DNA methylation signatures reveal that distinct combinations of transcription factors specify human immune cell epigenetic identity.

Epigenetic reprogramming underlies specification of immune cell lineages, but patterns that uniquely define immune cell types and the mechanisms by which they are established remain unclear. Here, we identified lineage-specific DNA methylation signatures of six immune cell types from human peripheral blood and determined their relationship to other epigenetic and transcriptomic patterns. Sites of lineage-specific hypomethylation were associated with distinct combinations of transcription factors in each cell type. By contrast, sites of lineage-specific hypermethylation were restricted mostly to adaptive immune cells. PU.1 binding sites were associated with lineage-specific hypo- and hypermethylation in different cell types, suggesting that it regulates DNA methylation in a context-dependent manner. These observations indicate that innate and adaptive immune lineages are specified by distinct epigenetic mechanisms via combinatorial and context-dependent use of key transcription factors. The cell-specific epigenomics and transcriptional patterns identified serve as a foundation for future studies on immune dysregulation in diseases and aging.

Enhanced In Vitro and In Vivo Potency of a T Cell Epitope in the Ebola Virus Glycoprotein Following Amino Acid Replacement at HLA-A*02:01 Binding Positions.

Studies on Ebola virus disease (EVD) survivors and clinical studies on Ebola virus (EBOV) vaccine candidates have pinpointed the importance of a strong antibody response in protection and survival from EBOV infection. However, little is known about the T cell responses to EBOV or EBOV vaccines. We used HLA-A*02:01 (HLA-A2) transgenic mice to study HLA-A2-specific T cell responses elicited following vaccination with EBOV glycoprotein (EBOV-GP) presented with three different systems: (i) recombinant protein (rEBOV-GP), (ii) vesicular stomatitis replication-competent recombinant virus (VSV-EBOV-GP), and (iii) modified vaccinia Ankara virus recombinant (MVA-EBOV-GP). T cells from immunized animals were analyzed using peptide pools representing the entire GP region and individual peptides. Regardless of the vaccine formulation, we identified a minimal 9mer epitope containing an HLA-A2 motif (FLDPATTS), which was confirmed through HLA-A2 binding affinity and immunization studies. Using binding prediction software, we identified substitutions surrounding position 9 (S9V, P10V, and Q11V) that predicted enhanced binding to the HLA-A2 molecule. This enhanced binding was confirmed through in vitro binding studies and enhanced potency was shown with in vivo immunization studies using the enhanced sequences and the wild-type sequence. Of note, in silico studies predicted the enhanced 9mer epitope carrying the S9V substitution as the best overall HLA-A2 epitope for the full-length EBOV-GP. These results suggest that EBOV-GP-S9V and EBOV-GP-P10V represent more potent in vivo immunogens. Identification and enhancement of EBOV-specific human HLA epitopes could lead to the development of tools and reagents to induce more robust T cell responses in human subjects. IMPORTANCE Vaccine efficacy and immunity to viral infection are often measured by neutralizing antibody titers. T cells are specialized subsets of immune cells with antiviral activity, but this response is variable and difficult to track. We showed that the HLA-A2-specific T cell response to the Ebola virus glycoprotein can be enhanced significantly by a single residue substitution designed to improve an epitope binding affinity to one of the most frequent MHC alleles in the human population. This strategy could be applied to improve T cell responses to Ebola vaccines designed to elicit antibodies and adapted to target MHC alleles of populations in regions where endemic infections, like Ebola virus disease, are still causing outbreaks with concerning pandemic potential.

A Novel NOX Inhibitor Treatment Attenuates Parkinson's Disease-Related Pathology in Mouse Models.

Parkinson's disease (PD) is a progressive neurodegenerative motor disorder without an available therapeutic to halt the formation of Lewy bodies for preventing dopaminergic neuronal loss in the nigrostriatal pathway. Since oxidative-stress-mediated damage has been commonly reported as one of the main pathological mechanisms in PD, we assessed the efficacy of a novel NOX inhibitor from AptaBio Therapeutics (C-6) in dopaminergic cells and PD mouse models. The compound reduced the cytotoxicity and enhanced the cell viability at various concentrations against MPP+ and α-synuclein preformed fibrils (PFFs). Further, the levels of ROS and protein aggregation were significantly reduced at the optimal concentration (1 µM). Using two different mouse models, we gavaged C-6 at two different doses to the PD sign-displaying transgenic mice for 2 weeks and stereotaxically PFF-injected mice for 5 weeks. Our results demonstrated that both C-6-treated mouse models showed alleviated motor deficits in pole test, hindlimb clasping, crossbeam, rotarod, grooming, and nesting analyses. We also confirmed that the compound treatment reduced the levels of protein aggregation, along with phosphorylated-α-synuclein, in the striatum and ventral midbrain and further dopaminergic neuronal loss. Taken together, our results strongly suggest that NOX inhibition can be a potential therapeutic target for PD.

CMOS-compatible Si metasurface at visible wavelengths prepared by low-temperature green laser annealing.

A low-temperature laser crystallization is newly devised for producing polycrystalline silicon (poly-Si) thin films of low-loss, low surface roughness enough for nanoscale patterning, applicable to practical Si metasurface elements on complementary metal-oxide semiconductor (CMOS) electronic architectures in visible lights. The method is based on dielectric encapsulation of an amorphous Si film and subsequent laser-induced local crystallization. Such poly-Si thin film yields order-of-magnitude smaller surface roughness and grain size than those obtained with the conventional laser annealing processes. The mechanism of the formation of small and uniform crystalline grains during solidification is studied to ensure the smooth surfaces enough for nanoscale patterning. By obtaining root mean square of surface roughness <2.49 nm and extinction coefficient <4.8 × 10-2 at 550 nm, visible metasurface color-filter elements are experimentally demonstrated with the resonant transmission-peak efficiency approaching ∼85%. This low-loss poly-Si metasurface is favorably compatible with embedded CMOS electronic architectures in contrast to the conventional thermal annealing processes that often cause failure of electrical device functionalities due to delamination and material-property degradation problems. The proposed fabrication in this study provides a practical method for further development of various Si metasurfaces in the visible domain and their integration with CMOS electronic devices as well.

Visible Wavelength Color Filters Using Dielectric Subwavelength Gratings for Backside-Illuminated CMOS Image Sensor Technologies.

We report transmissive color filters based on subwavelength dielectric gratings that can replace conventional dye-based color filters used in backside-illuminated CMOS image sensor (BSI CIS) technologies. The filters are patterned in an 80 nm-thick poly silicon film on a 115 nm-thick SiO2 spacer layer. They are optimized for operating at the primary RGB colors, exhibit peak transmittance of 60-80%, and have an almost insensitive response over a ± 20° angular range. This technology enables shrinking of the pixel sizes down to near a micrometer.

BetaCavityWeb: a webserver for molecular voids and channels.

Molecular cavities, which include voids and channels, are critical for molecular function. We present a webserver, BetaCavityWeb, which computes these cavities for a given molecular structure and a given spherical probe, and reports their geometrical properties: volume, boundary area, buried area, etc. The server's algorithms are based on the Voronoi diagram of atoms and its derivative construct: the beta-complex. The correctness of the computed result and computational efficiency are both mathematically guaranteed. BetaCavityWeb is freely accessible at the Voronoi Diagram Research Center (VDRC) (http://voronoi.hanyang.ac.kr/betacavityweb).

Tension assisted metal transfer of graphene for Schottky diodes onto wafer scale substrates.

We developed an effective graphene transfer method for graphene/silicon Schottky diodes on a wafer as large as 6 inches. Graphene grown on a large scale substrate was passivated and sealed with a gold layer, protecting graphene from any possible contaminant and keeping good electrical contact. The Au/graphene was transferred by the tension-assisted transfer process without polymer residues. The gold film itself was used directly as the electrodes of a Schottky diode. We demonstrated wafer-scale integration of graphene/silicon Schottky diode using the proposed transfer process. The transmission electron microscopy analysis and relatively low ideality factor of the diodes indicated fewer defects on the interface than those obtained using the conventional poly(methyl methacrylate)-assisted transfer method. We further demonstrated gas sensors as an application of graphene Schottky diodes.

Low-visibility patterning of transparent conductive silver-nanowire films.

A partial etching mechanism is proposed to meet the requirement for low-visibility patterning of silver nanowire (AgNW)-based transparent conductive electrodes (TCEs) by reducing the difference in optical properties between conductive and nonconductive regions of the pattern. Using the finite difference time domain (FDTD) method, etched geometries that provide the smallest difference in transmittance after etching are theoretically determined. A sodium hypochlorite-based etchant capable that allows the etched geometry to be varied by controlling the pH is used to create a low-visibility pattern with a transmittance and haze difference of 0.07 and 0.04%, respectively. To the best of our knowledge, this is the first time that a partial etching mechanism such as this has been studied in relation to AgNW-based TCEs.

Fascin regulates TLR4/PKC-mediated translational activation through miR-155 and miR-125b, which targets the 3' untranslated region of TNF-α mRNA.

Fascin is a well-known cytoskeletal regulatory protein that, as a substrate of protein kinase C (PKC), is involved in PKC-mediated translational regulation of TNF-α in macrophages stimulated with lipopolysaccharide (LPS). The regulatory effects of fascin targeted the 3'-untraslated region (UTR) of the TNF-α mRNA, and suppression of PKC activity or fascin expression resulted in specific blockage of the LPS-induced translational activation of the mRNA. In an effort to identify the molecular mechanism of this fascin-mediated translational regulation, the expression levels of micro-RNA (miRNA) after stimulation of the toll-like receptor 4 (TLR4) signaling pathways were analyzed in cells with down-regulation of fascin. The LPS-induced translation of TNF-α is known to be regulated by miR-155 and miR-125b, which have positive and negative effects, respectively. Interestingly, suppression of fascin expression reversed LPS-induced down-regulation of miR-125b and abolished the LPS-induced increase in miR-155. Furthermore, introduction of miR-155 precursor, blocking of miR-125b activity, or introduction of a mutation into the miR-125b binding site of the TNF-α 3'-UTR restored translational activation in cells with suppressed fascin expression. These data indicate that fascin regulates translation through miR-155 and miR-125b, which target 3' UTR in TNF-α mRNA.

BetaVoid: molecular voids via beta-complexes and Voronoi diagrams.

Molecular external structure is important for molecular function, with voids on the surface and interior being one of the most important features. Hence, recognition of molecular voids and accurate computation of their geometrical properties, such as volume, area and topology, are crucial, yet most popular algorithms are based on the crude use of sampling points and thus are approximations even with a significant amount of computation. In this article, we propose an analytic approach to the problem using the Voronoi diagram of atoms and the beta-complex. The correctness and efficiency of the proposed algorithm is mathematically proved and experimentally verified. The benchmark test clearly shows the superiority of BetaVoid to two popular programs: VOIDOO and CASTp. The proposed algorithm is implemented in the BetaVoid program which is freely available at the Voronoi Diagram Research Center (http://voronoi.hanyang.ac.kr).

Vaccine-associated respiratory pathology correlates with viral clearance and protective immunity after immunization with self-amplifying RNA expressing the spike (S) protein of SARS-CoV-2 in mouse models.

In this study, we evaluated the immunogenicity and protective immunity of in vitro transcribed Venezuelan equine encephalitis virus (VEEV TC-83 strain) self-amplifying RNA (saRNA) encoding the SARS-CoV-2 spike (S) protein in wild type (S-WT) and stabilized pre-fusion conformations (S-PP). Immunization with S-WT and S-PP saRNA induced specific neutralizing antibody responses in both K18-Tg hACE2 (K18) and BALB/c mice, as assessed using SARS-CoV-2 pseudotyped viruses. Protective immunity was assessed in challenge experiments. Two immunizations with S-WT and S-PP induced protective immunity, evidenced by lower mortality, lower weight loss and more than one log10 lower subgenomic virus RNA titers in the upper and lower respiratory tracts in both K18 and BALB/c mice. Histopathologic examination of lungs post-challenge showed that immunization with S-WT and S-PP resulted in a higher degree of immune cell infiltration and inflammatory changes, compared with control mice, characterized by high levels of T- and B-cell infiltration. No substantial differences were found in the presence and localization of eosinophils, macrophages, neutrophils, and natural killer cells. CD4 and CD8 T-cell depletion post immunization resulted in reduced lung inflammation post challenge but also prolonged virus clearance. These data indicate that immunization with saRNA encoding the SARS-CoV-2 S protein induces immune responses that are protective following challenge, that virus clearance is associated with pulmonary changes caused by T-cell and B-cell infiltration in the lungs, but that this T and B-cell infiltration plays an important role in viral clearance.

A beta-complex statistical four body contact potential combined with a hydrogen bond statistical potential recognizes the correct native structure from protein decoy sets.

We present a new four-body knowledge-based potential for recognizing the native state of proteins from their misfolded states. This potential was extracted from a large set of protein structures determined by X-ray crystallography using BetaMol, a software based on the recent theory of the beta-complex (ß-complex) and quasi-triangulation of the Voronoi diagram of spheres. This geometric construct reflects the size difference among atoms in their full Euclidean metric; property not accounted for in a typical 3D Delaunay triangulation. The ability of this potential to identify the native conformation over a large set of decoys was evaluated. Experiments show that this potential outperforms a potential constructed with a classical Delaunay triangulation in decoy discrimination tests. The addition of a statistical hydrogen bond potential to our four-body potential allows a significant improvement in the decoy discrimination, in such a way that we are able to predict successfully the native structure in 90% of cases.

GalaxyDock2: protein-ligand docking using beta-complex and global optimization.

In this article, an enhanced version of GalaxyDock protein-ligand docking program is introduced. GalaxyDock performs conformational space annealing (CSA) global optimization to find the optimal binding pose of a ligand both in the rigid-receptor mode and the flexible-receptor mode. Binding pose prediction has been improved compared to the earlier version by the efficient generation of high-quality initial conformations for CSA using a predocking method based on a beta-complex derived from the Voronoi diagram of receptor atoms. Binding affinity prediction has also been enhanced by using the optimal combination of energy components, while taking into consideration the energy of the unbound ligand state. The new version has been tested in terms of binding mode prediction, binding affinity prediction, and virtual screening on several benchmark sets, showing improved performance over the previous version and AutoDock, on which the GalaxyDock energy function is based. GalaxyDock2 also performs better than or comparable to other state-of-the-art docking programs. GalaxyDock2 is freely available at http://galaxy.seoklab.org/softwares/galaxydock.html.

A novel pathway responsible for lipopolysaccharide-induced translational regulation of TNF-α and IL-6 expression involves protein kinase C and fascin.

Fascin, as a substrate of protein kinase C (PKC), is a well-known cytoskeletal regulatory protein required for cell migration, invasion, and adhesion in normal and cancer cells. In an effort to identify the role of fascin in PKC-mediated cellular signaling, its expression was suppressed by stable transfection of specific short hairpin RNAs (shRNAs) in mouse monocytic leukemia RAW264.7 cells. Suppression of fascin expression resulted in impaired cellular migration and invasion through extracellular matrix proteins. Unexpectedly, the specific shRNA transfectants exhibited a marked reduction in LPS-induced expression of TNF-α and IL-6 by blocking the translation of their mRNAs. Transient transfection assay using a luciferase expression construct containing the 3' untranslated region of TNF-α or IL-6 mRNA revealed a significant reduction in both LPS- and PMA- (the direct activator of PKC) induced reporter activity in cells transfected with fascin-specific shRNA, indicating that fascin-mediated translational regulation targeted 3' untranslated region. Furthermore, LPS-induced translational activation of reporter expression was blocked by a pharmacological inhibitor of PKC, and the dominant-negative form of PKCα attenuated LPS-induced translational activation. The same type of regulation was also observed in the human monocytic leukemia cell line THP-1 and in mouse peritoneal macrophages. These data demonstrate the involvement of fascin in the PKC-mediated translational regulation of TNF-α and IL-6 expression during the LPS response.

Room temperature deposition of silicon nanodot clusters by plasma-enhanced chemical vapor deposition.

The formation of nanometer-scale (ns)-Si dots and clusters on p-GaN layers has been studied by controlling the early stage of growth during plasma-enhanced chemical vapor deposition (PECVD) at room temperature. We found that ns-Si dots and clusters formed on the p-GaN surface, indicating that growth was the Volmer-Weber mode. The deposition parameters such as radio frequency (RF) power and processing time mainly influenced the size of the ns-Si dots (40 nm-160 nm) and the density of the ns-Si dot clusters.

Ohmic contacts to N-face p-GaN using Ni/Au for the fabrication of polarization inverted light-emitting diodes.

The electrical properties of Ni-based ohmic contacts to N-face p-type GaN were investigated. The specific contact resistance of N-face p-GaN exhibits a linear decrease from 1.01 omega cm2 to 9.05 x 10(-3) omega cm2 for the as-deposited and the annealed Ni/Au contacts, respectively, with increasing annealing temperature. However, the specific contact resistance could be decreased down to 1.03 x 10(-4) omega cm2 by means of surface treatment using an alcohol-based (NH4)2S solution. The depth profile data measured from the intensity of O1s peak in the X-ray photoemission spectra showed that the alcohol-based (NH4)2S treatment was effective in removing the surface oxide layer of GaN.

Cp2Ti(II) Mediated Rearrangement of Cyclopropyl Imines.

Ti-catalyzed oxidative alkyne carboamination with alkenes and azo compounds can yield either α,ß-unsaturated imines or cyclopropyl imines through a common azatitanacyclohexene intermediate. Herein, we report the synthesis of a model azatitanacyclohexene complex (3) through the ring-opening of a cyclopropyl imine with Cp2Ti(BTMSA) (BTMSA = bis(trimethylsilyl)acetylene). 3 readily undergoes thermal or reductant-catalyzed ring contraction to an azatitanacyclopentene (4), analogous to the proposed mechanism for forming α,ß-unsaturated imines in the catalytic reaction. A cyclopropyl imine or an α,ß-unsaturated imine could be liberated via the oxidation of 3 or 4 with azobenzene, respectively, further implicating the role of these metallacycles in the Ti-catalyzed carboamination reaction.

Epigenetic signature of human immune aging in the GESTALT study.

Age-associated DNA methylation in blood cells convey information on health status. However, the mechanisms that drive these changes in circulating cells and their relationships to gene regulation are unknown. We identified age-associated DNA methylation sites in six purified blood-borne immune cell types (naive B, naive CD4+ and CD8+ T cells, granulocytes, monocytes, and NK cells) collected from healthy individuals interspersed over a wide age range. Of the thousands of age-associated sites, only 350 sites were differentially methylated in the same direction in all cell types and validated in an independent longitudinal cohort. Genes close to age-associated hypomethylated sites were enriched for collagen biosynthesis and complement cascade pathways, while genes close to hypermethylated sites mapped to neuronal pathways. In silico analyses showed that in most cell types, the age-associated hypo- and hypermethylated sites were enriched for ARNT (HIF1ß) and REST transcription factor (TF) motifs, respectively, which are both master regulators of hypoxia response. To conclude, despite spatial heterogeneity, there is a commonality in the putative regulatory role with respect to TF motifs and histone modifications at and around these sites. These features suggest that DNA methylation changes in healthy aging may be adaptive responses to fluctuations of oxygen availability.

Synthetic peptides containing ITIM-like domains block expression of inflammatory mediators and migration/invasion of cancer cells through activation of SHP-1 and PI3K.

Increasing evidence supports that inflammation is closely associated with the development of cancer. In an effort to develop synthetic peptides that can suppress the inflammatory activation of cancer cells, decapeptides representing immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences of immune receptor expressed on myeloid cells-1 (IREM-1) were tested for their anti-inflammatory effects in cancer cell lines. One (named TAT-YADL) out of the five synthetic peptides tested exhibited inhibitory effects on the expression of inflammatory mediators as well as invasion and migration. The inhibitory activities of the synthetic peptides required activation of SH2-containing protein tyrosine phosphatase-1 (SHP-1) and phosphoinositide 3-kinase (PI3K).