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Target sequencing

Neelanjan Mukherjee, Hans-Hermann Wessels, Svetlana Lebedeva, Marcin Sajek, Mahsa Ghanbari, Aitor Garzia, Alina Munteanu, Dilmurat Yusuf, Thalia Farazi, Jessica I Hoell, Kemal M Akat, Altuna Akalin, Thomas Tuschl, Uwe Ohler
RNA-binding proteins (RBPs) control and coordinate each stage in the life cycle of RNAs. Although in vivo binding sites of RBPs can now be determined genome-wide, most studies typically focused on individual RBPs. Here, we examined a large compendium of 114 high-quality transcriptome-wide in vivo RBP-RNA cross-linking interaction datasets generated by the same protocol in the same cell line and representing 64 distinct RBPs. Comparative analysis of categories of target RNA binding preference, sequence preference, and transcript region specificity was performed, and identified potential posttranscriptional regulatory modules, i...
December 5, 2018: Nucleic Acids Research
Arthur R Gorter de Vries, Lucas G F Couwenberg, Marcel van den Broek, Pilar de la Torre Cortés, Jolanda Ter Horst, Jack T Pronk, Jean-Marc G Daran
Targeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous genomes. However, genomes exhibiting polyploidy and/or high degrees of heterozygosity are less amenable to genetic modification. Here, we report an up to 99-fold lower gene editing efficiency when editing individual heterozygous loci in the yeast genome...
December 5, 2018: Nucleic Acids Research
Marius Gheorghe, Geir Kjetil Sandve, Aziz Khan, Jeanne Chèneby, Benoit Ballester, Anthony Mathelier
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most popular assay to identify genomic regions, called ChIP-seq peaks, that are bound in vivo by transcription factors (TFs). These regions are derived from direct TF-DNA interactions, indirect binding of the TF to the DNA (through a co-binding partner), nonspecific binding to the DNA, and noise/bias/artifacts. Delineating the bona fide direct TF-DNA interactions within the ChIP-seq peaks remains challenging. We developed a dedicated software, ChIP-eat, that combines computational TF binding models and ChIP-seq peaks to automatically predict direct TF-DNA interactions...
December 4, 2018: Nucleic Acids Research
Nikolaos Settas, Rebecca Persky, Fabio R Faucz, Nicole Sheanon, Antonis Voutetakis, Maya Lodish, Louise A Metherell, Constantine A Stratakis
Context: Multiple autosomal recessive genes have been etiologically linked to Primary Adrenal Insufficiency (PAI). Recently, SGPL1 gene mutations were recognized as causes of steroid-resistant nephrotic syndrome type 14 (NPHS14), a sphingolipidosis with multisystemic manifestations including PAI. Interestingly, this is the only monogenetic form of nephrotic syndrome (NS) and the sole sphingolipidosis causing adrenal disease. Objective: To understand if SGPL1 mutations are involved in the pathogenesis of PAI in patients who do not exhibit NS...
December 3, 2018: Journal of Clinical Endocrinology and Metabolism
Elena V Daoud, Megan Wachsmann, Timothy E Richardson, Divya Mella, Edward Pan, Anna Schwarzbach, Dwight Oliver, Kimmo J Hatanpaa
Pleomorphic xanthoastrocytoma (PXA) is a slow-growing neoplasm that predominantly affects the pediatric and young adult population. This neoplasm has a good prognosis, with a median 10-year survival rate of 70%. The majority of tumors are supratentorial and arise in the temporal lobe, while spinal tumors are extremely rare, with only 8 reported cases. Molecular perturbations involving the MAPK/ERK signaling pathway have been described in PXAs. The most common mutation is BRAF V600E in 60%-80% of cases. Other mechanisms activating this pathway in the absence of this mutation are rare and include CRAF (RAF1) fusion genes...
December 5, 2018: Journal of Neuropathology and Experimental Neurology
Takafumi Kawamura, Masayoshi Yamamoto, Katsunori Suzuki, Yuhi Suzuki, Megumu Kamishima, Mayu Sakata, Kiyotaka Kurachi, Mitsutoshi Setoh, Hiroyuki Konno, Hiroya Takeuchi
Background: Colitis-associated cancer (CAC) is one of the prognostic factors in inflammatory bowel disease (IBD), and prevention of CAC is a critical concern for patients with IBD. Component cells of the microenvironment, especially myofibroblasts, are known to affect tumor development, but the role of intestinal myofibroblasts (IMFs) in CAC has not been clarified. Here, we explored the role of IMFs in CAC and sought to identify candidate genes as novel therapeutic targets for the prevention of CAC...
December 4, 2018: Inflammatory Bowel Diseases
Indu Warrier, Nikhil Ram-Mohan, Zeyu Zhu, Ariana Hazery, Haley Echlin, Jason Rosch, Michelle M Meyer, Tim van Opijnen
Efficient and highly organized regulation of transcription is fundamental to an organism's ability to survive, proliferate, and quickly respond to its environment. Therefore, precise mapping of transcriptional units and understanding their regulation is crucial to determining how pathogenic bacteria cause disease and how they may be inhibited. In this study, we map the transcriptional landscape of the bacterial pathogen Streptococcus pneumoniae TIGR4 by applying a combination of high-throughput RNA-sequencing techniques...
December 5, 2018: PLoS Pathogens
Joel Pel, Amy Leung, Wendy W Y Choi, Milenko Despotovic, W Lloyd Ung, Gosuke Shibahara, Laura Gelinas, Andre Marziali
Targeted Next Generation Sequencing (NGS) is being adopted increasingly broadly in many research, commercial and clinical settings. Currently used target capture methods, however, typically require complex and lengthy (sometimes multi-day) workflows that complicates their use in certain applications. In addition, small panels for high sequencing depth applications such as liquid biopsy typically have low on-target rates, resulting in unnecessarily high sequencing cost. We have developed a novel targeted sequencing library preparation method, named Linked Target Capture (LTC), which replaces typical multi-day target capture workflows with a single-day, combined 'target-capture-PCR' workflow...
2018: PloS One
Gaurav Thareja, Sweety Mathew, Lisa S Mathew, Yasmin Ali Mohamoud, Karsten Suhre, Joel A Malek
Understanding the genetic diversity in a crop population is key to its targeted breeding for desired traits, such as higher yields, better fruit quality and resistance to disease and changing climates. Date fruits represent a major crop in the Middle East and are key to achieving future food independence in arid countries like Qatar. We previously determined the genome of the date palm Phoenix dactylifera and showed that date palm trees world-wide divide into two distinct subpopulations of Eastern and Western origins...
2018: PloS One
Vy T Duong, Megha H Unhelkar, John E Kelly, Suhn H Kim, Carter T Butts, Rachel W Martin
In plants, esterase/lipases perform transesterification reactions, playing an important role in the synthesis of useful molecules, such as those comprising the waxy coatings of leaf surfaces. Plant genomes and transcriptomes have provided a wealth of data about expression patterns and the circumstances under which these enzymes are upregulated, e.g. pathogen defense and response to drought; however, predicting their functional characteristics from genomic or transcriptome data is challenging due to weak sequence conservation among the diverse members of this group...
December 5, 2018: Integrative Biology: Quantitative Biosciences From Nano to Macro
Iryna Shakhmantsir, Soumyashant Nayak, Gregory R Grant, Amita Sehgal
Transcription-translation feedback loops that comprise eukaryotic circadian clocks rely upon temporal delays that separate the phase of active transcription of clock genes, such as Drosophila period (per) and timeless (tim), from negative feedback by the two proteins. However, our understanding of the mechanisms involved is incomplete. Through an RNA interference screen, we found that pre-mRNA processing 4 (PRP4) kinase, a component of the U4/U5.U6 triple small nuclear ribonucleoprotein (tri-snRNP) spliceosome, and other tri-snRNP components regulate cycling of the molecular clock as well as rest:activity rhythms...
December 5, 2018: ELife
Congshan Liu, Jiaqing Yao, Jianhai Yin, Jian Xue, Haobing Zhang
Echinococcosis, which causes a high disease burden and is of great public health significance, is caused by the larval stage of Echinococcus species. It has been suggested that tubulin is the target of benzimidazoles, the only drugs for the treatment of echinococcosis. This study evaluated the characteristics of tubulins from Echinococcus granulosus. The full-length cDNAs of E. granulosus α- and β-tubulin isoforms were cloned by reverse transcription PCR from protoscolex RNA. Then, these two tubulin isoforms (α9 and β4 ) were recombinantly expressed as insoluble inclusion bodies in Escherichia coli...
2018: Parasite: Journal de la Société Française de Parasitologie
Moran Amit, Shorook Na'ara, Eran Friedman, Euvgeni Vladovski, Tanya Wasserman, Neta Milman, Ziv Gil
Pancreatic ductal adenocarcinoma (PDA) remains a deadly disease, affecting about 40,000 individuals in the United States annually. We aimed to characterize the role of RET as a co-driver of pancreas tumorigenesis. To assess the role of RET as a co-driver of PDA; we generated a novel triple mutant transgenic mouse based on the cre-activated p53-R172H gene and a constitutively active RETM919T mutant (PRC). Survival analysis was performed using Kaplan-Meier analysis. Study of human PDA specimens and Pdx-1-Cre/KrasG12D /p53R172H (KPC) mice revealed that RET is upregulated during pancreas tumorigenesis, from inception through precursor lesions, to invasive cancer...
December 4, 2018: International Journal of Cancer. Journal International du Cancer
Wen-Yi Geng, Feng-Jiao Yao, Ting Tang, Shan-Shan Shi
The selection of a suitable reference gene is an important prerequisite for the precise analysis of target gene expression by real-time quantitative PCR (qPCR). The present study aims to explore the expression pattern of the Macrobrachium nipponense (M. nipponense) β-actin gene under Aeromonas hydrophila bacterial infection conditions. The complete sequence of the β-actin gene from M. nipponense was cloned by PCR. Identified and named β-actin genes were searched in the NCBI database, and the characteristics of the β-actin gene were analyzed using bioinformatics methods...
December 4, 2018: Molecular Biology Reports
Toshifumi Wakai, Pankaj Prasoon, Yuki Hirose, Yoshifumi Shimada, Hiroshi Ichikawa, Masayuki Nagahashi
Numerous technical and functional advances in next-generation sequencing (NGS) have led to the adoption of this technique in conventional clinical practice. Recently, large-scale genomic research and NGS technological innovation have revealed many more details of somatic and germline mutations in solid tumors. This development is allowing for the classification of tumor type sub-categories based on genetic alterations in solid tumors, and based on this information, new drugs and targeted therapies are being administered to patients...
December 4, 2018: International Journal of Clinical Oncology
Syed Shoeb Razvi, Hani Choudhry, Mohammed Nihal Hasan, Mohammed A Hassan, Said Salama Moselhy, Khalid Omer Abualnaja, Mazin A Zamzami, Taha Abduallah Kumosani, Abdulrahman Labeed Al-Malki, Majed A Halwani, Abdulkhaleg Ibrahim, Ali Hamiche, Christian Bronner, Tadao Asami, Mahmoud Alhosin
Natural polyamines such as putrescine, spermidine, and spermine are crucial in the cell proliferation and maintenance in all the eukaryotes. However, the requirement of polyamines in tumor cells is stepped up to maintain tumorigenicity. Many synthetic polyamine analogues have been designed recently to target the polyamine metabolism in tumors to induce apoptosis. N4 -Erucoyl spermidine (designed as N4 -Eru), a novel acylspermidine derivative, has been shown to exert selective inhibitory effects on both hematological and solid tumors, but its mechanisms of action are unknown...
2018: Epigenetics insights
Judith M Martin, Raghunandan Avula, Mary Patricia Nowalk, Chyongchiou Jeng Lin, William T Horne, Uma R Chandran, Jennifer P Nagg, Richard K Zimmerman, Kelly S Cole, John F Alcorn
Background: The reasons for differences in vaccine effectiveness between live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine (IIV) are not clear. Methods: Blood samples were obtained before vaccination and at days 7 and 21 postvaccination with 2015-2016 quadrivalent IIV or LAIV. Serologic response to the vaccine was measured by hemagglutination inhibition assay. Targeted RNA sequencing and serum cytokine analysis were performed. Paired analyses were used to determine gene expression and were compared between IIV and LAIV recipients...
November 2018: Open Forum Infectious Diseases
Zhen-Juan Yin, Xiao-Lin Dong, Kui Kang, Hao Chen, Xiao-Yan Dai, Guang-An Wu, Li Zheng, Yi Yu, Yi-Fan Zhai
Diapause is a complex physiological adaptation phenotype, and the transcription factor Forkhead-box O (FoxO) is a prime candidate for activating many of its diverse regulatory signaling pathways. Hormone signaling regulates nymphal diapause in Laodelphax striatellus . Here, the function of the FoxO gene isolated from L. striatellus was investigated. After knocking-down LsFoxO in diapausal nymphs using RNA interference, the titers of juvenile hormone III and some cold-tolerance substances decreased significantly, and the duration of the nymphal developmental period was severely shorted to 25...
2018: Frontiers in Physiology
Hongbo Zhao, Yutang Huang, Jingjing Shi, Yi Dai, Lanxiang Wu, Honghao Zhou
Gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (EGFR-TKI), is used clinically as first-line therapy in patients with advanced non-small cell lung cancer (NSCLC) with EGFR activating mutations, but the inevitable development of acquired resistance limits its efficacy. In up to 30-40% of NSCLC cases, the mechanism underlying acquired resistance remains unknown. ATP-binding cassette (ABC) transporters are a family of membrane proteins that can significantly influence the bioavailability of numerous drugs, and have confirmed to play an essential role in multidrug resistance (MDR) in cancer chemotherapy...
2018: Frontiers in Pharmacology
Michael Bartoschek, Nikolay Oskolkov, Matteo Bocci, John Lövrot, Christer Larsson, Mikael Sommarin, Chris D Madsen, David Lindgren, Gyula Pekar, Göran Karlsson, Markus Ringnér, Jonas Bergh, Åsa Björklund, Kristian Pietras
Cancer-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment, although their origin and roles in shaping disease initiation, progression and treatment response remain unclear due to significant heterogeneity. Here, following a negative selection strategy combined with single-cell RNA sequencing of 768 transcriptomes of mesenchymal cells from a genetically engineered mouse model of breast cancer, we define three distinct subpopulations of CAFs. Validation at the transcriptional and protein level in several experimental models of cancer and human tumors reveal spatial separation of the CAF subclasses attributable to different origins, including the peri-vascular niche, the mammary fat pad and the transformed epithelium...
December 4, 2018: Nature Communications
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