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.

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.

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.

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.

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.

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.

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.

Digital RNA allelotyping reveals tissue-specific and allele-specific gene expression in human.

We developed a digital RNA allelotyping method for quantitatively interrogating allele-specific gene expression. This method involves ultra-deep sequencing of padlock-captured single-nucleotide polymorphisms (SNPs) from the transcriptome. We characterized four cell lines established from two human subjects in the Personal Genome Project. Approximately 11-22% of the heterozygous mRNA-associated SNPs showed allele-specific expression in each cell line and 4.3-8.5% were tissue-specific, suggesting the presence of tissue-specific cis regulation. When we applied allelotyping to two pairs of sibling human embryonic stem cell lines, the sibling lines were more similar in allele-specific expression than were the genetically unrelated lines. We found that the variation of allelic ratios in gene expression among different cell lines was primarily explained by genetic variations, much more so than by specific tissue types or growth conditions. Comparison of expressed SNPs on the sense and antisense transcripts suggested that allelic ratios are primarily determined by cis-regulatory mechanisms on the sense transcripts.

A robust approach to identifying tissue-specific gene expression regulatory variants using personalized human induced pluripotent stem cells.

Normal variation in gene expression due to regulatory polymorphisms is often masked by biological and experimental noise. In addition, some regulatory polymorphisms may become apparent only in specific tissues. We derived human induced pluripotent stem (iPS) cells from adult skin primary fibroblasts and attempted to detect tissue-specific cis-regulatory variants using in vitro cell differentiation. We used padlock probes and high-throughput sequencing for digital RNA allelotyping and measured allele-specific gene expression in primary fibroblasts, lymphoblastoid cells, iPS cells, and their differentiated derivatives. We show that allele-specific expression is both cell type and genotype-dependent, but the majority of detectable allele-specific expression loci remains consistent despite large changes in the cell type or the experimental condition following iPS reprogramming, except on the X-chromosome. We show that our approach to mapping cis-regulatory variants reduces in vitro experimental noise and reveals additional tissue-specific variants using skin-derived human iPS cells.

Generation of functional human hepatic endoderm from human induced pluripotent stem cells.

UNLABELLED: With the advent of induced pluripotent stem cell (iPSC) technology, it is now feasible to generate iPSCs with a defined genotype or disease state. When coupled with direct differentiation to a defined lineage, such as hepatic endoderm (HE), iPSCs would revolutionize the way we study human liver biology and generate efficient "off the shelf" models of human liver disease. Here, we show the "proof of concept" that iPSC lines representing both male and female sexes and two ethnic origins can be differentiated to HE at efficiencies of between 70%-90%, using a method mimicking physiological relevant condition. The iPSC-derived HE exhibited hepatic morphology and expressed the hepatic markers albumin and E-cadherin, as assessed by immunohistochemistry. They also expressed alpha-fetoprotein, hepatocyte nuclear factor-4a, and a metabolic marker, cytochrome P450 7A1 (Cyp7A1), demonstrating a definitive endodermal lineage differentiation. Furthermore, iPSC-derived hepatocytes produced and secreted the plasma proteins, fibrinogen, fibronectin, transthyretin, and alpha-fetoprotein, an essential feature for functional HE. Additionally iPSC-derived HE supported both CYP1A2 and CYP3A4 metabolism, which is essential for drug and toxicology testing. CONCLUSION: This work is first to demonstrate the efficient generation of hepatic endodermal lineage from human iPSCs that exhibits key attributes of hepatocytes, and the potential application of iPSC-derived HE in studying human liver biology. In particular, iPSCs from individuals representing highly polymorphic variants in metabolic genes and different ethnic groups will provide pharmaceutical development and toxicology studies a unique opportunity to revolutionize predictive drug toxicology assays and allow the creation of in vitro hepatic disease models.

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.

Protective effects of neohesperidin and poncirin isolated from the fruits of Poncirus trifoliata on potential gastric disease.

The effects of Poncirus trifoliata (P. trifoliata) (Ponciri Fructus, PF) extract and its constituents such as neohesperidin and poncirin on gastritis in rats and human gastric cancer cells were investigated. The PF 70% ethanol extracts (1 g) showed approximately 11.38% of acid-neutralizing capacities and cytotoxicity (IC50=85.39 microg/mL) against human AGS gastric cancer cells. In addition, neohesperidin exhibited antioxidant activity (IC50=22.31 microg/mL) in the 1,1-diphenyl-2-picryldydrazyl (DPPH) radical-scavenging assay. Neohesperidin (50 mg/kg) and poncirin (100 mg/kg) significantly inhibited 55.0% and 60.0% of HCl/ethanol-induced gastric lesions, respectively, and increased the mucus content. In pylorus ligated rats, neohesperidin (50 mg/kg) significantly decreased the volume of gastric secretion and gastric acid output, and increased the pH. From these results, it could be suggested that neohesperidin and poncirin isolated from PF may be useful for the treatment and/or protection of gastritis.

Inhibition of the intestinal glucose transporter GLUT2 by flavonoids.

We tested whether the dominant intestinal sugar transporter GLUT2 was inhibited by intestinal luminal compounds that are inefficiently absorbed and naturally present in foods. Because of their abundance in fruits and vegetables, flavonoids were selected as model compounds. Robust inhibition of glucose and fructose transport by GLUT2 expressed in Xenopus laevis oocytes was produced by the flavonols myricetin, fisetin, the widely consumed flavonoid quercetin, and its glucoside precursor isoquercitrin [corrected]. IC50s for quercetin, myricetin, and isoquercitirin [corrected]were approximately 200- to 1000-fold less than glucose or fructose concentrations, and noncompetitive inhibition was observed. The two other major intestinal sugar transporters, GLUT5 and SGLT1, were unaffected by flavonoids. Sugar transport by GLUT2 overexpressed in pituitary cells and naturally present in Caco-2E intestinal cells was similarly inhibited by quercetin. GLUT2 was detected on the apical side of Caco-2E cells, indicating that GLUT2 was in the correct orientation to be inhibited by luminal compounds. Quercetin itself was not transported by the three major intestinal glucose transporters. Because the flavonoid quercetin, a food component with an excellent pharmacology safety profile, might act as a potent luminal inhibitor of sugar absorption independent of its own transport, flavonols show promise as new pharmacologic agents in the obesity epidemic.

Potential analgesic and anti-inflammatory activities of Panax ginseng head butanolic fraction in animals.

The present study reports the potential anti-rheumatoid activity of Panax ginseng head part. P. ginseng-head part BuOH fraction (PGHB) was safe in acute toxicity (LD(50)>5000mg/kg) and inhibited the partially acetic acid-induced writhes (approximately 32%, P<0.05) in mice. PGHB (500mg/kg) inhibited the acetic acid-induced extravasation of Evan's blue dye in mice by approximately 20.6% (P<0.05), and was similar to the suppressive effect of ibuprofen (27.7%) as a positive control drug. Also, PGHB reduced the carrageenan-induced paw edema at 3h after oral administration, and suppressed the production of serum IL-6 in CIA mice. This suggests that PGHB has potential analgesic and anti-inflammatory activities, and will be the supporting evidence for the potential anti-rheumatoid activity of Korean P. ginseng-head.

Anti-inflammatory mechanisms of apigenin: inhibition of cyclooxygenase-2 expression, adhesion of monocytes to human umbilical vein endothelial cells, and expression of cellular adhesion molecules.

The aim of this study was to clarify the anti-inflammatory mechanism of apigenin. Apigenin inhibited the collagenase activity involved in rheumatoid arthritis (RA) and suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) production in a dose dependent manner in RAW 264.7 macrophage cells. Pretreatment with apigenin also attenuated LPS-induced cyclooxygenase-2 (COX-2) expression. In addition, apigenin profoundly reduced the tumor necrosis factor-alpha (TNF-alpha)-induced adhesion of monocytes to HUVEC monolayer. Apigenin significantly suppressed the TNF-alpha-stimulated upregulation of vascular cellular adhesion molecule-1 (VCAM-1)-, intracellular adhesion molecule-1 (ICAM-1)-, and E-selectin-mRNA to the basal levels. Taken together, these results suggest that apigenin has significant anti-inflammatory activity that involves blocking NO-mediated COX-2 expression and monocyte adherence. These results further suggest that apigenin may be useful for therapeutic management of inflammatory diseases.

Moyamoya disease associated with Behcet's disease.

A 32-year-old woman with Behcet's disease suffered repeated transient ischemic attacks (TIA) consisting of left hemiparesis. Cerebral angiography revealed the typical findings of moyamoya disease, with occlusion of the supraclinoid portion of both internal carotid arteries, coupled with abnormal collateral vessels. She underwent right superficial temporal artery to middle cerebral artery (STA-MCA) anastomosis and encephalomyosynangiosis, due to decreased reserve capacity demonstrated on acetazolamide single positron emission computed tomography (SPECT). Postoperatively, the TIA symptoms subsided. This is the first report of moyamoya disease associated with Behcet's disease, and moyamoya disease should be considered in the differential diagnosis of cerebrovascular events in patients with Behcet's disease. Revascularization surgery is recommended for the prevention of ischemic insults resulting in permanent deficits.

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.