Establishment of a humanized animal model of systemic sclerosis in which T helper-17 cells from patients with systemic sclerosis infiltrate and cause fibrosis in the lungs and skin

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by inflammation, microangiopathy, and progressive fibrosis in the skin and internal organs. To evaluate the pathophysiologic mechanisms and efficacies of potential therapeutics for SSc, a preclinical model recapitulating the disease phenotypes is needed. Here, we introduce a novel animal model for SSc using immunodeficient mice injected with peripheral blood mononuclear cells (PBMCs) from SSc patients. Human PBMCs acquired from SSc patients and healthy controls were transferred into NOD.Cg-Prkdc^scidIl2rg^tm1Wjl (NSG) mice with concurrent bleomycin injection. Blood, skin, and lung tissues were acquired and analyzed after PBMC engraftment. In addition, we investigated whether the humanized murine model could be used to assess the efficacy of potential therapeutics for SSc. Human PBMCs from SSc patients and healthy controls were engrafted into the blood, skin, and lung tissues of NSG mice. Histological analysis of affected tissues from mice treated with SSc PBMCs (SSc hu-mice) demonstrated substantial inflammation, fibrosis and vasculopathy with human immune cell infiltration and increased expression of IL-17, TGF-β, CCL2, CCL3, and CXCL9. The proportions of circulating and tissue-infiltrating T helper 17 (Th17) cells were elevated in SSc hu-mice. These cells showed increased expression of CXCR3 and phosphorylated STAT3. SSc hu-mice treated with rebamipide and other potential Th17-cell-modulating drugs presented significantly reduced tissue fibrosis. Mice injected with patient-derived PBMCs show promise as an animal model of SSc.Subject terms: Autoimmunity, Autoimmune diseases     

BMS794833 inhibits macrophage efferocytosis by directly binding to MERTK and inhibiting its activity

Myeloid epithelial reproductive proto-oncogene tyrosine kinase (MERTK) plays an essential role in modulating cancer immune tolerance by regulating macrophage efferocytosis. Studies are underway to develop small-molecule chemicals that inhibit MERTK as cancer immunotherapeutic agents, but these efforts are in their early stages. This study identified BMS794833, whose primary targets are MET and VEGFR2, as a potent MERTK inhibitor and developed a real-time efferocytosis monitoring system. The X-ray cocrystal structure revealed that BMS794833 was in contact with the ATP-binding pocket and the allosteric back pocket, rendering MERTK inactive. Homogeneous time-resolved fluorescence kinetic and Western blotting analyses showed that BMS794833 competitively inhibited MERTK activity in vitro and inhibited the autophosphorylation of MERTK in macrophages. We developed a system to monitor MERTK-dependent efferocytosis in real time, and using this system, we confirmed that BMS794833 significantly inhibited the efferocytosis of differentiated macrophages. Finally, BMS794833 significantly inhibited efferocytosis in vivo in a mouse model. These data show that BMS794833 is a type II MERTK inhibitor that regulates macrophage efferocytosis. In addition, the real-time efferocytosis monitoring technology developed in this study has great potential for future applications.Subject terms: Biochemistry, Cell biology     

NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

Overcoming therapeutic resistance in glioblastoma (GBM) is an essential strategy for improving cancer therapy. However, cancer cells possess various evasion mechanisms, such as metabolic reprogramming, which promote cell survival and limit therapy. The diverse metabolic fuel sources that are produced by autophagy provide tumors with metabolic plasticity and are known to induce drug or radioresistance in GBM. This study determined that autophagy, a common representative cell homeostasis mechanism, was upregulated upon treatment of GBM cells with ionizing radiation (IR). Nuclear receptor binding factor 2 (NRBF2)—a positive regulator of the autophagy initiation step—was found to be upregulated in a GBM orthotopic xenograft mouse model. Furthermore, ATP production and the oxygen consumption rate (OCR) increased upon activation of NRBF2-mediated autophagy. It was also discovered that changes in metabolic state were induced by alterations in metabolite levels caused by autophagy, thereby causing radioresistance. In addition, we found that lidoflazine—a vasodilator agent discovered through drug repositioning—significantly suppressed IR-induced migration, invasion, and proliferation by inhibiting NRBF2, resulting in a reduction in autophagic flux in both in vitro models and in vivo orthotopic xenograft mouse models. In summary, we propose that the upregulation of NRBF2 levels reprograms the metabolic state of GBM cells by activating autophagy, thus establishing NRBF2 as a potential therapeutic target for regulating radioresistance of GBM during radiotherapy.Subject terms: Cancer metabolism, Prognostic markers     

Antimicrobial Activity of Smilax china L. Root Extracts against the Acne-Causing Bacterium,Cutibacterium acnes,and Its Active Compounds

The root of Smilax china L. is used in traditional Korean medicine. We found that the Smilax china L. root extract has strong antimicrobial activity against two Cutibacterium acnes strains (KCTC 3314 and KCTC 3320). The aim of this study was to identify the beneficial properties of Smilax china L. extracts for their potential use as active ingredients in cosmetics for the treatment of human skin acne. The high-performance liquid chromatography (HPLC) and liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC/QTOF/MS) methods were used to obtain the profile of secondary metabolites from the ethyl acetate-soluble fraction of the crude extract. Agar diffusion and resazurin-based broth microdilution assays were used to evaluate antimicrobial activity and minimum inhibitory concentrations (MIC), respectively. Among the 24 metabolites, quercetin, resveratrol, and oxyresveratrol were the most potent compounds against Cutibacterium acnes. Minimum inhibitory concentrations of quercetin, resveratrol, and oxyresveratrol were 31.25, 125, and 250 μg/mL, respectively.     

Structural and Functional Analysis of the Pyridoxal Phosphate Homeostasis Protein YggS from Fusobacterium nucleatum

Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B6, but it is highly reactive and poisonous in its free form. YggS is a PLP-binding protein found in bacteria and humans that mediates PLP homeostasis by delivering PLP to target enzymes or by performing a protective function. Several biochemical and structural studies of YggS have been reported, but the mechanism by which YggS recognizes PLP has not been fully elucidated. Here, we report a functional and structural analysis of YggS from Fusobacterium nucleatum (FnYggS). The PLP molecule could bind to native FnYggS, but no PLP binding was observed for selenomethionine (SeMet)-derivatized FnYggS. The crystal structure of FnYggS showed a type III TIM barrel fold, exhibiting structural homology with several other PLP-dependent enzymes. Although FnYggS exhibited low (<35%) amino acid sequence similarity with previously studied YggS proteins, its overall structure and PLP-binding site were highly conserved. In the PLP-binding site of FnYggS, the sulfate ion was coordinated by the conserved residues Ser201, Gly218, and Thr219, which were positioned to provide the binding moiety for the phosphate group of PLP. The mutagenesis study showed that the conserved Ser201 residue in FnYggS was the key residue for PLP binding. These results will expand the knowledge of the molecular properties and function of the YggS family.     

Performance Improvement of Acid Pretreated 3D-printing Composite for the Heavy Metal Ions Analysis

The performance enhancement of 3D-printed electrode comprised of polylactic acid (PLA) and graphite (Gr) doped with graphene oxide (GO) was studied to detect five heavy metal ions in trace level. The pretreatment of PLA/Gr/GO electrode with potential cycling in H₂SO₄ solution achieved the most sensitive response. The characteristics of the composite electrodes were verified using XPS, FE-SEM, EDXS, Raman, and impedance spectroscopy. The experimental variables affecting the response current were optimized with respect to pH, deposition time, ratio of PLA/Gr/GO, and supporting electrolytes. The pretreated 3D-PLA/Gr/GO electrode showed a wide dynamic range from 0.5 ppb to 1.0 ppm with low detection limits of 0.039–0.13 ppb. The reliability of the PLA/Gr/GO electrode was evaluated by analyzing the reference samples of European Reference Materials.     

Genetic analyzer-dependent DNA methylation detection and its application to existing age prediction models

DNA methylation is the most promising biomarker for estimating human age. There are various methods used for analyzing DNA methylation. Among those, the SNaPshot assay-based method provides a semi-quantitative measurement of DNA methylation using capillary electrophoresis on genetic analyzers. However, DNA methylation measures produced using different types of genetic analyzers have never been compared, although differences in methylation values can directly affect age estimates. To evaluate the differences between the results generated by different genetic analyzers, we analyzed the same blood, saliva, and control methylated DNA using three genetic analyzers—the Applied Biosystems 3130, 3500, and SeqStudio—and compared the methylation values at five CpG sites: ELOVL2, FHL2, KLF14, MIR29B2C, and TRIM59. The methylation value at each of the five CpG sites decreased in the order 3130, 3500, and SeqStudio. The differences in the results produced by the different genetic analyzers resulted in significant errors when applying the 3500 and SeqStudio data to a previous age estimation model constructed using the 3130 Genetic Analyzer data. Therefore, DNA methylation measurements from 3500 and SeqStudio were corrected using the regression functions obtained by plotting the DNA methylation data of one instrument versus the other to facilitate the application of DNA methylation data from one instrument to the age prediction model based on other instruments. The age prediction accuracy obtained by applying corrected 3500 and SeqStudio data to the existing age estimation model was as high as observed in the 3130 data.     

Material profile influences in bulk‐heterojunctions

The morphology in mixed bulk‐heterojunction films are compared using three different quantitative measurement techniques. We compare the vertical composition changes using high‐angle annular dark‐field scanning transmission electron microscopy with electron tomography and neutron and x‐ray reflectometry. The three measurement techniques yield qualitatively comparable vertical concentration measurements. The presence of a metal cathode during thermal annealing is observed to alter the fullerene concentration throughout the thickness of the film for all measurements. However, the absolute vertical concentration of fullerene is quantitatively different for the three measurements. The origin of the quantitative measurement differences is discussed. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2014 , 52, 1291–1300