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https://www.readbyqxmd.com/read/29791966/outcomes-of-bethesda-categories-iii-and-iv-thyroid-nodules-over-5-years-and-performance-of-the-afirma-gene-expression-classifier-a-single-institution-study
#1
Kelsi E Deaver, Bryan R Haugen, Nikita Pozdeyev, Carrie B Marshall
OBJECTIVE: The second edition Bethesda System for Reporting Thyroid Cytology estimates 6-18% malignancy rate of category III (B3) and 10-40% for category IV (B4) nodules; however, reported malignancy rates have considerable variability among institutions. Use of molecular classifiers (including Afirma Gene Expression Classifier, GEC) can be utilized in management of thyroid nodules. Our objective was to analyze malignancy rates of B3 and B4 nodules and determine clinical outcomes of GEC Benign nodules...
May 23, 2018: Clinical Endocrinology
https://www.readbyqxmd.com/read/29791835/nicotinic-cholinergic-receptors-in-vta-glutamate-neurons-modulate-excitatory-transmission
#2
Yijin Yan, Can Peng, Matthew C Arvin, Xiao-Tao Jin, Veronica J Kim, Matthew D Ramsey, Yong Wang, Sambashiva Banala, David L Wokosin, J Michael McIntosh, Luke D Lavis, Ryan M Drenan
Ventral tegmental area (VTA) glutamate neurons are important components of reward circuitry, but whether they are subject to cholinergic modulation is unknown. To study this, we used molecular, physiological, and photostimulation techniques to examine nicotinic acetylcholine receptors (nAChRs) in VTA glutamate neurons. Cells in the medial VTA, where glutamate neurons are enriched, are responsive to acetylcholine (ACh) released from cholinergic axons. VTA VGLUT2+ neurons express mRNA and protein subunits known to comprise heteromeric nAChRs...
May 22, 2018: Cell Reports
https://www.readbyqxmd.com/read/29790644/crispr-cas9-assisted-multiplex-genome-editing-technique-in-escherichia-coli
#3
Xu Feng, Dongdong Zhao, Xueli Zhang, Xiang Ding, Changhao Bi
Genome editing for site-specific chromosome modification is one of the most significant techniques in biological research. While conventional techniques usually deal with one genomic locus at a time, multiple genomic targets are often required to be modified to develop microbial cell factories. Thus, it is necessary to develop techniques for simultaneous editing of multiple loci. In this work, we developed a CRISPR/Cas9 assisted multiplex genome editing (CMGE) technique in Escherichia coli. With this editing method, all functional parts were assembled into replicable plasmids, and stringent inducible expression systems were used to control Cas9 gene expression, which was to decouple transformation from editing process to increase editing efficiency...
May 23, 2018: Biotechnology Journal
https://www.readbyqxmd.com/read/29790357/-control-editing-of-the-genome-within-reach-or-already-in-our-hands
#4
Radim Brdička, Radoslav Omelka
Although different genome editing tools have been around for decades, the recent emergence of cheap, quick, and accessible CRISPR/Cas9 technology has led to a revolution in this field. The technique has the potential to transform medicine from curative into preventive using a gene therapy. An application of genome editing has proven to be effective for both genetic and non-genetic (e.g. infectious) diseases. However, cancer and rare diseases treatment is at the forefront of interest. Concurrently, the legal and ethical frameworks should be discussed, especially as the technology moves towards a modification of the germ cells or embryos...
2018: Casopís Lékar̆ů C̆eských
https://www.readbyqxmd.com/read/29790280/cas9-versus-cas12a-cpf1-structure-function-comparisons-and-implications-for-genome-editing
#5
REVIEW
Daan C Swarts, Martin Jinek
Cas9 and Cas12a are multidomain CRISPR-associated nucleases that can be programmed with a guide RNA to bind and cleave complementary DNA targets. The guide RNA sequence can be varied, making these effector enzymes versatile tools for genome editing and gene regulation applications. While Cas9 is currently the best-characterized and most widely used nuclease for such purposes, Cas12a (previously named Cpf1) has recently emerged as an alternative for Cas9. Cas9 and Cas12a have distinct evolutionary origins and exhibit different structural architectures, resulting in distinct molecular mechanisms...
May 22, 2018: Wiley Interdisciplinary Reviews. RNA
https://www.readbyqxmd.com/read/29789579/endogenous-authentic-oct4a-proteins-directly-regulate-fos-ap-1-transcription-in-somatic-cancer-cells
#6
Yanwen Zhou, Xinyu Chen, Bo Kang, Shiqi She, Xiaobing Zhang, Cheng Chen, Wenxin Li, Wenjie Chen, Songsong Dan, Xiaoyun Pan, Xiaoli Liu, Jianqin He, Qingwei Zhao, Chenggang Zhu, Ling Peng, Haoyi Wang, Hangping Yao, Hongcui Cao, Lanjuan Li, Meenhard Herlyn, Ying-Jie Wang
OCT4A is well established as a master transcription factor for pluripotent stem cell (PSC) self-renewal and a pioneer factor for initiating somatic cell reprogramming, yet its presence and functionality in somatic cancer cells remain controversial and obscure. By combining the CRISPR-Cas9-based gene editing with highly specific PCR assays, highly sensitive immunoassays, and mass spectrometry, we provide unequivocal evidence here that full-length authentic OCT4A transcripts and proteins were both present in somatic cancer cells, and OCT4A proteins were heterogeneously expressed in the whole cell population and when expressed, they are predominantly localized in cell nucleus...
May 22, 2018: Cell Death & Disease
https://www.readbyqxmd.com/read/29789357/reactivation-of-%C3%AE-globin-in-adult-%C3%AE-yac-mice-after-ex-vivo-and-in-vivo-hematopoietic-stem-cell-genome-editing
#7
Chang Li, Nikoletta Psatha, Pavel Sova, Sucheol Gil, Hongjie Wang, Jiho Kim, Chandana Kulkarni, Cristina Valensisi, R David Hawkins, George Stamatoyannopoulos, André Lieber
Disorders involving β-globin gene mutations, mainly β-thalassemia and sickle cell disease, represent a major target for hematopoietic stem/progenitor cell (HSPC) gene therapy. This includes CRISPR/Cas9-mediated genome editing approaches in adult CD34+ cells aimed toward the re-activation of fetal γ-globin expression in red blood cells. Because models involving erythroid differentiation of CD34+ cells have limitations in assessing γ-globin re-activation, we focused on human β-globin locus transgenic (β-YAC) mice...
May 22, 2018: Blood
https://www.readbyqxmd.com/read/29787760/in-vivo-gene-correction-with-targeted-sequence-substitution-through-microhomology-mediated-end-joining
#8
Jeong Hong Shin, Soobin Jung, Suresh Ramakrishna, Hyongbum Henry Kim, Junwon Lee
Genome editing technology using programmable nucleases has rapidly evolved in recent years. The primary mechanism to achieve precise integration of a transgene is mainly based on homology-directed repair (HDR). However, an HDR-based genome-editing approach is less efficient than non-homologous end-joining (NHEJ). Recently, a microhomology-mediated end-joining (MMEJ)-based transgene integration approach was developed, showing feasibility both in vitro and in vivo. We expanded this method to achieve targeted sequence substitution (TSS) of mutated sequences with normal sequences using double-guide RNAs (gRNAs), and a donor template flanking the microhomologies and target sequence of the gRNAs in vitro and in vivo...
May 19, 2018: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29785946/-crispr-cas-systems-in-genome-engineering-of-bacteriophages
#9
Cai Jiao Liang, Fan Mei Meng, Yun Can Ai
Researches on CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) systems, that are adaptive immunity systems encoded by prokaryotes, have promoted the development of new genome-editing tools. Bacteriophages are not only the driving elements for the evolution of prokaryotes' CRISPR arrays, but also the targets of the CRISPR/Cas systems. Studies on functional genomics of bacteriophages have been lagging behind the discovery of new phage strains and the sequencing of their genomes...
May 20, 2018: Yi Chuan, Hereditas
https://www.readbyqxmd.com/read/29785489/trpc6-inactivation-confers-protection-in-a-model-of-severe-nephrosis-in-rats
#10
Eun Young Kim, Parisa Yazdizadeh Shotorbani, Stuart E Dryer
Mutations in canonical transient receptor potential-6 (TRPC6) channels give rise to rare familial forms of focal and segmental glomerulosclerosis (FSGS). Here we examined a possible role for TRPC6 in the progression of chronic puromycin aminonucleoside (PAN) nephrosis in Sprague-Dawley rats, a classic model of acquired nephrotic syndromes. We used CRISPR/Cas9 technology to delete a 239-bp region within exon 2 of the Trpc6 gene (Trpc6del allele). Trpc6del/del rats expressed detectable Trpc6 transcripts missing exon 2, and TRPC6 proteins could be detected by immunoblot of renal cortex...
May 22, 2018: Journal of Molecular Medicine: Official Organ of the "Gesellschaft Deutscher Naturforscher und Ärzte"
https://www.readbyqxmd.com/read/29785042/inducible-high-efficiency-crispr-cas9-targeted-gene-editing-and-precision-base-editing-in-african-trypanosomes
#11
Eva Rico, Laura Jeacock, Julie Kovářová, David Horn
The Cas9 endonuclease can be programmed by guide RNA to introduce sequence-specific breaks in genomic DNA. Thus, Cas9-based approaches present a range of novel options for genome manipulation and precision editing. African trypanosomes are parasites that cause lethal human and animal diseases. They also serve as models for studies on eukaryotic biology, including 'divergent' biology. Genome modification, exploiting the native homologous recombination machinery, has been important for studies on trypanosomes but often requires multiple rounds of transfection using selectable markers that integrate at low efficiency...
May 21, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29784797/mutations-in-a-subfamily-of-abscisic-acid-receptor-genes-promote-rice-growth-and-productivity
#12
Chunbo Miao, Lihong Xiao, Kai Hua, Changsong Zou, Yang Zhao, Ray A Bressan, Jian-Kang Zhu
Abscisic acid (ABA) is a key phytohormone that controls plant growth and stress responses. It is sensed by the pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/regulatory components of the ABA receptor (RCAR) family of proteins. Here, we utilized CRISPR/Cas9 technology to edit group I ( PYL1 - PYL6 and PYL12 ) and group II ( PYL7 - PYL11 and PYL13 ) PYL genes in rice. Characterization of the combinatorial mutants suggested that genes in group I have more important roles in stomatal movement, seed dormancy, and growth regulation than those in group II...
May 21, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29784672/genetic-screening-and-multipotency-in-rhesus-monkey-haploid-neural-progenitor-cells
#13
Haisong Wang, Wenhao Zhang, Jian Yu, Congyu Wu, Qian Gao, Xu Li, Yanni Li, Jinxin Zhang, Yaru Tian, Tao Tan, Weizhi Ji, Luyuan Li, Yang Yu, Ling Shuai
Haploid embryonic stem cells (haESCs) have been extensively applied in forward and reverse genetic screening. However, the mammalian haploid somatic cell line is difficult to achieve because of spontaneous diploidization in differentiation. As a non-human primate species, monkeys are widely used in basic and pre-clinical research in which haploid cells are restricted to ESCs. Here, we report that rhesus monkey haESCs in an optimized culture medium showed naïve-state pluripotency and stable haploidy. This model facilitated the derivation of haploid neural progenitor cells (haNPCs), which maintained haploidy and differentiation potential into neurons and glia for a long period in vitro High-throughput trapping mutations can be efficiently introduced into haNPCs via piggyBac transposons...
May 21, 2018: Development
https://www.readbyqxmd.com/read/29782018/novel-method-of-plasmid-dna-delivery-to-mouse-bladder-urothelium-by-electroporation
#14
Chuan Yu, Ofir Stefanson, Yueli Liu, Zhu A Wang
Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs...
May 3, 2018: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29781327/the-progress-of-aav-mediated-gene-therapy-in-neuromuscular-disorders
#15
Sara Aguti, Alberto Malerba, Haiyan Zhou
The well-defined genetic causes and monogenetic nature of many neuromuscular disorders, including Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA), present gene therapy as a prominent therapeutic approach. The novel variants of adeno-associated virus (AAV) can achieve satisfactory transduction efficiency of exogenous genes through the central nervous system and body-wide in skeletal muscle. Areas covered: In this review, we summarize the strategies of AAV gene therapy that are currently under preclinical and clinical evaluation for the treatment of degenerative neuromuscular disorders, with a focus on diseases such as DMD and SMA...
May 20, 2018: Expert Opinion on Biological Therapy
https://www.readbyqxmd.com/read/29780457/highly-efficient-base-editing-in-staphylococcus-aureus-using-an-engineered-crispr-rna-guided-cytidine-deaminase
#16
Tongnian Gu, Siqi Zhao, Yishuang Pi, Weizhong Chen, Chuanyuan Chen, Qian Liu, Min Li, Dali Han, Quanjiang Ji
Novel therapeutic means against Staphylococcus aureus infections are urgently needed due to the emergence of drug-resistant S. aureus . We report the development of a CRISPR RNA-guided cytidine deaminase (pnCasSA-BEC), enabling highly efficient gene inactivation and point mutations in S. aureus . We engineered a fusion of a Cas9 nickase (Cas9D10A) and a cytidine deaminase (APOBEC1) that can be guided to a target genomic locus for gene inactivation via generating a premature stop codon. The pnCasSA-BEC system nicks the non-edited strand of the genomic DNA, directly catalyzes the conversion of cytidine (C) to uridine (U), and relies on DNA replication to achieve C → T (G → A) conversion without using donor repair templates...
March 28, 2018: Chemical Science
https://www.readbyqxmd.com/read/29779890/high-throughput-screening-enhances-kidney-organoid-differentiation-from-human-pluripotent-stem-cells-and-enables-automated-multidimensional-phenotyping
#17
Stefan M Czerniecki, Nelly M Cruz, Jennifer L Harder, Rajasree Menon, James Annis, Edgar A Otto, Ramila E Gulieva, Laura V Islas, Yong Kyun Kim, Linh M Tran, Timothy J Martins, Jeffrey W Pippin, Hongxia Fu, Matthias Kretzler, Stuart J Shankland, Jonathan Himmelfarb, Randall T Moon, Neal Paragas, Benjamin S Freedman
Organoids derived from human pluripotent stem cells are a potentially powerful tool for high-throughput screening (HTS), but the complexity of organoid cultures poses a significant challenge for miniaturization and automation. Here, we present a fully automated, HTS-compatible platform for enhanced differentiation and phenotyping of human kidney organoids. The entire 21-day protocol, from plating to differentiation to analysis, can be performed automatically by liquid-handling robots, or alternatively by manual pipetting...
May 15, 2018: Cell Stem Cell
https://www.readbyqxmd.com/read/29779223/gene-editing-vectors-for-studying-nicotinic-acetylcholine-receptors-in-cholinergic-transmission
#18
Can Peng, Yijin Yan, Veronica J Kim, Staci E Engle, Jennifer N Berry, J Michael McIntosh, Rachael L Neve, Ryan M Drenan
Nicotinic acetylcholine receptors (nAChRs), prototype members of the cys-loop ligand gated ion channel family, are key mediators of cholinergic transmission in the central nervous system. Despite their importance, technical gaps exist in our ability to dissect the function of individual subunits in the brain. To overcome these barriers, we designed CRISPR/Cas9 small guide RNA sequences (sgRNAs) for production of loss-of-function alleles in mouse nAChR genes. These sgRNAs were validated in vitro via deep sequencing...
May 19, 2018: European Journal of Neuroscience
https://www.readbyqxmd.com/read/29779105/guide-rna-design-for-crispr-cas9-mediated-potato-genome-editing
#19
A V Khromov, V A Gushchin, V I Timerbaev, N O Kalinina, M E Taliansky, V V Makarov
The activity of the pool of sgRNA molecules designed for different regions of potato coilin and phytoene desaturase genes was compared in vitro. Due to the presence of nucleotides unpaired with DNA, sgRNA is able not only to inhibit but also to stimulate the activity of the Cas9-sgRNA complex in vitro. Although the first six nucleotides located in the DNA substrate proximally to the PAM site at the 3' end are the binding sites for cas9, they had no significant effect on the activity of the Cas9-sgRNA complex...
March 2018: Doklady. Biochemistry and Biophysics
https://www.readbyqxmd.com/read/29778643/inheritance-of-co-edited-genes-by-crispr-based-targeted-nucleotide-substitutions-in-rice
#20
Zenpei Shimatani, Ushio Fujikura, Hisaki Ishii, Yusuke Matsui, Minoru Suzuki, Yuki Ueke, Ken-Ichiro Taoka, Rie Terada, Keiji Nishida, Akihiko Kondo
The CRISPR/Cas9 system is a revolutionary genome-editing tool for directed gene editing in various organisms. Cas9 variants can be applied as molecular homing devices when combined with various functional effectors such as transcriptional activators or DNA modification enzymes. Target-AID is a synthetic complex of nuclease deficient Cas9 fused to an activation-induced cytidine deaminase (AID) that enables targeted nucleotide substitution (C to T or G to A). We previously demonstrated that the introduction of desired point mutations into target genes by Target-AID confers herbicide tolerance to rice callus...
April 25, 2018: Plant Physiology and Biochemistry: PPB
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