_wt= 0.22).</p> <p>Conclusions</p> <p>Individual tailored strategies that address family influences on alcohol expectancy may be needed in prevention programs targeting drinking behaviors in children.</p

Small Molecule Amiloride Modulates Oncogenic RNA Alternative Splicing to Devitalize Human Cancer Cells

Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could “normalize” the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of “normalized” oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics

Comparative Analysis of PvPAP Gene Family and Their Functions in Response to Phosphorus Deficiency in Common Bean

BACKGROUND: Purple acid phosphatases (PAPs) play a vital role in adaptive strategies of plants to phosphorus (P) deficiency. However, their functions in relation to P efficiency are fragmentary in common bean. PRINCIPAL FINDINGS: Five PvPAPs were isolated and sequenced in common bean. Phylogenetic analysis showed that PvPAPs could be classified into two groups, including a small group with low molecular mass, and a large group with high molecular mass. Among them, PvPAP3, PvPAP4 and PvPAP5 belong to the small group, while the other two belong to the large group. Transient expression of 35S:PvPAPs-GFP on onion epidermal cells verified the variations of subcellular localization among PvPAPs, suggesting functional diversities of PvPAPs in common bean. Quantitative PCR results showed that most PvPAPs were up-regulated by phosphate (Pi) starvation. Among them, the expression of the small group PvPAPs responded more to Pi starvation, especially in the roots of G19833, the P-efficient genotype. However, only overexpressing PvPAP1 and PvPAP3 could result in significantly increased utilization of extracellular dNTPs in the transgenic bean hairy roots. Furthermore, overexpressing PvPAP3 in Arabidopsis enhanced both plant growth and total P content when dNTPs were supplied as the sole external P source. CONCLUSIONS: The results suggest that PvPAPs in bean varied in protein structure, response to P deficiency and subcellular localization. Among them, both PvPAP1 and PvPAP3 might function as utilization of extracellular dNTPs

MicroRNAs Differentially Expressed in Postnatal Aortic Development Downregulate Elastin via 3′ UTR and Coding-Sequence Binding Sites

Elastin production is characteristically turned off during the maturation of elastin-rich organs such as the aorta. MicroRNAs (miRNAs) are small regulatory RNAs that down-regulate target mRNAs by binding to miRNA regulatory elements (MREs) typically located in the 3′ UTR. Here we show a striking up-regulation of miR-29 and miR-15 family miRNAs during murine aortic development with commensurate down-regulation of targets including elastin and other extracellular matrix (ECM) genes. There were a total of 14 MREs for miR-29 in the coding sequences (CDS) and 3′ UTR of elastin, which was highly significant, and up to 22 miR-29 MREs were found in the CDS of multiple ECM genes including several collagens. This overrepresentation was conserved throughout mammalian evolution. Luciferase reporter assays showed synergistic effects of miR-29 and miR-15 family miRNAs on 3′ UTR and coding-sequence elastin constructs. Our results demonstrate that multiple miR-29 and miR-15 family MREs are characteristic for some ECM genes and suggest that miR-29 and miR-15 family miRNAs are involved in the down-regulation of elastin in the adult aorta

Base-Pair Resolution DNA Methylation Sequencing Reveals Profoundly Divergent Epigenetic Landscapes in Acute Myeloid Leukemia

We have developed an enhanced form of reduced representation bisulfite sequencing with extended genomic coverage, which resulted in greater capture of DNA methylation information of regions lying outside of traditional CpG islands. Applying this method to primary human bone marrow specimens from patients with Acute Myelogeneous Leukemia (AML), we demonstrated that genetically distinct AML subtypes display diametrically opposed DNA methylation patterns. As compared to normal controls, we observed widespread hypermethylation in IDH mutant AMLs, preferentially targeting promoter regions and CpG islands neighboring the transcription start sites of genes. In contrast, AMLs harboring translocations affecting the MLL gene displayed extensive loss of methylation of an almost mutually exclusive set of CpGs, which instead affected introns and distal intergenic CpG islands and shores. When analyzed in conjunction with gene expression profiles, it became apparent that these specific patterns of DNA methylation result in differing roles in gene expression regulation. However, despite this subtype-specific DNA methylation patterning, a much smaller set of CpG sites are consistently affected in both AML subtypes. Most CpG sites in this common core of aberrantly methylated CpGs were hypermethylated in both AML subtypes. Therefore, aberrant DNA methylation patterns in AML do not occur in a stereotypical manner but rather are highly specific and associated with specific driving genetic lesions

Dynamically-Driven Inactivation of the Catalytic Machinery of the SARS 3C-Like Protease by the N214A Mutation on the Extra Domain

Despite utilizing the same chymotrypsin fold to host the catalytic machinery, coronavirus 3C-like proteases (3CLpro) noticeably differ from picornavirus 3C proteases in acquiring an extra helical domain in evolution. Previously, the extra domain was demonstrated to regulate the catalysis of the SARS-CoV 3CLpro by controlling its dimerization. Here, we studied N214A, another mutant with only a doubled dissociation constant but significantly abolished activity. Unexpectedly, N214A still adopts the dimeric structure almost identical to that of the wild-type (WT) enzyme. Thus, we conducted 30-ns molecular dynamics (MD) simulations for N214A, WT, and R298A which we previously characterized to be a monomer with the collapsed catalytic machinery. Remarkably, three proteases display distinctive dynamical behaviors. While in WT, the catalytic machinery stably retains in the activated state; in R298A it remains largely collapsed in the inactivated state, thus implying that two states are not only structurally very distinguishable but also dynamically well separated. Surprisingly, in N214A the catalytic dyad becomes dynamically unstable and many residues constituting the catalytic machinery jump to sample the conformations highly resembling those of R298A. Therefore, the N214A mutation appears to trigger the dramatic change of the enzyme dynamics in the context of the dimeric form which ultimately inactivates the catalytic machinery. The present MD simulations represent the longest reported so far for the SARS-CoV 3CLpro, unveiling that its catalysis is critically dependent on the dynamics, which can be amazingly modulated by the extra domain. Consequently, mediating the dynamics may offer a potential avenue to inhibit the SARS-CoV 3CLpro

Stem Cell Factor SALL4 Represses the Transcriptions of PTEN and SALL1 through an Epigenetic Repressor Complex

Background The embryonic stem cell (ESC) factor, SALL4, plays an essential role in both development and leukemogenesis. It is a unique gene that is involved in self-renewal in ESC and leukemic stem cell (LSC).Methodology/Principal Findings To understand the mechanism(s) of SALL4 function(s), we sought to identify SALL4-associated proteins by tandem mass spectrometry. Components of a transcription repressor Mi-2/Nucleosome Remodeling and Deacetylase (NuRD) complex were found in the SALL4-immunocomplexes with histone deacetylase (HDAC) activity in ESCs with endogenous SALL4 expression and 293T cells overexpressing SALL4. The SALL4-mediated transcriptional regulation was tested on two potential target genes: PTEN and SALL1. Both genes were confirmed as SALL4 downstream targets by chromatin-immunoprecipitation, and their expression levels, when tested by quantitative reverse transcription polymerase chain reaction (qRT-PCR), were decreased in 293T cells overexpressing SALL4. Moreover, SALL4 binding sites at the promoter regions of PTEN and SALL1 were co-occupied by NuRD components, suggesting that SALL4 represses the transcriptions of PTEN and SALL1 through its interactions with the Mi-2/NuRD complex. The in vivo repressive effect(s) of SALL4 were evaluated in SALL4 transgenic mice, where decreased expressions of PTEN and SALL1 were associated with myeloid leukemia and cystic kidneys, respectively.Conclusions/Significance In summary, we are the first to demonstrate that stem cell protein SALL4 represses its target genes, PTEN and SALL1, through the epigenetic repressor Mi-2/NuRD complex. Our novel finding provides insight into the mechanism(s) of SALL4 functions in kidney development and leukemogenesis