Enhancing recombinant protein and viral vector production in mammalian cells by targeting the YTHDF readers of N6–methyladenosine in Mrna

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Controlling insect pests with bacterial genes.

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Rolling cycle translation of circularized infinite open reading frames; fooling the ribosome

Poster Number 156 ROLLING CYCLE TRANSLATION OF CIRCULARIZED INFINITE OPEN READING FRAMES; FOOLING THE RIBOSOME Alan Costello, National Institute for Cellular Biotechnology, Dublin City University [email protected] Nga Lao, National Institute for Cellular Biotechnology, Dublin City University Niall Barron, National Institute for Bioprocessing Research and Training, University College Dublin Martin Clynes, National Institute for Cellular Biotechnology, Dublin City University Key Words: Circular RNA, Translation, RNA structure, Cell engineering, Chinese Hamster Ovary (CHO) cell Recent. Please click Additional Files below to see the full abstract

miR-CATCH identifies biologically active miRNA regulators of the pro-survival gene XIAP, in Chinese hamster ovary cells

Genetic engineering of mammalian cells, in particular Chinese hamster ovary (CHO) cells, is of critical interest to the biopharmaceutical industry as a means to further boost the yields of therapeutic proteins. Complimentary to already in place advanced bioprocesses, stable overexpression of the pro-survival X-linked inhibitor of apoptosis (XIAP) is one example of the successful manipulation of CHO cell genetics resulting in prolonged culture survival, ultimately increasing recombinant protein productivity. However, saturation or burdening of the cells translational machinery can occur in instances of forced expression of a trans-gene thereby achieving the anticipated cellular phenotype without the associated improvement in productivity. Ribosomal footprint sequencing has demonstrated that ~15% of an IgG-producing CHO cell translatome is occupied by the Neomycin selection marker. microRNAs (miRNAs) have the ability to fine tune endogenous gene expression thereby achieving elevated gene levels without the excess that could negatively impact global gene expression. Additionally, not only does a single miRNA have the capacity to regulate multiple mRNA transcripts simultaneously but individual mRNAs can be regulated by a multitude of miRNAs at the post-transcriptional level. This can facilitate the maximal translation of an endogenous gene without surpassing the superphysiological threshold associated with diminished productivity. The promiscuous nature of miRNA represented by the variety of binding patterns associated with mRNA targeting limits the predictability of high confidence miRNA regulators of attractive engineering candidates. This results in a lengthy list of falsely predicted in-silico miRNA regulators for a single gene. We explored the identification of direct miRNA regulators of the pro-survival endogenous XIAP gene in CHO-K1 cells by using a miR-CATCH1 protocol. A biotin-tagged antisense DNA oligonucleotide was designed for an exposed predicted secondary structure loop of endogenous CHO XIAP. This mRNA anchor resulted in the pulldown of XIAP and all associated RNA/protein complexes thereby enriching for all bound miRNAs. Two miRNAs were chosen out of the 14 miRNAs identified for further validation, miR-124-3p and miR-19b-3p. Transient transfection of mimics for both resulted in the diminished translation of endogenous CHO XIAP protein whereas their inhibition increased XIAP protein levels (Fig. 1). Please click Additional Files below to see the full abstract

Homeologous Plastid DNA Transformation in Tobacco Is Mediated by Multiple Recombination Events

Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids

A complete workflow for single cell mtDNAseq in CHO cells, from cell culture to bioinformatic analysis

Chinese hamster ovary (CHO) cells have a long history in the biopharmaceutical industry and currently produce the vast majority of recombinant therapeutic proteins. A key step in controlling the process and product consistency is the development of a producer cell line derived from a single cell clone. However, it is recognized that genetic and phenotypic heterogeneity between individual cells in a clonal CHO population tends to arise over time. Previous bulk analysis of CHO cell populations revealed considerable variation within the mtDNA sequence (heteroplasmy), which could have implications for the performance of the cell line. By analyzing the heteroplasmy of single cells within the same population, this heterogeneity can be characterized with greater resolution. Such analysis may identify heterogeneity in the mitochondrial genome, which impacts the overall phenotypic performance of a producer cell population, and potentially reveal routes for genetic engineering. A critical first step is the development of robust experimental and computational methods to enable single cell mtDNA sequencing (termed scmtDNAseq). Here, we present a protocol from cell culture to bioinformatic analysis and provide preliminary evidence of significant mtDNA heteroplasmy across a small panel of single CHO cells

Versatile Dual Reporter Gene Systems for Investigating Stop Codon Readthrough in Plants

Background Translation is most often terminated when a ribosome encounters the first in-frame stop codon (UAA, UAG or UGA) in an mRNA. However, many viruses (and some cellular mRNAs) contain ?stop? codons that cause a proportion of ribosomes to terminate and others to incorporate an amino acid and continue to synthesize a ?readthrough?, or C-terminally extended, protein. This dynamic redefinition of codon meaning is dependent on specific sequence context. Methodology We describe two versatile dual reporter systems which facilitate investigation of stop codon readthrough in vivo in intact plants, and identification of the amino acid incorporated at the decoded stop codon. The first is based on the reporter enzymes NAN and GUS for which sensitive fluorogenic and histochemical substrates are available; the second on GST and GFP. Conclusions We show that the NAN-GUS system can be used for direct in planta measurements of readthrough efficiency following transient expression of reporter constructs in leaves, and moreover, that the system is sufficiently sensitive to permit measurement of readthrough in stably transformed plants. We further show that the GST-GFP system can be used to affinity purify readthrough products for mass spectrometric analysis and provide the first definitive evidence that tyrosine alone is specified in vivo by a `leaky? UAG codon, and tyrosine and tryptophan, respectively, at decoded UAA, and UGA codons in the Tobacco mosaic virus (TMV) readthrough context

An Analysis of Hypertension Patients’ Overlapped Medical Utilization —Using National Health Insurance Registry for Beneficiaries Claims Data Files of 2005

本研究之目的為分析高血壓病人西醫門急診之重複醫療資源利用情形。研究資料以全民健保資料庫2005年承保抽樣歸人檔第一組至第四組共20萬人為基礎，定義國際疾病分類號第一、二、三欄中(主、次診斷碼)任一欄前三碼為401至405之高血壓病人(共20,209名)為研究對象，分析其重複醫療資源利用情形。 分析結果發現重複使用醫療資源者共8,050名，重複使用率為39.83%，重複用藥率為41.62%，用藥日數重複率為2.75%。重複使用醫療資源者中97.81%有重複用藥，重複用藥者平均每人累計重複用藥13.93天，平均每人累計重複藥費為508.46點。重複使用醫療資源者中19.65%有重複使用非藥品醫令，平均每人累計重複非藥品醫令費用為883.06點。有重複使用醫療資源之高血壓病人，平均每人重複的總醫令費用為670.89點，總醫療費用為3,331.19點。 性別方面，女性重複使用醫療資源之機率較男性高，但男性重複使用醫療資源之程度卻較女性高。年齡方面，年齡愈大者愈容易重複使用醫療資源，重複使用的程度也愈高。有重複使用醫療資源者中，免部分負擔者重複使用醫療資源的程度較需部分負擔者高。 病人之C.C.I.越高，會重複使用醫療資源的機率及程度越高，病人因高血壓而看診之醫師數、醫療機構數越多，重複使用醫療資源的機率及程度也越高。 病人的就醫選擇方面，無固定醫療機構權屬別及特約類別者重複使用醫療資源的機率較低，無固定醫療機構權屬別者重複使用醫療資源的程度也較低。論：絕大部分有重複使用醫療資源之高血壓病人有重複用藥。後續研究者可結合問卷，瞭解高血壓病人重複使用醫療資源之原因。The purpose of the study was to analyze hypertension patients’ overlapped medical utilization by using the 2005 National Health Insurance Registry for Beneficiaries Claims Data files, the medical service utilization data of 200,000 persons. This study identified 20,209 hypertension patients visited western outpatient and emergency department. The percentage of overlapped medical resources utilization, overlapped medication and overlapped days of prescriptions was 39.83%, 41.62% and 2.75%, respectively. Among hypertension patients who used overlapped medical resources, 97.81% also had overlapped medication. The average overlapped days of prescriptions and overlapped medication expenses were 13.93 days and 508.46 points. Nineteen point six percent hypertension patients who used overlapped medical resources also had overlapped non-drug orders. The average overlapped non-drug orders expenses were 883.06 points. The average overlapped total orders expenses of 8,050 hypertension patients who used overlapped medical resources were 670.89 points and the overlapped total medical expenses were 3,331.19 points. Females were more likely to use overlapped medical resources, but the degree was lower than males. Hypertension patients’ medical demand and overlapped medical resources utilization increased as age increased. Once the patients used overlapped medical resources, subjects who did not need to pay copayments had higher degree of overlapped medical resources utilization than their counterpart. The probability and the degree of overlapped medical resources utilization were higher when the Charlson comorbidity index was higher. The more the number of doctors and medical facilities patients visited because of hypertension, the higher the probability and the degree of overlapped medical resources utilization. Subjects who did not have a regular place of care were less likely to overlap medical resources utilization than their counterparts and the degree of overlapped medical resources utilization was lower. onclusions: The majority of the hypertension patients who had overlapped medical resources utilization also had overlapped medication. Future researchers can incorporate questionnaire to investigate the reasons of overlapped medical resources utilization.致謝.............................................I文摘要.........................................IIbstract.........................................III一章 前言.....................................1一節 研究緣起................................1 第二節 研究目的...............................2 第三節 研究之重要性...........................2二章 文獻探討.................................3 第一節 高血壓之定義、分類及其臨床治療指引.....3 第二節 國內外高血壓病人之分佈及醫療資源使用情形.. 11 第三節 就醫行為之相關研究.................... 17 第四節 重複醫療資源利用之相關研究.............23 第五節 綜合討論...............................31三章 研究材料與方法...........................34 第一節 名詞定義...............................34 第二節 研究架構...............................38 第三節 研究假說...............................39 第四節 研究對象及材料.........................39 第五節 研究變項...............................39 第六節 資料處理與統計分析.....................43四章 研究結果.................................46 第一節 描述性統計之結果.......................46 第二節 推論性統計之結果.......................48五章 討論.....................................54 第一節 研究結果之討論.........................54 第二節 研究限制...............................61六章 結論與建議...............................62 第一節 結論...................................62 第二節 建議...................................64考文獻.........................................6