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Supplementary Material 1

Additional file 1. Additional Methods: The methods of qPCR and NGS assay. Additional Discussion: Evaluation of PCR step in CRISPR-CPPC using cfDNA from patients with NSCLC. Table S1: LOB and LOD of CRISPR-CPPC assay for T790M mutation. Table S2: Application of CRISPR-CPPC assay on patient samples. Table S3: Analytical performance of assays for detecting T790M mutation in cfDNA from patients with NSCLC presenting disease progression on 1st or 2nd generation EGFR-TKI. Table S4: Comparison of test results of qPCR to those of CRISPR-CPPC assay and ddPCR for EGFR T790M in cell-free plasma DNA. Table S5: Comparison of the test results of ddPCR to those of CRISPR-CPPC assay for EGFR T790M in cell-free plasma DNA. Table S6: Cases with different ddPCR and CRISPR-CPPC assay results. Table S7: Application of CRISPR-CPPC assay on follow-up patient samples. Table S8: Evaluation of PCR step in CRISPR-CPPC using cfDNA from patients with NSCLC. Fig S1: Study flow chart. Fig S2: Schematic illustration of the CRISPR target site around the human EGFR T790 locus. Fig S3: Distribution of T790M copies from sixty samples in this study.

Table S1. Genes included in the hereditary cancer panel. Table S2. Primers used in the MEMO-PCR to confirm low-level variants in APC. Table S3. Pathogenic or likely pathogenic germline APC variants in patients suspicious for familial adenomatous polyposis. Table S4. All variants identified from NGS hereditary cancer panel. (DOCX 58 kb)

This file includes the methods detecting pathogenic variants and lage deletion in this study; depth of coverage and method for detection of large insertion-deletion of exon using next-generation sequencing, and confirmation of deleterious mutations using Sanger sequencing or MLPA in four patients. (PDF 1477 kb)

This file includes two tables regarding baseline characteristics of study participants, possibly pathogenic variants and the classification according to ACMG guidelines mentied in the main manuscript. (DOCX 24 kb)

Water shortages are gradually accelerating because higher standards of living are required and water resources are more heavily utilized. Therefore, effective water treatment is necessary in order to retain the required qualify and amount of water. General treatment includes coagulation, flocculation, filtering, and disinfection. coagulation, flocculation, and disinfection are major components of water treatment processes. In this paper, a new automatic decision algorithm is proposed for coagulation. The proposed method shows how to determine the coagulant type and amount using data mining techniques.

While the cytogenetic and genetic characteristics of childhood acute lymphoblastic leukemias (ALL) are well studied, less clearly understood are the contributing epigenetic mechanisms that influence the leukemia phenotype. Our previous studies and others identified gene mutation (RAS) and DNA methylation (FHIT) to be associated with the most common cytogenetic subgroup of childhood ALL, high hyperdiploidy (having 5 more chromosomes). We screened DNA methylation profiles, using a genome-wide high dimension platform, of 166 childhood ALLs and 6 normal pre-B cell samples and observed a strong association of DNA methylation status at the PTPRG locus in human samples with levels of PTPRG gene expression as well as with RAS gene mutation status. In the 293 cell line, we found that PTPRG expression induces de-phosphorylation of ERK, a downstream RAS target which may be critical for mutant RAS-induced cell growth. In addition, PTPRG expression is up-regulated by RAS activation under DNA hypomethylating conditions. An element within the PTPRG promoter is bound by the RAS-responsive transcription factor RREB1, also under hypomethylating conditions. In conclusion, we provide evidence that DNA methylation of the PTPRG gene is a complementary event in oncogenesis induced by RAS mutations. Evidence for additional roles for PTPR family member genes is also suggested. This provides a potential therapeutic target for RAS-related leukemias as well as insight into childhood ALL etiology and pathophysiology.

Glioma is the most common cancer of the central nervous system but with few confirmed risk factors. It has been inversely associated with chicken pox, shingles and seroreactivity to varicella virus (VZV), as well as to allergies and allergy-associated IgE. The role of antibody reactivity against individual VZV antigens has not been assessed. Ten VZV-related proteins, selected for high immunogenicity or known function, were synthesized and used as targets for antibody measurements in the sera of 143 glioma cases and 131 healthy controls selected from the San Francisco Bay Area Adult Glioma Study. Glioma cases exhibited significantly reduced seroreactivity compared to controls for six antigens, including proteins IE63 [odds ratio (OR) = 0.26, 95% confidence interval (CI): 0.12-0.58, comparing lowest quartile to highest) and the VZV-unique protein ORF2p (OR = 0.44, 95% CI: 0.21-0.96, lowest quartile to highest). When stratifying the study population into those with low and high self-reported allergy history, VZV protein seroreactivity was only associated inversely with glioma among individuals self-reporting more than two allergies. The data provide insight into both allergy and VZV effects on glioma: strong anti-VZV reactions in highly allergic individuals are associated with reduced occurrence of glioma. This result suggests a role for specificity in the anti-VZV immunity in brain tumor suppression for both individual VZV antigens and in the fine-tuning of the immune response by allergy. Anti-VZV reactions may also be a biomarker of effective CNS immunosurveillance owing to the tropism of the virus.

The development of an in vitro culture system, comparable to the in vivo environment in terms of effects on oocyte growth and development, could provide a valuable experimental tool for studying the mechanisms governing oocyte development as well as practical clinical, agricultural, zoological, and biotechnological applications. This study reports on the importance of the microenvironment for the ovarian folliculogenesis process. The complexity of such a microenvironment was approached with a strategy based on functionalized PEG-hydrogels. The PEG matrix not only serves as a scaffold, but it is also used a reservoir of immobilized cues. Using tethered integrin-binding peptides in combination with other signaling factors, we aimed at better understanding the interactions of the oocyte and its surrounding granulosa that may determine the efficiency of the actual and the future in vitro mature oocyte production. In a first step, the mechanical properties of PEG-hydrogel were optimized for producing secondary follicles, in which the oocyte is surrounded by two layers of granulosa cells. Follicle growth was highly dependent on the mechanical properties of the surrounding environment, with the optimal elastic modulus being approx. 1 kPa. The effects of key soluble factors were also investigated to confirm their compatibility with the established 3-D culture system and to further qualitatively and quantitatively improve the produced mature oocytes. Accordingly, various combinations of the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were tested. Interestingly, the effects of the gonadotropins in the 3-D PEG system were close to their known in vivo effects. In conclusion, this study demonstrates the efficiency and the flexibility of a novel 3-D culture system, PEG-hydrogel. Circumventing problems inherent to the "on-plastic" standard culture, such as the loss of the granulosa-oocyte interactions, allowed the emergence of a culture system tailored for investigating fundamental folliculogenesis-related questions. Furthermore, the reported culture system may serve as a platform for developing clinical and biotechnology applications.

We present development and use of a 3D synthetic extracellular matrix (ECM) analog with integrin- specific adhesion ligands to characterize the microenvironmental influences in embryonic stem cell (ESC) self-renewal. Transcriptional analysis of 24 integrin subunits followed by confirmation at the trans- lational and functional levels suggested that integrins a5b1, avb5, a6b1 and a9b1 play important roles in maintenance of stemness in undifferentiated mouse ESCs. Using the well-defined matrix as a tool to activate integrins a5b1 plus avb5, a6b1 and a9b1, individually and in combination, differential integrin activation was demonstrated to exert exquisite control over ESC fate decisions. Simultaneous ligation of these four integrin heterodimers promoted self-renewal, as evidence by prolonged SSEA-1, Oct4 and Nanog expression, and induced Akt1 kinase signaling along with translational regulation of other stemness-related genes. The biofunctional network we have designed based on this knowledge may be useful as a defined niche for regulating ESC pluripotency through selective cell–matrix interactions, and the method we present may be more generally useful for probing matrix interactions in stem cell self- renewal and differentiation.

We engineered an acellular biomimetic microenvironment to regulate stem cell fate and applied it to maintain mouse embryonic stem (ES) cell self-renewal. In the 3D environment formed using hydrogel scaffolds in which specific integrin ligation was provided. Stat3 activation by exogenous leukemia inhibitory factor (LIF) no longer acted as a limiting factor for stem cell self-renewal. Instead, simultaneous stimulation of integrins alpha(5)beta(1), alpha(v)beta(5), alpha(6)beta(1) and alpha(9)beta(1) within the 3D scaffold greatly increased Akt1 and Smad 1/5/8 activation, which resulted in prolonged self-renewal of the ES cells. The ES cells exposed to the combined stimulation of the integrins for 4 wk in LIF-free 3D scaffolds maintained the spherical morphology of cell colonies without losing any activity of pluripotency. In conclusion, cell niche-specific integrin signaling within the 3D environment supported mouse ES cell self-renewal, and the resulting integrin signaling replaced Stat3 with Akt1 and Smad 1/5/8 as critical signals for mouse ES cell self-renewal. (c) 2012 Elsevier Ltd. All rights reserved.

We evaluated whether the genetic background of embryonic stem cells (ESCs) affects the properties suitable for three-dimensional (3D) synthetic scaffolds for cell self-renewal. Inbred R1 and hybrid B6D2F1 mouse ESC lines were cultured for 7 days in hydrogel scaffolds with different properties derived from conjugating 7.5, 10, 12.5, or 15% (wt/vol) vinylsulfone-functionalized three-, four-, or eight-arm polyethylene glycol (PEG) with dicysteine-containing crosslinkers with an intervening matrix metalloproteinase-specific cleavage sites. Cell proliferation and expression of self-renewal-related genes and proteins by ESCs cultured in feeder-free or containing 2D culture plate or 3D hydrogel were monitored. As a preliminary experiment, the E14 ESC-customized synthetic 3D microenvironment did not maintain self-renewal of either the R1 or B6D2F1 ESCs. The best R1 cell proliferation (10.04 vs. 0.16-4.39; p

This study was undertaken to examine how the softness of poly(ethylene) glycol (PEG)-based hydrogels, creating a three-dimensional (3D) microenvironment, influences the in vitro growth of mouse ovarian follicles. Early secondary, preantral follicles of 2week-old mice were cultured in a crosslinked four-arm PEG hydrogel. The hydrogel swelling ratio, which relates to softness, was modified within the range 25.7-15.5 by increasing the reactive PEG concentration in the precursor solution from 5% to 15% w/v, but it did not influence follicular growth to form the pseudoantrum (60-80%; p=0.76). Significant (p21.4 yielded better oocyte maturation than other levels, while the highest competence to develop pronuclear and blastocyst formation was detected at 20.6. In conclusion, gel softness, as reflected in swelling ratio, was one of the essential factors for supporting folliculogenesis in vivo within a hydrogel-based, 3D microenvironment. (c) 2014 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.