Pan-cancer analysis reveals multifaceted roles of retrotransposon-fusion RNAs.

Transposon-derived transcripts are abundant in RNA sequences, yet their landscape and function, especially for fusion transcripts derived from unannotated or somatically acquired transposons, remains underexplored. Here, we developed a new bioinformatic tool to detect transposon-fusion transcripts in RNA-sequencing data and performed a pan-cancer analysis of 10,257 cancer samples across 34 cancer types as well as 3,088 normal tissue samples. We identified 52,277 cancer-specific fusions with ~30 events per cancer and hotspot loci within transposons vulnerable to fusion formation. Exonization of intronic transposons was the most prevalent genic fusions, while somatic L1 insertions constituted a small fraction of cancer-specific fusions. Source L1s and HERVs, but not Alus showed decreased DNA methylation in cancer upon fusion formation. Overall cancer-specific L1 fusions were enriched in tumor suppressors while Alu fusions were enriched in oncogenes, including recurrent Alu fusions in EZH2 predictive of patient survival. We also demonstrated that transposon-derived peptides triggered CD8+ T-cell activation to the extent comparable to EBV viruses. Our findings reveal distinct epigenetic and tumorigenic mechanisms underlying transposon fusions across different families and highlight transposons as novel therapeutic targets and the source of potent neoantigens.

Synergistic effects of arginine-glycine-aspartic acid and phosphatidylserine on the surface immunomodulation and osseointegration of titanium implants.

The control of macrophage polarization is important in bone tissue regeneration such as osseointegration. In this study, a coating method was developed to improve the osseointegration of titanium (Ti) implants by generating an immunomodulatory effect. The surface of the Ti discs was coated with a poly(lactide-co-glycolide)(PLGA) polymer, phosphatidylserine (PS), and arginine-glycine-aspartic acid (RGD) peptide conjugated phospholipid. In in vitro assay using mouse bone marrow-derived macrophages (BMDMs), the most significant expression of the M2 marker genes (Arg-1, YM-1, FIZZ1) and CD206, an M2 surface marker, was obtained with coatings containing 6 mol% RGD conjugates and phospholipids consisting of 50 mol% PS. The M2-inducing effect of RGD and PS was also verified in rat femurs where coated Ti rods were implanted. The RGD and PS coating significantly enhanced the osseointegration of the Ti implants. Moreover, a biomechanical push-out test showed that the RGD and PS coating increased the interfacial binding force between the bone and implants. These results indicate that PS and RGD can be applied to the solid surface of implantable biomedical devices to improve immunomodulation and tissue regeneration.

Enhancing effect of sodium butyrate on phosphatidylserine-liposome-induced macrophage polarization.

OBJECT: Phosphatidylserine-containing liposomes (PSLs) can mimic the immunomodulatory effects of apoptotic cells by binding to the phosphatidylserine receptors of macrophages. Sodium butyrate, an antiinflammatory short-chain fatty acid, is known to facilitate the M2 polarization of macrophages. This study aimed to investigate the effect of sodium butyrate on PSLs-induced macrophage polarization. METHODS: PSLs physical properties and cellular uptake tests, reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence staining, and flow cytometry analysis were performed to assess the polarization-related indicators of M1/M2 macrophages. RESULTS: The results showed that sodium butyrate did not affect the size and cellular uptake of PSLs. For M1 macrophage polarization, sodium butyrate significantly intensified the antiinflammatory function of PSLs, inhibiting LPS-induced proinflammatory genes expression, cytokines and enzyme release (tumor necrosis factor-alpha, interleukin (IL)-1ß, IL-6, and inducible nitric oxide synthase), as well as CD86 (M1 marker) expression. In addition to the enhancing effect of antiinflammation, sodium butyrate also promoted PSL-induced M2 macrophages polarization, especially elevated thymus and activation-regulated chemokine (TARC) and arginase-1 (Arg-1) enzyme levels which are involved in tissue repair. CONCLUSION: Sodium butyrate enhanced antiinflammatory properties and M2-polarization inducing effect of PSLs. Therefore, sodium butyrate may represent a novel approach to enhance PSL-induced macrophage polarization.

Effects of RGD-grafted phosphatidylserine-containing liposomes on the polarization of macrophages and bone tissue regeneration.

Phosphatidylserine-containing liposomes (PSLs) can mimic the anti-inflammatory effects of apoptotic cells by binding to the phosphatidylserine receptors of macrophages. MGF-E8, a bridge molecule between phosphatidylserine and macrophages, can promote M2 polarization by activating macrophage integrin with its arginine-glycine-aspartic acid (RGD) motif. In this study, to mimic MGF-E8, PSLs presenting RGD peptide (RGD-PSLs) were prepared, and their immunomodulatory effects on macrophages and the bone tissue regeneration of rat calvarial defects were investigated. RGD peptides enhanced the phagocytosis of PSLs by macrophages, especially when the PSLs contained 3% RGD. RGD-PSLs were also more effective than PSLs for the suppression of lipopolysaccharide-induced gene expression of proinflammatory cytokines (i.e., IL-1ß, IL-6, and TNF-α) as well as CD86 (M1 marker) expression. Furthermore, RGD promoted PSL-induced M2 polarization: 3%-RGD-PSLs significantly enhanced the mRNA expression of Arg-1, FIZZ1, and YM-1, as well as CD206 (M2 marker) expression. In a calvarial defect model, a significant increase in M2 with a decrease in M1 macrophages was observed with 3%-RGD-PSL treatment compared with the effects of PSLs alone. Finally, new bone formation was also accelerated by 3%-RGD-PSLs. Thus, these results suggest that the intensive immunomodulatory effect of RGD-PSLs led to the enhancement of bone tissue regeneration.

Dumbbell-Shaped 2,2'-Bipyridines: Controlled Metal Monochelation and Application to Ni-Catalyzed Cross-Couplings.

2,2'-Bipyridine ligands (dsbpys) with dumbbell-like shapes and differently substituted triarylmethyl groups at the C5 and C5' positions showed high ligand performance in the Ni-catalyzed cross-electrophile coupling and the Ni/photoredox-synergistically catalyzed decarboxylative coupling reactions. The superior ligand effects of dsbpys compared to the conventional bpy ligands were attributed to the monochelating nature of dsbpys.

Reduction of fibrous encapsulation by polyethylene glycol-grafted liposomes containing phosphatidylserine.

Biomedical implants tend to induce fibrous encapsulation which can cause malfunction of devices and local discomfort of patients. The purpose of this study was to reduce foreign body-induced fibrous capsule formation by immunomodulation of macrophages. Polyethylene-glycol-grafted liposomes containing phosphatidylserine (PEG-PSLs) were used to modulate macrophages. Mixed cellulose ester (MCE) membranes coated with a PEG-PSLs-entrapped alginate-gelatin matrix were subcutaneously implanted into rats, and the thickness of the fibrous capsule around each MCE membrane was analyzed after four weeks. PEG-PSLs significantly reduced fibrous capsule thickness, while liposomes containing phosphatidylserine (PSLs) did not affect fibrosis. In in vitro assays, PEG-PSLs suppressed TGF-ß1 secretion and multinucleated giant cell (MGC) formation in IL-4-treated RAW 264.7, a murine macrophage cell line. Although PSLs inhibited MGC formation, they exerted no effect on the secretion of TGF- ß1, which is known to be an important factor in tissue fibrosis. Therefore, our results suggest that PEG-PSLs reduce fibrous capsule formation by mediating the suppression of TGF-ß1 secretion from macrophages.

Modulation of Macrophage Polarization by Phospholipids on the Surface of Titanium.

Macrophage polarization has become increasingly important for the improvement of the biocompatibility of biomaterials. In this study, we coated Ti discs with phospholipids (phosphatidylserine/phosphatidylcholine [4:1 mole/mole]) by evaporating the solvent under vacuum, and observed the polarization of RAW 264.7 cells cultured on the discs. The coated discs were hydrated before cell culture was added. The shape of the hydrated phospholipids varied with the concentration of loaded phospholipids: a perforated layer (0.1 mM), tubules and spheres (1 mM), and spheres (10 mM). RAW 264.7 cells exhibited different morphologies, depending on the concentration of phospholipids. On the coated discs, the gene expression and protein release of TGF-ß1, VEGF, Arg-1, and TNF-α were downregulated, especially with 10 mM phospholipids. The stimulation of mRNA expression and the protein release of those genes by IL-4 and LPS were also disturbed on the phospholipid-coated discs. In conclusion, the polarization of RAW 264.7 cells was prevented by hydrated phospholipids on Ti discs.

Streamlined Synthesis of C(sp3)-Rich N-Heterospirocycles Enabled by Visible-Light-Mediated Photocatalysis.

We report a general visible-light-mediated strategy that enables the construction of complex C(sp3)-rich N-heterospirocycles from feedstock aliphatic ketones and aldehydes with a broad selection of alkene-containing secondary amines. Key to the success of this approach was the utilization of a highly reducing Ir-photocatalyst and orchestration of the intrinsic reactivities of 1,4-cyclohexadiene and Hantzsch ester. This methodology provides streamlined access to complex C(sp3)-rich N-heterospirocycles displaying structural and functional features relevant to fragment-based lead identification programs.

Plasmid Display for Stabilization of Enzymes Inside the Cell to Improve Whole-Cell Biotransformation Efficiency.

Recombinant whole-cell biocatalysts are widely used for biotransformation of valuable products. However, some key enzymes involved in biotransformation processes are unstable and cannot be easily expressed in the functional form. In this study, we describe a versatile platform for enzyme stabilization inside the cell: Intracellularly Immobilized Enzyme System (IIES). A 1,2-fucosyltransferase from Pedobactor saltans (PsFL) and a 1,3-fucosyltransferase from Helicobacter pylori (HpFL), chosen as model proteins, were fused with Oct-1 DNA-binding domain, which mediated the formation of a plasmid-protein complex. Oct-1 fusion enabled both soluble and stable expression of recombinant proteins in the cytoplasm because the fusion proteins were stabilized on the plasmid like immobilized enzymes bound to solid surface. As a result, Oct-1-fusion proteins exhibited significantly greater product titer and yield than non-fusion proteins. Use of fusion proteins PsFL-Oct-1 with C-terminal Oct-1 and Oct-1-PsFL with N-terminal Oct-1 resulted in ~3- and ~2-fold higher 2'-fucosyllactose titers, respectively, than with the use of PsFL alone. When Oct-1 was fused to HpFL, which requires dimerization through heptad repeats, almost two times more 3-fucosyllactose was produced. Fucosyllactose has been used as a food additive because it has various beneficial effects on human health. We anticipate that IIES using Oct-1 fusion protein developed in this study can be applied to stabilize other unstable enzymes.

Immunomodulation of Biomaterials by Controlling Macrophage Polarization.

Macrophages are key players in innate immune responses to foreign substances. They participate in the phagocytosis of biomaterial-derived particles, angiogenesis, recruitment of fibroblasts, and formation of granulation tissues. Most macrophage functions are achieved through the release of various cytokines and chemokines; the release profile of cytokines is dependent on the phenotype of macrophages, namely proinflammatory M1 or antiinflammatory M2. M1 and M2 macrophages coexist during an inflammatory phase, and the M1/M2 ratio is considered to be an important factor for wound-healing or tissue regeneration. This ratio depends on the chemical and physical properties of biomaterials. To obtain a favorable foreign body reaction to biomaterials, the phenotypes of the macrophages can be modulated by cytokines, antibodies, small chemicals, and microRNAs. Geometrical surface fabrication of biomaterials can also be used for modulating the phenotype of macrophages.

Effects of phosphatidylserine-containing supported lipid bilayers on the polarization of macrophages.

Placement of dental implants initiates inflammatory foreign body response, in which macrophages play a central role and affect the subsequent tissue healing process such as bone formation. The purpose of this study was to fabricate phosphatidylserine (PS)-containing supported lipid bilayers (SLBs) on a titanium surface to regulate the polarization of macrophages, a critical factor that affects following tissue healing and regeneration. The fluorescent recovery after photobleaching images showed that the percentage of PS had a critical influence on the fluidity, and 20% PS had the highest fluidity. Furthermore, more expanded and elongated cells were observed in the SLB-coated groups. transforming growth factor-ß1 and vascular endothelial growth factor, the key cytokine markers of M2 macrophage polarization, were increased in the SLB-coated groups, especially in the 20% PS group. Consistently, cells cultured on the SLB-coated titanium exhibited the distribution of CD206+ , which is a M2 macrophage specific maker. The results of this study demonstrated M2 polarization of macrophages on PS-SLB-coated titanium discs, which suggests the application of PS-SLB as an immune-regulating coating material to improve tissue reactions to dental implants. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2625-2633, 2018.

The Effects of M1 and M2 Macrophages on Odontogenic Differentiation of Human Dental Pulp Cells.

INTRODUCTION: M1 (classically activated) and M2 (alternatively activated) macrophages are known to play primary roles in inflammation and tissue regeneration. To investigate the role of macrophages in dentin regeneration, this study examined the effects of M1 and M2 macrophages on the odontogenic/osteogenic differentiation of human dental pulp cells (HDPCs) using the conditioned media (CM) of the activated human monocyte cell line THP-1. METHODS: M1 and M2 macrophages were induced by lipopolysaccharide/interferon-γ and interleukin-4, respectively, and the phenotypes were confirmed by flow cytometry. Macrophage CM was prepared at 2-day intervals for a period of 6 days, which included the first 2 days of activation. The CM obtained on days 4 (M1CM-4 day and M2CM-4 day) and 6 (M1CM-6 day and M2CM-6 day) were tested for their ability to promote the alkaline phosphatase (ALP) activity of HDPCs. M2CM-4 day was also examined for its effects on the messenger RNA expression of dentin sialophosphoprotein and osteocalcin genes and the matrix mineralization of HDPCs. Tumor necrosis factor alpha and transforming growth factor beta 1 (TGF-ß1) in M1CM and M2CM, respectively, were quantified by an enzyme-linked immunosorbent assay. To verify the role of TGF-ß1, M2CM-4 day was pretreated by a TGF-ß blocking antibody and was examined for its effect on the ALP activity of HDPCs. RESULTS: M2CM-4 day and M2CM-6 day enhanced the ALP activity of HDPCs (P < .05). Furthermore, M2CM-4 day promoted the messenger RNA expression of the dentin sialophosphoprotein gene and matrix mineralization (P < .05), whereas M1CM did not affect ALP activity. The enzyme-linked immunosorbent assay detected large amounts of TGF-ß1 in M2CM-4 day and M2CM-6 day. The TGF-ß blocking antibody suppressed the ALP-enhancing activity of M2CM-4 day (P < .05). Furthermore, the same amount of TGF-ß1 as in M2CM-4 day increased ALP activity to a similar level as M2CM-4day-treated HDPCs. CONCLUSIONS: The CM of M2 macrophages enhanced the odontogenic/osteogenic differentiation of HDPCs. M1CM did not affect the ALP activity of HDPCs at least in the absence of M1-type inducers. The effects of M2CM on HDPCs were likely caused by TGF-ß1. Therefore, M2 macrophages are expected to support dentin regeneration in dental pulps.

Modulation of the anti-inflammatory effects of phosphatidylserine-containing liposomes by PEGylation.

Inhibiting liposome uptake by macrophages using polyethylene glycol (PEG) surface modifications is a widely used approach for extending the half-life of liposomes circulating in the blood. However, the biological effects of PEGylated liposomes on macrophages have not yet been thoroughly investigated. The purpose of this study was to examine the effects of PEGylated phosphatidylserine-containing liposomes (PEG-PSLs) on the expression of two inflammation-associated cytokines, tumor necrosis factor-α (TNF-α) and transforming growth factor-ß (TGF-ß), in the murine macrophage-like cell line RAW 264.7. Previous studies have demonstrated that PSLs inhibit TNF-α secretion and enhance TGF-ß synthesis in macrophages by mimicking apoptotic cells. We found that PEGylation differentially affected the TNF-α and TGF-ß levels. The PSL-mediated inhibitory effect on TNF-α secretion was enhanced by PEGylation, and PEG-PSLs decreased TGF-ß levels compared with non-PEGylated PSLs. Fluorescence-activated cell sorting analysis demonstrated that 1% PEGylation disturbed the incorporation of PSLs into macrophages. The interference of uptake is thought to extend the binding interaction between PS to PS receptors for PSL-mediated inhibition of TNF-α expression. Together, these findings indicate that PEG-PSLs can prevent TNF-α secretion without increasing TGF-ß levels in macrophages, and they support the potential clinical use of PEG-PSLs as anti-inflammatory agents with a relatively low potential to induce tissue fibrosis. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1479-1486, 2017.

Over-expression of methionine sulfoxide reductase A in the endoplasmic reticulum increases resistance to oxidative and ER stresses.

MsrA and MsrB catalyze the reduction of methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, to methionine in different cellular compartments of mammalian cells. One of the three MsrBs, MsrB3, is an endoplasmic reticulum (ER)-type enzyme critical for stress resistance including oxidative and ER stresses. However, there is no evidence for the presence of an ER-type MsrA or the ER localization of MsrA. In this work, we developed an ER-targeted recombinant MsrA construct and investigated the potential effects of methionine-S-sulfoxide reduction in the ER on stress resistance. The ER-targeted MsrA construct contained the N-terminal ER-targeting signal peptide of human MsrB3A (MSPRRSLPRPLSLCLSLCLCLCLAAALGSAQ) and the C-terminal ER-retention signal sequence (KAEL). The over-expression of ER-targeted MsrA significantly increased cellular resistance to H2O2-induced oxidative stress. The ER-targeted MsrA over-expression also significantly enhanced resistance to dithiothreitol-induced ER stress; however, it had no positive effects on the resistance to ER stresses induced by tunicamycin and thapsigargin. Collectively, our data suggest that methionine-S-sulfoxide reduction in the ER compartment plays a protective role against oxidative and ER stresses.

Interleukin-1ß induced by Helicobacter pylori infection enhances mouse gastric carcinogenesis.

Interleukin-1ß (Il1b) is considered to be involved in Helicobacter pylori (HP)-induced human gastric carcinogenesis, while the role of its polymorphisms in gastric cancer susceptibility remains controversial. Here, we aimed to clarify the role of HP infection-induced IL1B in gastric inflammation and carcinogenesis using Il1b(-/-) (Il1b-null) mice. In gastric mucosa of the Il1b(+/+) (WT) mice, HP infection induced Il1b expression and severe inflammation. In contrast, in Il1b-null mice, recruitment of neutrophils and macrophages by HP infection was markedly suppressed. In a carcinogenicity test, the multiplicity of gastric tumors was significantly suppressed in theIl1b-null mice (58% of WT; P<0.005). Mechanistically, HP infection induced NF-κB activation both in the inflammatory and epithelial cells in gastric mucosae, and the activation was attenuated in the Il1b-null mice. Accordingly, increased proliferation and decreased apoptosis of gastric epithelial cells induced by HP infection in the WT mice were attenuated in the Il1b-null mice. These results demonstrated that the IL1B physiologically induced by HP infection enhanced gastric carcinogenesis by affecting both inflammatory and epithelial cells.

Identification of an antimicrobial peptide from human methionine sulfoxide reductase B3.

Human methionine sulfoxide reductase B3A (hMsrB3A) is an endoplasmic reticulum (ER) reductase that catalyzes the stereospecific reduction of methionine-R-sulfoxide to methionine in proteins. In this work, we identified an antimicrobial peptide from hMsrB3A protein. The N-terminal ER-targeting signal peptide (amino acids 1-31) conferred an antimicrobial effect in Escherichia coli cells. Sequence and structural analyses showed that the overall positively charged ER signal peptide had an Argand Pro-rich region and a potential hydrophobic α-helical segment that contains 4 cysteine residues. The potential α-helical region was essential for the antimicrobial activity within E. coli cells. A synthetic peptide, comprised of 2-26 amino acids of the signal peptide, was effective at killing Gram-negative E. coli, Klebsiella pneumoniae, and Salmonella paratyphi, but had no bactericidal activity against Gram-positive Staphylococcus aureus.