Poly-L-Lactic Acid Fillers Improved Dermal Collagen Synthesis by Modulating M2 Macrophage Polarization in Aged Animal Skin.

Poly-L-lactic acid (PLLA) fillers correct cutaneous volume loss by stimulating fibroblasts to synthesize collagen and by augmenting the volume. PLLA triggers the macrophage-induced activation of fibroblasts that secrete transforming growth factor-ß (TGF-ß). However, whether M2 macrophage polarization is involved in PLLA-induced collagen synthesis via fibroblast activation in aged skin is not known. Therefore, we evaluated the effect of PLLA on dermal collagen synthesis via M2 polarization in an H2O2-induced cellular senescence model and aged animal skin. H2O2-treated macrophages had increased expression levels of the M1 marker CD80 and decreased expression levels of the M2 marker CD163, which were reversed by PLLA. The expression levels of interleukin (IL)-4 and IL-13, which mediate M2 polarization, were decreased in H2O2-treated macrophages and increased upon the PLLA treatment. CD163, IL-4, and IL-13 expression levels were decreased in aged skin, but increased after the PLLA treatment. The expression levels of TGF-ß, pSMAD2/SMAD2, connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA), collagen type 1A1 (COL1A1), and COL3A1 were also decreased in aged skin, but increased after the PLLA treatment. Moreover, PLLA upregulated phosphatidylinositol 3-kinase p85α (PI3-kinase p85α)/protein kinase B (AKT) signaling, leading to fibroblast proliferation. PLLA decreased the expression of matrix metalloproteinase (MMP) 2 and MMP3, which destroy collagen and elastin fibers in aged skin. The amount of collagen and elastin fibers in aged skin increased following the PLLA treatment. In conclusion, PLLA causes M2 polarization by increasing IL-4 and IL-13 levels and upregulating TGF-ß expression and collagen synthesis in aged skin.

The Extracellular Matrix Vitalizer RATM Increased Skin Elasticity by Modulating Mitochondrial Function in Aged Animal Skin.

Oxidative stress-induced cellular senescence and mitochondrial dysfunction result in skin aging by increasing ECM levels-degrading proteins such as MMPs, and decreasing collagen synthesis. MMPs also destroy the basement membrane, which is involved in skin elasticity. The extracellular matrix vitalizer RATM (RA) contains various antioxidants and sodium hyaluronate, which lead to skin rejuvenation. We evaluated whether RA decreases oxidative stress and mitochondrial dysfunction, eventually increasing skin elasticity in aged animals. Oxidative stress was assessed by assaying NADPH oxidase activity, which is involved in ROS generation, and the expression of SOD, which removes ROS. NADPH oxidase activity was increased in aged skin and decreased by RA injection. SOD expression was decreased in aged skin and increased by RA injection. Damage to mitochondrial DNA and mitochondrial fusion markers was increased in aged skin and decreased by RA. The levels of mitochondrial biogenesis markers and fission markers were decreased in aged skin and increased by RA. The levels of NF-κB/AP-1 and MMP1/2/3/9 were increased in aged skin and decreased by RA. The levels of TGF-ß, CTGF, and collagen I/III were decreased in aged skin and increased by RA. The expression of laminin and nidogen and basement membrane density were decreased in aged skin and increased by RA. RA increased collagen fiber accumulation and elasticity in aged skin. In conclusion, RA improves skin rejuvenation by decreasing oxidative stress and mitochondrial dysfunction in aged skin.

Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses.

We present barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel insitu analyses (BOLORAMIS), a reverse transcription-free method for spatially-resolved, targeted, in situ RNA identification of single or multiple targets. BOLORAMIS was demonstrated on a range of cell types and human cerebral organoids. Singleplex experiments to detect coding and non-coding RNAs in human iPSCs showed a stem-cell signature pattern. Specificity of BOLORAMIS was found to be 92% as illustrated by a clear distinction between human and mouse housekeeping genes in a co-culture system, as well as by recapitulation of subcellular localization of lncRNA MALAT1. Sensitivity of BOLORAMIS was quantified by comparing with single molecule FISH experiments and found to be 11%, 12% and 35% for GAPDH, TFRC and POLR2A, respectively. To demonstrate BOLORAMIS for multiplexed gene analysis, we targeted 96 mRNAs within a co-culture of iNGN neurons and HMC3 human microglial cells. We used fluorescence in situ sequencing to detect error-robust 8-base barcodes associated with each of these genes. We then used this data to uncover the spatial relationship among cells and transcripts by performing single-cell clustering and gene-gene proximity analyses. We anticipate the BOLORAMIS technology for in situ RNA detection to find applications in basic and translational research.

Correlation between Solid Content and Antioxidant Activities in Umbelliferae Salad Plants.

The aim of this study is to evaluate the antioxidant properties of 70% methanolic extracts and the correlation between several antioxidant activities in selected Umbelliferae plants, based on total phenolic content (TPC) and total flavonoid content (TFC). For Umbelliferae plants extracts, the IC50 of DPPH radical (100 µM) quenching activities for extract, TPC, and TFC were 39∼179 µg dry weight (DW)/mL, 14.08∼38.11 µg TPC/mL, and 0.36∼1.51 µg TFC/mL, respectively. The oxygen radical absorbance capacity (ORAC) of extracts ranged from 11.44 to 42.88 mg Trolox equivalent (TE)/g DW extract, whereas ORAC for TPC and TFC was 47.40∼240.19 mg TE/g and 0.72∼11.22 g TE/g, respectively. The TPC had a superior linear correlation (r2=0.817) with 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) values. Of the 14 Umbelliferae plant extracts, Sanicula rubiflora, Sanicula chinensis, Torilis japonica, Torilis scabra, and Angelica fallax showed the strongest antioxidant activity.

National Cancer Institute Think-Tank Meeting Report on Proteomic Cartography and Biomarkers at the Single-Cell Level: Interrogation of Premalignant Lesions.

A Think-Tank Meeting was convened by the National Cancer Institute (NCI) to solicit experts' opinion on the development and application of multiomic single-cell analyses, and especially single-cell proteomics, to improve the development of a new generation of biomarkers for cancer risk, early detection, diagnosis, and prognosis as well as to discuss the discovery of new targets for prevention and therapy. It is anticipated that such markers and targets will be based on cellular, subcellular, molecular, and functional aberrations within the lesion and within individual cells. Single-cell proteomic data will be essential for the establishment of new tools with searchable and scalable features that include spatial and temporal cartographies of premalignant and malignant lesions. Challenges and potential solutions that were discussed included (i) The best way/s to analyze single-cells from fresh and preserved tissue; (ii) Detection and analysis of secreted molecules and from single cells, especially from a tissue slice; (iii) Detection of new, previously undocumented cell type/s in the premalignant and early stage cancer tissue microenvironment; (iv) Multiomic integration of data to support and inform proteomic measurements; (v) Subcellular organelles-identifying abnormal structure, function, distribution, and location within individual premalignant and malignant cells; (vi) How to improve the dynamic range of single-cell proteomic measurements for discovery of differentially expressed proteins and their post-translational modifications (PTM); (vii) The depth of coverage measured concurrently using single-cell techniques; (viii) Quantitation - absolute or semiquantitative? (ix) Single methodology or multiplexed combinations? (x) Application of analytical methods for identification of biologically significant subsets; (xi) Data visualization of N-dimensional data sets; (xii) How to construct intercellular signaling networks in individual cells within premalignant tumor microenvironments (TME); (xiii) Associations between intrinsic cellular processes and extrinsic stimuli; (xiv) How to predict cellular responses to stress-inducing stimuli; (xv) Identification of new markers for prediction of progression from precursor, benign, and localized lesions to invasive cancer, based on spatial and temporal changes within individual cells; (xvi) Identification of new targets for immunoprevention or immunotherapy-identification of neoantigens and surfactome of individual cells within a lesion.

Efficient in situ barcode sequencing using padlock probe-based BaristaSeq.

Cellular DNA/RNA tags (barcodes) allow for multiplexed cell lineage tracing and neuronal projection mapping with cellular resolution. Conventional approaches to reading out cellular barcodes trade off spatial resolution with throughput. Bulk sequencing achieves high throughput but sacrifices spatial resolution, whereas manual cell picking has low throughput. In situ sequencing could potentially achieve both high spatial resolution and high throughput, but current in situ sequencing techniques are inefficient at reading out cellular barcodes. Here we describe BaristaSeq, an optimization of a targeted, padlock probe-based technique for in situ barcode sequencing compatible with Illumina sequencing chemistry. BaristaSeq results in a five-fold increase in amplification efficiency, with a sequencing accuracy of at least 97%. BaristaSeq could be used for barcode-assisted lineage tracing, and to map long-range neuronal projections.

Antiobesity Effects of Salvia plebeia R. Br. Extract in High-Fat Diet-Induced Obese Mice.

This study was designed to investigate the antiobesity effects of Salvia plebeia R. Br. ethanolic extracts (SPE) in mice fed high-fat diets (HFD). Male C57BL/6J mice were randomly assigned to four groups: normal diet (Chow), high-fat diet (HFD, 45% fat), HFD+SPE 200 (200 mg/kg b.w.), and HFD+SPE 400 (400 mg/kg b.w.). Extracts were administered orally every day for 8 weeks. Increases in body/fat weight and feed efficiency ratio were monitored in all mice. In addition, obesity resulting from feeding HFD to the mice was confirmed by the increase of glucose level, aspartate transaminase, alanine transaminase, triglyceride (TG), high-density lipoprotein cholesterol, very low-density lipoprotein-c, leptin, and adiponectin in blood. The SPE-treated mice gained less body and mesenteric/subcutaneous adipose tissues weights and had lower TG, very low-density lipoprotein cholesterol, leptin, and glucose level in serum, compared to the HFD group. Moreover, histopathological examinations revealed that the size of adipocytes in liver and adipose tissue was significantly decreased by SPE, compared to the HFD group. The expression of adipogenesis transcription factors (e.g., peroxisome proliferator activated receptor γ and CCAAT/enhancer binding protein α) and lipogenesis-related target genes (adipocyte fatty acid-binding protein 2, lipoprotein lipase, fatty acid synthase, and sterol regulatory element-binding transcription factor 1c) in HFD-induced obese mice was decreased by SPE treatment. These results suggest that SPE attenuates the fat accumulation in HFD-induced obese mice by suppressing the expressions of genes related to adipogenesis and lipogenesis activity. Therefore, SPE could be developed as a potential therapy for reduction of body weight and antiobesity intervention.

Aglycone Isoflavones and Exopolysaccharides Produced by Lactobacillus acidophilus in Fermented Soybean Paste.

Bioconversion of aglycone-formed isoflavones from glycoside-formed isoflavones by commercial lactic acid bacteria in fermented soybean paste was evaluated. Enterococcus faecium KCTC 13410 showed the most resistant capacity and Lactobacillus acidophilus KCTC 3925 had a sensitive susceptibility at a high NaCl concentration (13.2%) in fermented soybean paste. Among the 5 strains tested, Lac. acidophilus KCTC 3925 showed the highest relative ratio of aglycone-formed isoflavones to total isoflavones in fermented soybean paste. Production of exopolysaccarides (EPS) by lactic acid bacteria was compared using de Man, Rogosa, and Sharpe medium containing 1% sucrose at 37°C for 48 h. Among the 5 lactic acid bacteria, Lac. acidophilus KCTC 3925 and Lactobacillus rhamnosus KCTC 3929 were investigated to produce EPS. Based on the results concerning growing susceptibility and conversion of aglycone-formed isoflavones/EPS production, it is anticipated that Lac. acidophilus KCTC 3925 may be used for preparation of Cheonggukjang, which contains relative low NaCl content.

Eating Habits and Food Preferences of Elementary School Students in Urban and Suburban Areas of Daejeon.

This study investigated the dietary habits and food preferences of elementary school students. The survey was conducted by means of a questionnaire distributed to 4th and 5th grade elementary school students (400 boys and 400 girls) in urban and suburban areas of Daejeon. The results of this study were as follows: male students in urban areas ate breakfast, unbalanced diets, and dairy products more frequently than male students in suburban areas (p < 0.05). Female students in urban areas ate dairy products (p < 0.01) and fruits (p < 0.001) more frequently than female students in suburban areas. Students had the high preferences for boiled rice and noodles with black bean sauce, beef rib soup, steamed beef rib, steamed egg, beef boiled in soy sauce, egg roll, bulgogi, pork cutlet, deep-fried pork covered with sweet and sour starchy sauce, and honeyed juice mixed with fruit as a punch. All students preferred kimchi, although students in the suburban areas preferred kimchi-fried rice (p < 0.05), and those in the urban areas preferred bean-paste soup (p < 0.01). Students in suburban areas showed a greater preference for seasoned bean sprouts and Altari kimchi. All of the students preferred fruits, rice cake made with glutinous rice, and pizza among other foods. Overall, there were distinct differences in the eating habits and food preferences of elementary school students according to the place of residence.

Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues.

RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d.

Analysis of six elements (Ca, Mg, Fe, Zn, Cu, and Mn) in several wild vegetables and evaluation of their intakes based on Korea National Health and Nutrition Examination Survey 2010-2011.

Wild vegetables, those edible among naturally grown vegetables, have been reported to contain many bioactive substances, dietary fibers, vitamins, and minerals. The purpose of this study is to examine the six elements of the wild vegetables frequently consumed by Koreans and assess the element intakes through them. Contents of six kinds of elements (Ca, Mg, Fe, Zn, Cu, and Mn) in 11 wild vegetables were analyzed by inductively coupled plasma optical emission spectroscopy. Using these analysis data, the 6-element intakes from the wild vegetables were evaluated in healthy Korean adults aged 19-64 years from the Korea National Health and Nutrition Examination Survey (2010-2011). Sedum and shepherd's purse contained over 100 mg of Ca in 100 g of their edible portion. The Mg content per 100 g of the 11 wild vegetables ranged from 12.1 mg to 43.4 mg. The wild vegetable with the highest mineral content per 100 g was sedum for Ca, spinach for Mg, shepherd's purse for Fe, spinach for Zn, bracken for Cu, and fragrant edible wild aster for Mn. The element intakes from the 11 wild vegetables compared with dietary reference intakes in the healthy Koreans were 1.0 % for Ca, 2.1 % for Mg, 5.3 % for Fe, 1.4 % for Zn, 0.3 % for Cu, and 1.8 % for Mn. Considering the low intake ratio (1.2 %) of the wild vegetable to total food intake, wild vegetables may contribute to some element intakes. Our results show the nutritional value of the wild vegetables in the aspect of mineral nutrition; however, further research is needed to evaluate the bioavailability of various elements in wild vegetables.

Highly multiplexed subcellular RNA sequencing in situ.

Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.

High-resolution antibody dynamics of vaccine-induced immune responses.

The adaptive immune system confers protection by generating a diverse repertoire of antibody receptors that are rapidly expanded and contracted in response to specific targets. Next-generation DNA sequencing now provides the opportunity to survey this complex and vast repertoire. In the present work, we describe a set of tools for the analysis of antibody repertoires and their application to elucidating the dynamics of the response to viral vaccination in human volunteers. By analyzing data from 38 separate blood samples across 2 y, we found that the use of the germ-line library of V and J segments is conserved between individuals over time. Surprisingly, there appeared to be no correlation between the use level of a particular VJ combination and degree of expansion. We found the antibody RNA repertoire in each volunteer to be highly dynamic, with each individual displaying qualitatively different response dynamics. By using combinatorial phage display, we screened selected VH genes paired with their corresponding VL library for affinity against the vaccine antigens. Altogether, this work presents an additional set of tools for profiling the human antibody repertoire and demonstrates characterization of the fast repertoire dynamics through time in multiple individuals responding to an immune challenge.

Barcoding cells using cell-surface programmable DNA-binding domains.

We report an approach to barcode cells through cell-surface expression of programmable zinc-finger DNA-binding domains (surface zinc fingers, sZFs). We show that sZFs enable sequence-specific labeling of living cells by dsDNA, and we develop a sequential labeling approach to image more than three cell types in mixed populations using three fluorophores. We demonstrate the versatility of sZFs through applications in which they serve as surrogate reporters, function as selective cell capture reagents and facilitate targeted cellular delivery of viruses.

Quality characteristics and ginsenosides composition of ginseng-yakju according to the particle size of ginseng powder.

The aim of this study was to develop rice wine (Yakju) containing various amounts and particle sizes of ginseng powder and to analyze the physicochemical characteristics and content of ginsenosides in ginseng-Yakju. Soluble solid content, pH, ethanol concentration, acidity, amino acid content, and evaluation of preference showed no difference between four kinds of Yakju groups, regardless of ginseng supplementation and particle size of the ginseng powder. During fermentation of Yakju containing ginseng, the contents of ginsenosides Rb1, Rb2, Rb3, and Rc were decreased. Otherwise, the content of ginsenoside Rh1 was increased highly by brewing microorganisms in Yakju. Recovery ratios of ginsenosides in ginseng-Yakju were approximately 25.4% (coarse ginseng power) and 23.8% (fine ginseng powder), which were superior to the recovery ratio of ginsenosides in Yakju containing ginseng slices (5%).

A public resource facilitating clinical use of genomes.

Rapid advances in DNA sequencing promise to enable new diagnostics and individualized therapies. Achieving personalized medicine, however, will require extensive research on highly reidentifiable, integrated datasets of genomic and health information. To assist with this, participants in the Personal Genome Project choose to forgo privacy via our institutional review board- approved "open consent" process. The contribution of public data and samples facilitates both scientific discovery and standardization of methods. We present our findings after enrollment of more than 1,800 participants, including whole-genome sequencing of 10 pilot participant genomes (the PGP-10). We introduce the Genome-Environment-Trait Evidence (GET-Evidence) system. This tool automatically processes genomes and prioritizes both published and novel variants for interpretation. In the process of reviewing the presumed healthy PGP-10 genomes, we find numerous literature references implying serious disease. Although it is sometimes impossible to rule out a late-onset effect, stringent evidence requirements can address the high rate of incidental findings. To that end we develop a peer production system for recording and organizing variant evaluations according to standard evidence guidelines, creating a public forum for reaching consensus on interpretation of clinically relevant variants. Genome analysis becomes a two-step process: using a prioritized list to record variant evaluations, then automatically sorting reviewed variants using these annotations. Genome data, health and trait information, participant samples, and variant interpretations are all shared in the public domain-we invite others to review our results using our participant samples and contribute to our interpretations. We offer our public resource and methods to further personalized medical research.

Accurate whole-genome sequencing and haplotyping from 10 to 20 human cells.

Recent advances in whole-genome sequencing have brought the vision of personal genomics and genomic medicine closer to reality. However, current methods lack clinical accuracy and the ability to describe the context (haplotypes) in which genome variants co-occur in a cost-effective manner. Here we describe a low-cost DNA sequencing and haplotyping process, long fragment read (LFR) technology, which is similar to sequencing long single DNA molecules without cloning or separation of metaphase chromosomes. In this study, ten LFR libraries were made using only ∼100 picograms of human DNA per sample. Up to 97% of the heterozygous single nucleotide variants were assembled into long haplotype contigs. Removal of false positive single nucleotide variants not phased by multiple LFR haplotypes resulted in a final genome error rate of 1 in 10 megabases. Cost-effective and accurate genome sequencing and haplotyping from 10-20 human cells, as demonstrated here, will enable comprehensive genetic studies and diverse clinical applications.

Anti-gastric actions of eugenol and cinnamic acid isolated from Cinnamomi Ramulus.

We investigated the evidence of gastric protection for ulcer and gastritis by Cinnamomi Ramulus (Cinnamomum cassia Blume, Geiji, CR) extract and its several constituents. CR ethanolic extract showed the potent antioxidant activity and cytotoxicity of Helicobacter pylori (H. pylori) and acid-neutralizing capacity. Especially, eugenol exerted a significant antioxidant activity and inhibited the colonization of H. pylori. In vivo test, eugenol and cinnamic acid significantly inhibited HCl/ethanol-induced gastric lesions and increased the mucus content though they didn't inhibit gastric secretion effectively. Taken together, eugenol and cinnamic acid, which were isolated from CR, exhibited the antioxidant activity in vitro and protective effect against gastric damage in vivo through stimulation of mucus secretion and so on. It suggested that they are useful as the neutraceuticals for gastritis.

Somatic coding mutations in human induced pluripotent stem cells.

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use.

Evaluation of antioxidant and inhibitory activities for different subclasses flavonoids on enzymes for rheumatoid arthritis.

Antioxidant activities of flavonoids were decreased in the order of flavonols > flavanones > flavones. Inhibitory intensities for hyaluronidase and collagenase reaction differed clearly according to flavonoid subclasses. Kaempferol, quercetin, myricetin, and rutin in flavonols inhibited hyaluronidase reaction specifically, while apigenin, luteolin, baicalin, and baicalein in flavones showed specific inhibition to collagenase reaction. In addition, the flavonoids, except baicalin and catechin, inhibited potently LPS-induced nitrite production in a dose-dependent manner, which might be mainly due to the suppression of inducible nitric oxide (NO) synthase. Quercetin and luteolin showed the strongest inhibitory activities on 15-lipoxygenase (LOX), and quercetin showed relatively potent inhibition on cyclooxygenase-1 (COX-1) reaction. Otherwise, all tested flavonoids possessed the inhibitory activity to COX-2 reaction, and especially luteolin, kaempferol, hesperetin, and naringin showed relatively the potent inhibition on COX-2 reaction. This report elucidated the anti-inflammatory activities, such as the antioxidant property, inhibition of NO production, and inhibition of inflammatory enzymes (hyaluronidase, collagenase, LOX, and COXs) of several subclass flavonoids.