Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth.

KRAS is the most frequently mutated oncogene in human tumors, and its activating mutations represent important therapeutic targets. The combination of Cas9 and guide RNA from the CRISPR-Cas system recognizes a specific DNA sequence and makes a double-strand break, which enables editing of the relevant genes. Here, we harnessed CRISPR to specifically target mutant KRAS alleles in cancer cells. We screened guide RNAs using a reporter system and validated them in cancer cells after lentiviral delivery of Cas9 and guide RNA. The survival, proliferation, and tumorigenicity of cancer cells in vitro and the growth of tumors in vivo were determined after delivery of Cas9 and guide RNA. We identified guide RNAs that efficiently target mutant KRAS without significant alterations of the wild-type allele. Doxycycline-inducible expression of this guide RNA in KRAS-mutant cancer cells transduced with a lentiviral vector encoding Cas9 disrupted the mutant KRAS gene, leading to inhibition of cancer cell proliferation both in vitro and in vivo. Intra-tumoral injection of lentivirus and adeno-associated virus expressing Cas9 and sgRNA suppressed tumor growth in vivo, albeit incompletely, in immunodeficient mice. Expression of Cas9 and the guide RNA in cells containing wild-type KRAS did not alter cell survival or proliferation either in vitro and in vivo. Our study provides a proof-of-concept that CRISPR can be utilized to target driver mutations of cancers in vitro and in vivo.

Active Compound of Pharbitis Semen (Pharbitis nil Seeds) Suppressed KRAS-Driven Colorectal Cancer and Restored Muscle Cell Function during Cancer Progression.

Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven colorectal cancer (CRC) is notorious to target with drugs and has shown ineffective treatment response. The seeds of Pharbitis nil, also known as morning glory, have been used as traditional medicine in East Asia. We focused on whether Pharbitis nil seeds have a suppressive effect on mutated KRAS-driven CRC as well as reserving muscle cell functions during CRC progression. Seeds of Pharbitis nil (Pharbitis semen) were separated by chromatography and the active compound of Pharbitis semen (PN) was purified by HPLC. The compound PN efficiently suppressed the proliferation of mutated KRAS-driven CRC cells and their clonogenic potentials in a concentration-dependent manner. It also induced apoptosis of SW480 human colon cancer cells and cell cycle arrest at the G2/M phase. The CRC related pathways, including RAS/ERK and AKT/mTOR, were assessed and PN reduced the phosphorylation of AKT and mTOR. Furthermore, PN preserved muscle cell proliferation and myotube formation in cancer conditioned media. In summary, PN significantly suppressed mutated KRAS-driven cell growth and reserved muscle cell function. Based on the current study, PN could be considered as a promising starting point for the development of a nature-derived drug against KRAS-mutated CRC progression.

Genome editing: a robust technology for human stem cells.

Human pluripotent stem cells comprise induced pluripotent and embryonic stem cells, which have tremendous potential for biological and therapeutic applications. The development of efficient technologies for the targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. Genome editing of stem cells is possible with the help of synthetic nucleases that facilitate site-specific modification of a gene of interest. Recent advances in genome editing techniques have improved the efficiency and speed of the development of stem cells for human disease models. Zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system are powerful tools for editing DNA at specific loci. Here, we discuss recent technological advances in genome editing with site-specific nucleases in human stem cells.

Sensing Properties of ZnO Nanoparticles for Detection of 2-Chloroethyl Ethyl Sulfide as a Mustard Simulant.

In this work, we present the fabrication and characterization of a 2-chloroethyl ethyl sulfide (2-CEES) gas sensor based on ZnO nanoparticles (NPs) synthesized by a hydrothermal method. We confirmed that synthesized ZnO NPs adopt a polycrystalline phase. Partially aggregated ZnO-NPs revealed spherical or ellipsoidal nanocrystalline particles in a size range of 30-50 nm, as observed by field-emission scanning electron microscopy (FE-SEM). The maximum response of the ZnO NPs was 15 at 1 ppm 2-CEES concentration, and a low detection limit of 0.4 ppm was observed at an optimal operating temperature of 250 °C. The lowest response time was 6 s in 20 ppm at 250 °C. The linearity response with correlation coefficient (R2) was 0.9887 at 2-CEE concentrations of 0.4-1 ppm at the operating temperature of 250 °C. The enhanced sensing performance and a decrease in the operating temperature were attributed to a high specific surface area and more active sites in the ZnO NPs after exposure to 2-CEES.

Pediococcus pentosaceus-Fermented Cordyceps militaris Inhibits Inflammatory Reactions and Alleviates Contact Dermatitis.

Cordyceps militaris is a medicinal mushroom used to treat immune-related diseases in East Asia. We investigated the anti-inflammatory effect of the extract of C. militaris grown on germinated Rhynchosia nulubilis (GRC) fermented with Pediococcus pentosaceus ON89A isolated from onion (GRC-ON89A) in vivo as well as in vitro. The anti-inflammatory effect of GRC-ON89A was investigated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The total polyphenol content (TPC) and total flavonoid content (TFC) in the GRC-ON89A ethanol extract were significantly increased compared to that in GRC. GRC-ON89A hexane fraction (GRC-ON89A-Hex) inhibited the release of nitric oxide (NO) compared to that of the LPS-treated control without cytotoxicity in LPS-stimulated RAW 264.7 macrophages. GRC-ON89A-Hex decreased the inducible NO synthase (iNOS), cyclooxygenase 2 (COX2), and tumor necrosis factor (TNF)-α mRNA expression in LPS-stimulated RAW 264.7 macrophages. In addition, pre-treatment with GRC-ON89A-Hex significantly inhibited LPS-stimulated phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB. To induce allergic contact dermatitis (ACD), 1-fluoro-2, 4-dinitrofluorobenzene (DNFB) was applied to the surface of the right ears of C57BL/6N mice. GRC-ON89A reduced the ear swelling and thickness in DNFB-induced ACD mice. This study demonstrates the potential usefulness of GRC-ON89A as an anti-inflammatory dietary supplement or drug.

Phellinus linteus Grown on Germinated Brown Rice Increases Cetuximab Sensitivity of KRAS-Mutated Colon Cancer.

Colon cancer is one of the most common types of cancer, and it has recently become a leading cause of death worldwide. Among colon cancers, the v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated form is notorious for its non-druggable features. Cetuximab, a monoclonal antibody that binds to the epidermal growth factor receptor, has been introduced as an antitumor therapy; however, secondary resistance and side effects significantly limit its effective use in these cancers. In this study, we prepared Phellinuslinteus on germinated brown rice (PBR) extracts to increase the sensitivity of KRAS-mutated colon cancers to cetuximab. The combined treatment of PBR extract and cetuximab suppressed SW480 cell viability/proliferation, with the cells exhibiting altered cellular morphology and clonogenic potential. AnnexinV-fluorescein isothiocyanate/propidium iodide-stained flow cytometry and Western blotting were performed, and PBR extract combined with cetuximab treatment increased apoptosis of the SW480 cells and suppressed their KRAS protein expression. The potential of PBR as a synergistic anticancer agent was further investigated in a tumor-xenografted mouse model. Tumor growth was significantly suppressed with PBR extract and cetuximab co-treatment. In conclusion, PBR increased the sensitivity of KRAS-mutated colon cancer cells to cetuximab, which indicates the potential use of PBR as a medical food against colon cancer.

Evaluation of Surface Cleaning Procedures in Terms of Gas Sensing Properties of Spray-Deposited CNT Film: Thermal- and O2 Plasma Treatments.

The effect of cleaning the surface of single-walled carbon nanotube (SWNT) networks by thermal and the O2 plasma treatments is presented in terms of NH3 gas sensing characteristics. The goal of this work is to determine the relationship between the physicochemical properties of the cleaned surface (including the chemical composition, crystal structure, hydrophilicity, and impurity content) and the sensitivity of the SWNT network films to NH3 gas. The SWNT networks are spray-deposited on pre-patterned Pt electrodes, and are further functionalized by heating on a programmable hot plate or by O2 plasma treatment in a laboratory-prepared plasma chamber. Cyclic voltammetry was employed to semi-quantitatively evaluate each surface state of various plasma-treated SWNT-based electrodes. The results show that O2 plasma treatment can more effectively modify the SWNT network surface than thermal cleaning, and can provide a better conductive network surface due to the larger number of carbonyl/carboxyl groups, enabling a faster electron transfer rate, even though both the thermal cleaning and the O2 plasma cleaning methods can eliminate the organic solvent residues from the network surface. The NH3 sensors based on the O2 plasma-treated SWNT network exhibit higher sensitivity, shorter response time, and better recovery of the initial resistance than those prepared employing the thermally-cleaned SWNT networks.

Paternal Somatic Mosaicism of a Novel Frameshift Mutation in ELANE Causing Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) is a bone marrow failure disease with an autosomal dominant inheritance from mutations in ELANE. Here, we report a 7-week-old Korean male with SCN. His elder sister died from pneumonia at 2 years. Direct sequencing of ELANE in the proband identified a heterozygous novel frameshift mutation: c.658delC (p.Arg220Glyfs20*). Family study involving his asymptomatic parents with normal cell counts revealed that his father had the same mutation, but at a lower burden than expected in a typical heterozygous state. Further molecular investigation demonstrated somatic mosaicism with ~18% mutant alleles. We concluded the proband inherited the mutation from his somatic mosaic father.

CB307: A Dual Targeting Costimulatory Humabody VH Therapeutic for Treating PSMA-Positive Tumors.

PURPOSE: CD137 is a T- and NK-cell costimulatory receptor involved in consolidating immunologic responses. The potent CD137 agonist urelumab has shown clinical promise as a cancer immunotherapeutic but development has been hampered by on-target off-tumor toxicities. A CD137 agonist targeted to the prostate-specific membrane antigen (PSMA), frequently and highly expressed on castration-resistant metastatic prostate cancer (mCRPC) tumor cells, could bring effective immunotherapy to this immunologically challenging to address disease. EXPERIMENTAL DESIGN: We designed and manufactured CB307, a novel half-life extended bispecific costimulatory Humabody VH therapeutic to elicit CD137 agonism exclusively in a PSMA-high tumor microenvironment (TME). The functional activity of CB307 was assessed in cell-based assays and in syngeneic mouse antitumor pharmacology studies. Nonclinical toxicology and toxicokinetic properties of CB307 were assessed in a good laboratory practice (GLP) compliant study in cynomolgus macaques. RESULTS: CB307 provides effective CD137 agonism in a PSMA-dependent manner, with antitumor activity both in vitro and in vivo, and additional activity when combined with checkpoint inhibitors. A validated novel PSMA/CD137 IHC assay demonstrated a higher prevalence of CD137-positive cells in the PSMA-expressing human mCRPC TME with respect to primary lesions. CB307 did not show substantial toxicity in nonhuman primates and exhibited a plasma half-life supporting weekly clinical administration. CONCLUSIONS: CB307 is a first-in-class immunotherapeutic that triggers potent PSMA-dependent T-cell activation, thereby alleviating toxicologic concerns against unrestricted CD137 agonism.

Dispersion of nanodiamond on chemical mechanical polishing performance for Ge1Sb6Te3 film.

This study describes the effect of surfactant concentration on the chemical mechanical polishing process of Ge1Sb6Te3 film using nanodiamond-based slurry. Aggregated diamond nanoparticles were dispersed in a slurry solution containing anionic poly(sodium 4-styrene sulfonate) using milling system. The zeta-potential, particle size and transmission electron microscopy image of the dispersed nanodiamond particles were analyzed for slurries having varying surfactant concentrations to identify the effect of the surfactant concentration on the milling process. The cationic nanodiamond particles were covered with the anionic poly(sodium 4-styrene sulfonate) polymer, and the polymer acted as a dispersion agent on account of the electrostatic repulsion. By increasing the surfactant concentration in the milling process, the average particle size of the nanodiamond particle decreased until the concentration reached 0.9 wt%. In addition, the surface roughness and material removal rate of the Ge1Sb6Te3 film in the polishing process strongly-depended on the surfactant concentration. Both surface roughness and material removal rate decreased with an increase in the surfactant concentration. Excess poly(sodium 4-styrene sulfonate) acted as a passivation layer, resulting in a decrease in the surface roughness and material removal rate of the Ge1Sb6Te3 film.

Analysis of impacts of exogenous pollutant bisphenol-A penetration on soybeans roots and their biological growth.

Bisphenol A (BPA) is a common chemical used in plastic production. BPA, which has the potential to be poisonous to plants, has lately emerged as a serious environmental concern owing to its extensive usage and release patterns. Prior study has only looked at how BPA affects plants up to a certain stage in their growth. The precise mechanism of toxicity, penetration of BPA, and damage to internal root tissues remains unknown. Therefore, the goal of this study was to examine the hypothesized mechanism for BPA-induced root cells by studying the effects of bisphenol A (BPA) on the ultrastructure and function of root tip cells of soybean plants. We looked at plant changes in root cell tissues after BPA exposure. Further, the biological characteristics that responded to BPA stress were investigated, and the accumulation of BPA in the root, stem, and leaf of the soybean plant was systematically investigated by using FTIR and SEM analysis. The uptake of BPA is a key internal factor that contributes to changes in biological characteristics. Our findings provide insight into how BPA could alter plant root growth, which might contribute new knowledge toward a better scientific appraisal of the possible dangers of BPA exposure for plants.

Estimation of carrier frequencies of six autosomal-recessive Mendelian disorders in the Korean population.

Although many studies have been performed to identify mutations in Korean patients with various autosomal-recessive Mendelian disorders (AR-MDs), little is known about the carrier frequencies of AR-MDs in the Korean population. Twenty common mutations from six AR-MDs, including Wilson disease (WD), non-syndromic hearing loss (NSHL), glycogen storage disease type Ia (GSD Ia), phenylketonuria (PKU), congenital hypothyroidism (CH), and congenital lipoid adrenal hyperplasia (CLAH) were selected to screen for based on previous studies. A total of 3057 Koreans were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry followed by confirmation using the Sanger sequencing. We found 201 and 8 carriers with either one or two mutations in different genes, respectively, yielding a total carrier frequency of 1 in 15 (6.7%). Of the six AR-MDs, NSHL has the highest carrier frequency followed by WD, CH, CLAH, GSD Ia, and PKU. As carrier screening tests are becoming prevalent and the number of mutations known and tested is rising, a priori data on the carrier frequencies in different ethnic groups is mandatory to plan a population screening program and to estimate its efficiency. In light of this, the present results can be used as a basis to establish a screening policy for common AR-MRs in the Korean population.

Heterogeneous lengths of copy number mutations in human coagulopathy revealed by genome-wide high-density SNP array.

BACKGROUND: The recent advent of genome-wide molecular platforms has facilitated our understanding of the human genome and disease, particularly copy number aberrations. We performed genome-wide single nucleotide polymorphism-array in hereditary coagulopathy to delineate the extent of copy number mutations and to assess its diagnostic utility. DESIGN AND METHODS: The study subjects were 17 patients with hereditary coagulopathy from copy number mutations in coagulation genes detected by multiple ligation-dependent probe amplification. Eleven had hemophilia (7 hemophilia A and 4 hemophilia B) and 6 had thrombophilia (4 protein S deficiency and 2 antithrombin deficiency). Single nucleotide polymorphism-array experiments were performed using Affymetrix Genome-Wide Human SNP arrays 6.0. RESULTS: Copy number mutations were identified by single nucleotide polymorphism-array in 9 patients, which ranged in length from 51 Kb to 6,288 Kb harboring 2 to ~160 genes. Single nucleotide polymorphism-array showed a neutral copy number status in 8 patients including 7 with either a single-exon copy number mutation or duplication mutations of PROS1. CONCLUSIONS: This study revealed unexpectedly heterogeneous lengths of copy number mutations underlying human coagulopathy. Single nucleotide polymorphism-array had limitations in detecting copy number mutations involving a single exon or those of a gene with homologous sequences such as a pseudogene.

A novel initiation codon mutation in the ribosomal protein S17 gene (RPS17) in a patient with Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by pure red cell aplasia, various congenital anomalies, and cancer predisposition. We report a novel mutation in the RPS17 gene in a Korean patient with DBA. The mutation occurred in the translation initiation codon, changing Atg to Gtg (c.1A>G), thus disrupting the natural start of the RPS17 protein biosynthesis. This is the third case of DBA from a RPS17 mutation in the literature and is the second case of a RPS17 mutation in the translation initiation codon, following c.2T>G.

Direct deposition of patterned nanocrystalline CVD diamond using an electrostatic self-assembly method with nanodiamond particles.

Micron-sized and precise patterns of nanocrystalline CVD diamond were fabricated successfully on substrates using dispersed nanodiamond particles, charge connection by electrostatic self-assembly, and photolithography processes. Nanodiamond particles which had been dispersed using an attritional milling system were attached electrostatically on substrates as nuclei for diamond growth. In this milling process, poly sodium 4-styrene sulfonate (PSS) was added as an anionic dispersion agent to produce the PSS/nanodiamond conjugates. Ultra dispersed nanodiamond particles with a ζ-potential and average particle size of - 60.5 mV and ∼ 15 nm, respectively, were obtained after this milling process. These PSS/nanodiamond conjugates were attached electrostatically to a cationic polyethyleneimine (PEI) coated surface on to which a photoresist had been patterned in an aqueous solution of the PSS/nanodiamond conjugated suspension. A selectively seeded area was formed successfully using the above process. A hot filament chemical vapor deposition system was used to synthesize the nanocrystalline CVD diamond on the seeded area. Micron-sized, thin and precise nanocrystalline CVD diamond patterns with a high nucleation density (3.8 ± 0.4 × 10(11) cm(-2)) and smooth surface were consequently fabricated.

The common NF-κB essential modulator (NEMO) gene rearrangement in Korean patients with incontinentia pigmenti.

Incontinentia pigmenti (IP) is a rare X-linked dominant disorder characterized by highly variable abnormalities of the skin, eyes and central nervous system. A mutation of the nuclear factor-κB essential modulator (NEMO) located at Xq28 is believed to play a role in pathogenesis and the mutation occurs mostly in female patients due to fatal consequence of the mutation in males in utero. This study was designed to identify the common NEMO rearrangement in four Korean patients with IP. Deletion of exons 4 to 10 in the NEMO, the most common mutation in IP patients, was detected in all of the patients by the use of long-range PCR analysis. This method enabled us to discriminate between NEMO and pseudogene rearrangements. Furthermore, all of the patients showed skewed XCI patterns, indicating pathogenicity of IP was due to cells carrying the mutant X chromosome. This is the first report of genetically confirmed cases of IP in Korea.

Mesenchymal Stem Cell Therapy and Alzheimer's Disease: Current Status and Future Perspectives.

Alzheimer's disease (AD) is the most common progressive neurodegenerative disease worldwide, but its cause remains unclear. Although a few drugs can provide temporary and partial relief of symptoms in some patients, no curative treatment is available. Therefore, attention has been focused on research using stem cells to treat AD. Among stem cells, mesenchymal stem cells (MSCs) have been used to treat the related pathologies in animal models of AD, and other neurodegenerative disease. This review describes latest research trends on the use of MSC-based therapies in AD and its action of mechanism. MSCs have several beneficial effects. They would be specified as the reduction of neuroinflammation, the elimination of amyloid-ß, neurofibrillary tangles, and abnormal protein degradation, the promotion of autophagy-associated and blood-brain barrier recoveries, the upregulation of acetylcholine levels, improved cognition, and the recovery of mitochondrial transport. Therefore, this review describes the latest research trends in MSC-based therapy for AD by demonstrating the importance of MSC-based therapy and understanding of its mechanisms in AD and discusses the limitations and perspectives of stem cell therapy in AD.

Synthesis of hierarchically structured ɤ-Fe2O3-PPy nanocomposite as effective adsorbent for cationic dye removal from wastewater.

Industrial dye effluents, which are a major wastage component that enter the natural environment, pose a significant health risk to human and aquatic life. Therefore, the effective removal of dye effluents is a major concern. Against this backdrop, in this study, a low-cost, earth-abundant, and ecofriendly ɤ-Fe2O3-PPy nanocomposite was prepared employing the conventional hydrothermal method. The morphology, functional groups, and elemental composition of ɤ-Fe2O3-PPy were characterized by XRD, SEM, XPS, and FTIR studies. Under optimized conditions, the prepared novel ɤ-Fe2O3-PPy nanocomposite showed a high methylene blue (MB) adsorption capacity of 464 mg/g, which is significantly higher than that of existing adsorbents such as CNTs and polymer-modified CNTs. The adsorption parameters such as pH, adsorbent dosage, and ionic strength were optimized to enhance the MB adsorption capacity. The adsorption results revealed that MB is adsorbed onto the adsorbent surface via electrostatic interactions, hydrogen bonding, and chemical binding interactions. In terms of practical application, the adsorbent's adsorption-desorption ability in conjunction with magnetic separation was investigated; the prepared ɤ-Fe2O3-PPy nanocomposite exhibited excellent adsorption and desorption efficiencies over more than seven adsorption-desorption cycles.

New Analytical Approach for The Determination of Calcium Phosphate Dibasic and Tribasic in Processed Food by Comparison of Ion Chromatography With High-Performance Liquid Chromatography.

An analytical method to measure solubilized orthophosphate ions (HPO42- and PO43- ) from the water-insoluble food additives calcium phosphate dibasic (DCP) and calcium phosphate tribasic (TCP) in processed foods was optimized by comparing ion chromatography (IC) coupled with DS6 conductivity detector (Cond.) and high-performance liquid chromatography (HPLC) with Evaporative light scattering detector (ELSD) methods. The ion-pairing HPLC method could analyze calcium and phosphate ions successively. However, this method exhibited low reproducibility after approximately 48 hours of measurements. The IC method was established as an effective method of measuring orthophosphate ions with high reproducibility using distilled water and KOH solution as the mobile phase with a Dionex column. Matrix-based limit of detections (LOD) and limit of quantifications (LOQ) for snacks and cereals were estimated in the range of 0.01-0.91 µg/mL and 0.21-2.74 µg/mL, respectively. In inter-day and intra-day tests, the calculated precision (%RSD) and accuracy (recovery %) ranged from 0.5% to 6.6% and 82% to 117%, respectively, in both food samples. The levels of DCP or TCP could be analyzed in various positive food samples, and the developed IC method demonstrated good applicability in the analysis of DCP and TCP in collected processed foods.

An electrochemical biosensor array for rapid detection of alanine aminotransferase and aspartate aminotransferase.

An increment of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) in human serum indicates an abnormal symptom of the liver. Hence, an electrochemical biosensor array that uses micro electro mechanical systems technology is required for rapid and integrated measurement of ALT/AST. Here we describe a biosensor array consisting of two glutamate sensors. It turned out that porous silicon layers formed on each working electrode were useful to increase the effective surface area. This biosensor array was constructed with platinum electrodes and a polydimethylsiloxane (PDMS) microchannel. Electrodes in sampling wells minimized a cross-interference effect and permitted multiple sampling by immobilization with glutamate oxidase using a silanization technique. The device sensitivities derived from semi-logarithmic plots were 0.145 microA/(U/l) for ALT and 0.463 microA/(U/l) for AST over a range of 1.3 U/l to 250 U/l. Hene, this ALT/AST biosensor array can be applied in diagnostic and home use.