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1
Wolf, Steven E., and Kenneth J. Woodside. "Transgenic and gene knock-out techniques and burn research." Journal of Surgical Research 123, no. 2 (February 2005): 328–39. http://dx.doi.org/10.1016/j.jss.2004.06.001.
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Monahan, Paul E. "Factor IX: Insights from knock-out and genetically engineered mice." Thrombosis and Haemostasis 100, no. 10 (2008): 563–75. http://dx.doi.org/10.1160/th08-04-0262.
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SummaryThe study of coagulation factors has been rapidly advanced by studies performed in genetically engineered mouse strains. Investigation of factor IX (FIX) has benefited from excellent genedeleted mouse models that recapitulate many of the features of human haemophilia B. Moreover, advanced positional cloning techniques and availability of technology to allow not only knock-out mice, but also knock-in and knock-down mice, provide new opportunities to observe genotype-phenotype and structure-function correlations regarding FIX, as well as the interaction of FIX with inflammatory, immune, and tissue repair systems. In this paper, available FIX knock-out mice and additional haemophilia B mouse models are reviewed specifically in regards to observations these models have facilitated concerning: factor IX gene expression and factor IX protein pharmacokinetics; the role of FIX in haemostasis, thrombosis and wound healing; insights into coagulation FIX arising out of gene therapy applications in haemophilia mouse models; immunology of tolerance or loss of tolerance of FIX and inhibitor antibody formation.
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Ohara, Hiroki, and Toru Nabika. "Genetic Modifications to Alter Blood Pressure Level." Biomedicines 10, no. 8 (August 1, 2022): 1855. http://dx.doi.org/10.3390/biomedicines10081855.
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Genetic manipulation is one of the indispensable techniques to examine gene functions both in vitro and in vivo. In particular, cardiovascular phenotypes such as blood pressure cannot be evaluated in vitro system, necessitating the creation of transgenic or gene-targeted knock-out and knock-in experimental animals to understand the pathophysiological roles of specific genes on the disease conditions. Although genome-wide association studies (GWAS) in various human populations have identified multiple genetic variations associated with increased risk for hypertension and/or its complications, the causal links remain unresolved. Genome-editing technologies can be applied to many different types of cells and organisms for creation of knock-out/knock-in models. In the post-GWAS era, it may be more worthwhile to validate pathophysiological implications of the risk variants and/or candidate genes by creating genome-edited organisms.
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Yang, Caiting, Yu Lei, Tinglin Ren, and Mingze Yao. "The Current Situation and Development Prospect of Whole-Genome Screening." International Journal of Molecular Sciences 25, no. 1 (January 4, 2024): 658. http://dx.doi.org/10.3390/ijms25010658.
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High-throughput genetic screening is useful for discovering critical genes or gene sequences that trigger specific cell functions and/or phenotypes. Loss-of-function genetic screening is mainly achieved through RNA interference (RNAi), CRISPR knock-out (CRISPRko), and CRISPR interference (CRISPRi) technologies. Gain-of-function genetic screening mainly depends on the overexpression of a cDNA library and CRISPR activation (CRISPRa). Base editing can perform both gain- and loss-of-function genetic screening. This review discusses genetic screening techniques based on Cas9 nuclease, including Cas9-mediated genome knock-out and dCas9-based gene activation and interference. We compare these methods with previous genetic screening techniques based on RNAi and cDNA library overexpression and propose future prospects and applications for CRISPR screening.
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Syarifuddin, Syarifuddin, Ade Ulansari, and Iftahurrahmah Iftahurrahmah. "ARCHITECTURAL DESIGN AND WOODEN SRUCTURES OF TRADITIONAL OGAN ILIR BUILDINGS AS THE LOCAL CULTURAL WEALTH OF THE OGAN ILIR COMMUNITY." Sosiohumaniora 24, no. 2 (July 4, 2022): 256. http://dx.doi.org/10.24198/sosiohumaniora.v24i2.36893.
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South Sumatra has a lot of traditional houses across its region. Each house has its own characteristics. One notable design is houses built on stilts. In Ogan Ilir, especially Tanjung Batu, it is famous for the production of knock-down houses. Knock-down houses in Tanjung Batu are a stilt house with an architectural design that almost resembles a Limas house, only that these knock-down houses are specially designed to make it easy to disassemble and reassemble them elsewhere. This study aims to give a closer look at the design, structure and wood choices of knock-down houses that have become unique, cultural houses of Ogan Ilir people. This study will look at how the architectural design of the houses’ characteristics and wooden structures are adapted to the surrounding environment. This study was conducted qualitatively with an anthropological approach through ethnological techniques. To dig up information about knock-down houses, this was done through observation and data collection by conducting interviews and collecting written data such as journals and articles related to the topic. Observations and interviews were carried out in Tanjung Batu, which is the center for knock-down house craftsmen. The results obtained in this study are: (a) the daily activities of carpenters in the manufacture of knock-down houses, (b) tools and materials used in the construction of the knock-down houses, (c) sales of knock-down houses, (d) the development of wood tools and materials following the development of increasingly sophisticated technology, and (e) information about knock-down houses as a local, cultural building typical of Ogan Ilir.
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Riyandi, Ages, and Desy Misnawati. "STRATEGI PEMASARAN PRODUK RUMAH KNOCK DOWN PADA MASYARAKAT TANJUNG BATU SEBERANG." Jurnal Inovasi 15, no. 1 (May 12, 2021): 11–27. http://dx.doi.org/10.33557/ji.v15i1.2200.
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The production of knock down houses is in a great demand especially in Tanjung BatuSeberang.The craft of knxok down houses was passed down from the era of Sriwijaya Kingdom untilnow. The demand of the craft is inscreasing from year to year this has turned out to be anopportunity so that many young people are engaged in this business. Kotler and Armstrong'sMarketing Strategy Theory (2004 in Varadarajan 2009). The research method uses qualitativeresearch. Collecting data through observation as the main source, interviews and documentation.While data analysis techniques, data presentation and data collection. The research refers to themain question: What is the knock down marketing strategy for the people of Tanjung Batu Seberang?. The subjects of this study were 4 knock down house entrepreneurs in Tanjung Batu District.Equipped with these 4 information so as to enrich the data, the results of this study do not know thatthe marketing strategy is used by all informants in marketing knock down house production
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Fabbri, Mattia, and Pier Giuseppe Giribone. "Design, implementation and validation of advanced lattice techniques for pricing EAKO — European American Knock-Out option." International Journal of Financial Engineering 06, no. 04 (December 2019): 1950032. http://dx.doi.org/10.1142/s2424786319500324.
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The paper presents a series of advanced lattice methods aimed at evaluating an EAKO European-American Knock-Out contract. The first part of the paper deals with the numerical methods implemented for pricing: Binomial and Trinomial Stochastic trees, Adaptive Mesh Model, Pentanomial and Heptanomial lattice. In the second part, specific tests are designed to validate the code written in Matlab language. The study concludes by applying the most performing model to a real market case.
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Mittal, Vikram. "A Review of Recent Advancements in Knock Detection in Spark Ignition Engines." Signals 5, no. 1 (March 21, 2024): 165–80. http://dx.doi.org/10.3390/signals5010009.
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In gasoline engines, the combustion process involves a flame’s propagation from the spark plug to the cylinder walls, resulting in the localized heating and pressurization of the cylinder content ahead of the flame, which can lead to the autoignition of the gasoline and air. The energy release from the autoignition event causes the engine cylinder to resonate, causing an unpleasant noise and eventual engine damage. This process is termed as knock. Avoiding knock has resulted in limiting the maximum engine pressures, and hence limiting the maximum efficiencies of the engine. Modern engines employ knock sensors to detect resonances, adjusting the spark plug timing to reduce pressures and temperatures, albeit at the expense of engine performance. This paper sets out to review the different signals that can be measured from an engine to detect the start of knock. These signals traditionally consist of the in-cylinder pressure, the vibrations of the engine block, and acoustic noise. This paper reviews each of these techniques, with a focus on recent advances. A number of novel methods are also presented, including identifying perturbations in the engine speed or exhaust temperature; measuring the ion charge across the spark plug leads; and using artificial intelligence to build models based on engine conditions. Each of these approaches is also reviewed and compared to the more traditional approaches. This review finds that in-cylinder pressure measurements remain as the most accurate for detecting knock in modern engines; however, their usage is limited to research settings. Meanwhile, new sensors and processing techniques for vibration measurements will more accurately detect knock in modern vehicles in the short term. Acoustic measurements and other novel approaches are showing promise in the long term.
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Balla, Beata, Florin Tripon, and Claudia Banescu. "From Descriptive to Functional Genomics of Leukemias Focusing on Genome Engineering Techniques." International Journal of Molecular Sciences 22, no. 18 (September 17, 2021): 10065. http://dx.doi.org/10.3390/ijms221810065.
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Genome engineering makes the precise manipulation of DNA sequences possible in a cell. Therefore, it is essential for understanding gene function. Meganucleases were the start of genome engineering, and it continued with the discovery of Zinc finger nucleases (ZFNs), followed by Transcription activator-like effector nucleases (TALENs). They can generate double-strand breaks at a desired target site in the genome, and therefore can be used to knock in mutations or knock out genes in the same way. Years later, genome engineering was transformed by the discovery of clustered regularly interspaced short palindromic repeats (CRISPR). Implementation of CRISPR systems involves recognition guided by RNA and the precise cleaving of DNA molecules. This property proves its utility in epigenetics and genome engineering. CRISPR has been and is being continuously successfully used to model mutations in leukemic cell lines and control gene expression. Furthermore, it is used to identify targets and discover drugs for immune therapies. The descriptive and functional genomics of leukemias is discussed in this study, with an emphasis on genome engineering methods. The CRISPR/Cas9 system’s challenges, viewpoints, limits, and solutions are also explored.
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Li, Meng, Xufang Niu, Shuang Li, Shasha Fu, Qianfang Li, Meizhi Xu, Chunhua Wang, and Shuang Wu. "CRISPR/Cas9 Based Cell-Type Specific Gene Knock-Out in Arabidopsis Roots." Plants 12, no. 12 (June 19, 2023): 2365. http://dx.doi.org/10.3390/plants12122365.
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CRISPR/Cas9 (hereafter Cas9)-mediated gene knockout is one of the most important tools for studying gene function. However, many genes in plants play distinct roles in different cell types. Engineering the currently used Cas9 system to achieve cell-type-specific knockout of functional genes is useful for addressing the cell-specific functions of genes. Here we employed the cell-specific promoters of the WUSCHEL RELATED HOMEOBOX 5 (WOX5), CYCLIND6;1 (CYCD6;1), and ENDODERMIS7 (EN7) genes to drive the Cas9 element, allowing tissue-specific targeting of the genes of interest. We designed the reporters to verify the tissue-specific gene knockout in vivo. Our observation of the developmental phenotypes provides strong evidence for the involvement of SCARECROW (SCR) and GIBBERELLIC ACID INSENSITIVE (GAI) in the development of quiescent center (QC) and endodermal cells. This system overcomes the limitations of traditional plant mutagenesis techniques, which often result in embryonic lethality or pleiotropic phenotypes. By allowing cell-type-specific manipulation, this system has great potential to help us better understand the spatiotemporal functions of genes during plant development.
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Taylor, R., A. F. Steinberg, J. Pasternak, R. B. Appleby, S. L. Sheehy, and E. Benedetto. "Slow Extraction Techniques from Fixed Field Accelerators." Journal of Physics: Conference Series 2687, no. 2 (January 1, 2024): 022022. http://dx.doi.org/10.1088/1742-6596/2687/2/022022.
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Abstract Fixed Field Accelerators are a candidate for future hadron cancer therapy facilities as their high repetition rate and large energy acceptance enables novel treatment modalities such as high dose rate FLASH. However, conventional dose delivery mechanisms are still necessary, requiring continuous beam delivery over 1–30s. This work is the first study of slow extraction from a scaling Fixed Field Accelerator, using the LhARA facility for baseline parameters. At a horizontal tune of 10/3, the intrinsic sextupole strength of the nonlinear FFA magnetic field is sufficient to excite the resonance, although extraction is better controlled using an additional excitation sextupole at a tune close to 8/3, with radiofrequency knock-out extraction. Including considerations of issues due to nonlinear fields and limitations required to keep the tune energy-independent, slow extraction from Fixed Field Accelerators is successfully demonstrated.
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Solo, Katherine M., Sara B. Collins, Liesel G. Schneider, M. R. Hajimorad, Frank A. Hale, John B. Wilkerson, Alan S. Windham, and Mark T. Windham. "Evaluation of Floral Cuts on Eriophyid Mite Retention on Knock Out and Multiflora Rose Cuttings." Plant Health Progress 20, no. 2 (January 1, 2019): 83–87. http://dx.doi.org/10.1094/php-12-18-0080-rs.
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Current eriophyid mite quantification techniques require transportation of the Rosa spp. cuttings to the laboratory. It is thought that the change in xylem hydraulic conductance within the cut cane could trigger the mites to abandon their host, owing to the changes to the microenvironments that these mites are inhabiting. An experiment was conducted to determine the necessity of floral cuts (reducing stem embolisms by an additional cut underwater) for the retention of eriophyid mites during transit. Four groups of plants (rose rosette virus (RRV)-free Knock Out roses, RRV-infected Knock Out roses, RRV-free multiflora roses, and RRV-infected multiflora roses) were evaluated at different time intervals (0.5, 2, 4, 8, 24, 48, 72, and 96 h postharvest) to assess mite populations on each plant (number of mites per gram of tissue). Cut type (floral or dry cut) and rose species were found not to have a significant effect on the number of mites per gram of tissue found, indicating that floral cuts are not needed for accurately estimating eriophyid mite populations. Rose cuttings infected with RRV were found to have an average of 46 times more mites per gram in comparison with RRV-free cuttings.
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Waheed, Usama, and Muhammad Naveed. "Enhancing Wheat Crop Yield and Quality through Genetic Engineering Technologies: A Comprehensive Review." Journal of Global Innovations in Agricultural Sciences 11, no. 4 (December 22, 2023): 481–89. http://dx.doi.org/10.22194/jgias/11.1089.
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The increasing world population has created an alarming situation to meet the demand for food. Ultimately wheat yield is required to be increased proportionally to the world’s population. An estimated increase of 05 tonnes per hectare is direly needed from the current production of 3.3 tonnes by the end of 2050. To achieve this goal it is required to adopt new, emerging, and efficient breeding techniques which caused a recent breakthrough in wheat genome sequencing and provided sufficient information for the traits which are directly related to quality and yield of crops. This review provides a comprehensive overview of tools and techniques which are widely used for discovering the functionality of genes that results in specific phenotypes of Wheat. It gives a deep historical account of the development of wheat transformation techniques and provides an idea about in which way these techniques have been adapted to produce gain-of-function phenotypes through gene overexpression, loss-of-function phenotypes through the expression of antisense RNAs (RNA interference or RNAi). Recently, gene structure and expression manipulation using site-specific nucleases such as CRISPR/Cas9 for genome editing. The review summarizes recent successes in the application of wheat genetic manipulation to enhance yield, improve wheat's nutritional and health-promoting qualities, and boost the crop's resistance to various biotic and abiotic stresses. Keywords: Transformation, gene knock-Out, gene knock-In, RNAI, overexpression.
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Ostrowski, Martin, Sasha Tetu, Karl Hassan, Anahit Penesyan, Kent Lim, Liam Elbourne, Liping Li, Deepa Varkey, and Ian Paulsen. "From omics to systems biology: Exploring the mystery box of microbial life." Microbiology Australia 32, no. 4 (2011): 147. http://dx.doi.org/10.1071/ma11147.
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Microbial molecular biology has traditionally used very reductionist approaches; for example, find a gene of interest, clone it or knock it out and see if you can detect a phenotype. The genomics era has opened up the possibility of analysing microbes and communities at a systems level by combining high-throughput experimental data from genomic, transcriptomic, proteomic and phenomic techniques. This parallels earlier reductionist approaches by going from DNA to RNA to protein to phenotype, albeit on a global rather than individual gene scale.
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Allen, Daniel, Nechama Kalter, Michael Rosenberg, and Ayal Hendel. "Homology-Directed-Repair-Based Genome Editing in HSPCs for the Treatment of Inborn Errors of Immunity and Blood Disorders." Pharmaceutics 15, no. 5 (April 24, 2023): 1329. http://dx.doi.org/10.3390/pharmaceutics15051329.
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Genome engineering via targeted nucleases, specifically CRISPR-Cas9, has revolutionized the field of gene therapy research, providing a potential treatment for diseases of the blood and immune system. While numerous genome editing techniques have been used, CRISPR-Cas9 homology-directed repair (HDR)-mediated editing represents a promising method for the site-specific insertion of large transgenes for gene knock-in or gene correction. Alternative methods, such as lentiviral/gammaretroviral gene addition, gene knock-out via non-homologous end joining (NHEJ)-mediated editing, and base or prime editing, have shown great promise for clinical applications, yet all possess significant drawbacks when applied in the treatment of patients suffering from inborn errors of immunity or blood system disorders. This review aims to highlight the transformational benefits of HDR-mediated gene therapy and possible solutions for the existing problems holding the methodology back. Together, we aim to help bring HDR-based gene therapy in CD34+ hematopoietic stem progenitor cells (HSPCs) from the lab bench to the bedside.
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Russo, Emma E., Lola E. Zovko, Reza Nazari, Hendrik Steenland, Amy J. Ramsey, and Ali Salahpour. "Evaluation and Validation of Commercially Available Dopamine Transporter Antibodies." eneuro 10, no. 5 (May 2023): ENEURO.0341–22.2023. http://dx.doi.org/10.1523/eneuro.0341-22.2023.
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AbstractWith a wide variety of dopamine transporter (DAT) antibodies available commercially, it is important to validate which antibodies provide sufficient immunodetection for reproducibility purpose and for accurate analysis of DAT levels and/or location. Commercially available DAT antibodies that are commonly used were tested in western blotting (WB) on wild-type (WT) and DAT-knock-out (DAT-KO) brain tissue and with immunohistology (IH) techniques against coronal slices of unilaterally lesioned 6-OHDA rats, in addition to wild-type and DAT-knock-out mice. DAT-KO mice and unilateral 6-OHDA lesions in rats were used as a negative control for DAT antibody specificity. Antibodies were tested at various concentrations and rated based on signal detection varying from no signal to optimal signal detection. Commonly used antibodies, including AB2231 and PT-22 524-1-AP, did not provide specific DAT signals in WB and IH. Although certain antibodies provided a good DAT signal, such as SC-32258, D6944, and MA5-24796, they also presented nonspecific bands in WB. Many DAT antibodies did not detect the DAT as advertised, and this characterization of DAT antibodies may provide a guide for immunodetection of DAT for molecular studies.
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DUCOS, A., B. BED'HOM, H. ACLOQUE, and B. PAIN. "Modifications ciblées des génomes : apports et impacts pour les espèces d’élevage." INRA Productions Animales 30, no. 1 (June 14, 2018): 3–18. http://dx.doi.org/10.20870/productions-animales.2017.30.1.2226.
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L’avènement des nucléases programmables, et de CRISPR-Cas9 en particulier, constitue une réelle rupture technologique dans le domaine de l’ingénierie génétique. Le principe de ces méthodes est relativement simple. Il consiste en premier lieu à générer des cassures double-brin au niveau de régions très précises d’une séquence cible (ADN d’une cellule somatique, germinale, embryonnaire, iPS...). Ces cassures sont ensuite réparées par des mécanismes cellulaires pouvant conduire à l’inactivation des régions modifiées (knock-out, ou KO), ou à l’insertion, par recombinaison homologue, d’un fragment de séquence modèle apporté à la cellule (knock-in, ou KI). Les domaines d’application de ces techniques sont très nombreux : recherche fondamentale, thérapie génique, ingénierie écologique, biotechnologies industrielles, agriculture, etc. Elles ont d’ores et déjà été utilisées à de multiples reprises dans les espèces animales d’élevage. Des exemples d’applications visant à améliorer la santé des animaux (induction de résistances pour des maladies infectieuses à fort impact économique, et/ou à fort potentiel zoonotique), à éviter des pratiques d’élevage compromettant le bien-être animal (écornage, élimination systématique de poussins mâles), ou à modifier les produits (lait, œufs, viande) dans le but d’améliorer leur valeur santé (réduction des allergies), ou nutritionnelle, sont présentés. Le déploiement de ces méthodes soulève de multiples questions (techniques, réglementaires, économiques, éthiques...) qui sont discutées.
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Salman, Ahmed, Samuel B. Hutton, Tutte Newall, Jennifer A. Scott, Helen L. Griffiths, Helena Lee, Diego Gomez-Nicola, Andrew J. Lotery, and Jay E. Self. "Characterization of the Frmd7 Knock-Out Mice Generated by the EUCOMM/COMP Repository as a Model for Idiopathic Infantile Nystagmus (IIN)." Genes 11, no. 10 (September 30, 2020): 1157. http://dx.doi.org/10.3390/genes11101157.
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In this study, we seek to exclude other pathophysiological mechanisms by which Frmd7 knock-down may cause Idiopathic Infantile Nystagmus (IIN) using the Frmd7.tm1a and Frmd7.tm1b murine models. We used a combination of genetic, histological and visual function techniques to characterize the role of Frmd7 gene in IIN using a novel murine model for the disease. We demonstrate that the Frmd7.tm1b allele represents a more robust model of Frmd7 knock-out at the mRNA level. The expression of Frmd7 was investigated using both antibody staining and X-gal staining confirming previous reports that Frmd7 expression in the retina is restricted to starburst amacrine cells and demonstrating that X-gal staining recapitulates the expression pattern in this model. Thus, it offers a useful tool for further expression studies. We also show that gross retinal morphology and electrophysiology are unchanged in these Frmd7 mutant models when compared with wild-type mice. High-speed eye-tracking recordings of Frmd7 mutant mice confirm a specific horizontal optokinetic reflex defect. In summary, our study confirms the likely role for Frmd7 in the optokinetic reflex in mice mediated by starburst amacrine cells. We show that the Frmd7.tm1b model provides a more robust knock-out than the Frmd7.tm1a model at the mRNA level, although the functional consequence is unchanged. Finally, we establish a robust eye-tracking technique in mice that can be used in a variety of future studies using this model and others. Although our data highlight a deficit in the optiokinetic reflex as a result of the starburst amacrine cells in the retina, this does not rule out the involvement of other cells, in the brain or the retina where Frmd7 is expressed, in the pathophysiology of IIN.
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Murwaningtyas, Chatarina Enny, William Saputra Haryono, Maria Andriani Uge, and Tedi Kristofel. "Perbandingan Metode Monte Carlo Antithetic Variate dan Control Variate dalam Penentuan Harga Opsi Barrier Knock-Out." Euler : Jurnal Ilmiah Matematika, Sains dan Teknologi 12, no. 1 (June 1, 2024): 37–44. http://dx.doi.org/10.37905/euler.v12i1.25128.
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This study aims to examine the effectiveness of the Monte Carlo antithetic variate and control variate methods in pricing knock-out barrier options compared to the standard Monte Carlo method. The main problem in barrier option pricing is the high variance of estimates, which can reduce the accuracy and efficiency of results. The standard Monte Carlo method often requires a very large number of simulations to achieve stable results, which is computationally inefficient. To address this issue, this study employs variance reduction techniques, antithetic variate, and control variate. The findings indicate that both methods offer higher accuracy in price estimation compared to the standard Monte Carlo method. Further analysis reveals that the control variate method is more effective for pricing up and out barrier call options and down and out barrier call options, while the antithetic variate method excels in pricing up and out barrier put options and down and out barrier put options. This study underscores the importance of selecting the appropriate method according to the type of option involved to achieve accurate and efficient estimations.
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Taylor, R., E. Benedetto, M. Sapinski, and J. Pasternak. "Slow extraction modelling for NIMMS hadron therapy synchrotrons." Journal of Physics: Conference Series 2420, no. 1 (January 1, 2023): 012101. http://dx.doi.org/10.1088/1742-6596/2420/1/012101.
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Abstract The Next Ion Medical Machine Study (NIMMS) is an umbrella R&D programme for CERN accelerator technologies targeting advanced accelerator options for proton and light ion therapy. In collaboration with the European programme HITRIplus, one area of study is slow extraction which is required to deliver a uniform beam spill for radiotherapy treatment. Several techniques use the third-order resonance to extract hadrons; these include betatron core driven extraction and radiofrequency knock-out. Flexible simulation tools using these techniques were prepared and initially benchmarked with results from the literature that used the Proton-Ion Medical Machine Study (PIMMS) design. The limits of the current PIMMS design were then pushed to evaluate its compatibility to deliver 10x higher intensity ion beams, and using increased extraction rates.
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Hédou, Gaël, and Isabelle M. Mansuy. "Inducible molecular switches for the study of long-term potentiation." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 358, no. 1432 (April 29, 2003): 797–804. http://dx.doi.org/10.1098/rstb.2002.1245.
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This article reviews technical and conceptual advances in unravelling the molecular bases of long-term potentiation (LTP), learning and memory using genetic approaches. We focus on studies aimed at testing a model suggesting that protein kinases and protein phosphatases balance each other to control synaptic strength and plasticity. We describe how gene ‘knock-out’ technology was initially exploited to disrupt the Ca 2+ /calmodulin-dependent protein kinase II α (CaMKII α ) gene and how refined knock-in techniques later allowed an analysis of the role of distinct phosphorylation sites in CaMKII. Further to gene recombination, regulated gene expression using the tetracycline-controlled transactivator and reverse tetracycline-controlled transactivator systems, a powerful new means for modulating the activity of specific molecules, has been applied to CaMKII α and the opposing protein phosphatase calcineurin. Together with electro-physiological and behavioural evaluation of the engineered mutant animals, these genetic methodologies have helped gain insight into the molecular mechanisms of plasticity and memory. Further technical developments are, however, awaited for an even higher level of finesse.
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AZAIEZ, F. "SHELL STRUCTURE EVOLUTION IN NUCLEI: NEW PARADIGM." International Journal of Modern Physics E 18, no. 10 (November 2009): 1986–91. http://dx.doi.org/10.1142/s0218301309014135.
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Shell structure evolution in nuclei situated at the extremes of neutron and proton excess are investigated using in-beam gamma spectroscopy techniques with radioactive beams at GANIL. A selection of results obtained very recently is presented: i) The reduced transition probabilities [Formula: see text] of the neutron-rich 74 Zn and 70 Ni nuclei have been measured using Coulomb excitation at intermediate energy. An unexpected large proton core polarization has been found in 70 Ni and interpreted as being due to the monopole interaction between the neutron g 9/2 and protons f 7/2 and f 5/2 spin-orbit partner orbitals. ii) Two proton knock-out reactions has been performed in order to study the most neutron-rich nuclei at the N =28 shell closure. Gamma rays spectra and momentum distribution have been obtained for 42 Si and neighboring nuclei. Evidences has been found for a deformed structure for 42 Si and for the disappearance of the spherical N =28 shell effect. iii) The in-beam gamma spectroscopy of 36 Ca performed using neutron knock-out reactions revealed that N =16 is as large sub-shell closure as large as Z =16 in 36 S . The uniquely large excitation energy difference of the first 2+ state in these mirror nuclei turns out to be a consequence of the relatively pure neutron (in 36 Ca ) or proton (in 36 S ) 1 p ( d 3/2)-1 h ( s 1/2) nature.
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Jin, Binbin, Ding Zhao, Fei Liang, Lufang Liu, Dongli Liu, Pan Wang, and Min Qiu. "Electron-Beam Irradiation Induced Regulation of Surface Defects in Lead Halide Perovskite Thin Films." Research 2021 (June 4, 2021): 1–11. http://dx.doi.org/10.34133/2021/9797058.
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Organic-inorganic hybrid perovskites (OIHPs) have been intensively studied due to their fascinating optoelectronic performance. Electron microscopy and related characterization techniques are powerful to figure out their structure-property relationships at the nanoscale. However, electron beam irradiation usually causes damage to these beam-sensitive materials and thus deteriorates the associated devices. Taking a widely used CH3NH3PbI3 film as an example, here, we carry out a comprehensive study on how electron beam irradiation affects its properties. Interestingly, our results reveal that photoluminescence (PL) intensity of the film can be significantly improved along with blue-shift of emission peak at a specific electron beam dose interval. This improvement stems from the reduction of trap density at the CH3NH3PbI3 surface. The knock-on effect helps expose a fresh surface assisted by the surface defect-induced lowering of displacement threshold energy. Meanwhile, the radiolysis process consistently degrades the crystal structure and weaken the PL emission with the increase of electron beam dose. Consequently, the final PL emission comes from a balance between knock-on and radiolysis effects. Taking advantage of the defect regulation, we successfully demonstrate a patterned CH3NH3PbI3 film with controllable PL emission and a photodetector with enhanced photocurrent. This work will trigger the application of electron beam irradiation as a powerful tool for perovskite materials processing in micro-LEDs and other optoelectronic applications.
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Lee, Gloria, Annie Lo, Sarah A. Short, Tosti J. Mankelow, Frances Spring, Stephen F. Parsons, Karina Yazdanbakhsh, Narla Mohandas, David J. Anstee, and Joel Anne Chasis. "Targeted gene deletion demonstrates that the cell adhesion molecule ICAM-4 is critical for erythroblastic island formation." Blood 108, no. 6 (September 15, 2006): 2064–71. http://dx.doi.org/10.1182/blood-2006-03-006759.
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AbstractErythroid progenitors differentiate in erythroblastic islands, bone marrow niches composed of erythroblasts surrounding a central macrophage. Evidence suggests that within islands adhesive interactions regulate erythropoiesis and apoptosis. We are exploring whether erythroid intercellular adhesion molecule 4 (ICAM-4), an immunoglobulin superfamily member, participates in island formation. Earlier, we identified αV integrins as ICAM-4 counterreceptors. Because macrophages express αV, ICAM-4 potentially mediates island attachments. To test this, we generated ICAM-4 knock-out mice and developed quantitative, live cell techniques for harvesting intact islands and for re-forming islands in vitro. We observed a 47% decrease in islands reconstituted from ICAM-4 null marrow compared to wild-type marrow. We also found a striking decrease in islands formed in vivo in knock-out mice. Further, peptides that block ICAM-4/αV adhesion produced a 53% to 57% decrease in reconstituted islands, strongly suggesting that ICAM-4 binding to macrophage αV functions in island integrity. Importantly, we documented that αV integrin is expressed in macrophages isolated from erythroblastic islands. Collectively, these data provide convincing evidence that ICAM-4 is critical in erythroblastic island formation via ICAM-4/αV adhesion and also demonstrate that the novel experimental strategies we developed will be valuable in exploring molecular mechanisms of erythroblastic island formation and their functional role in regulating erythropoiesis.
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Ghribi, Manel, Serge Basile Nouemssi, Fatma Meddeb-Mouelhi, and Isabel Desgagné-Penix. "Genome Editing by CRISPR-Cas: A Game Change in the Genetic Manipulation of Chlamydomonas." Life 10, no. 11 (November 20, 2020): 295. http://dx.doi.org/10.3390/life10110295.
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Microalgae are promising photosynthetic unicellular eukaryotes among the most abundant on the planet and are considered as alternative sustainable resources for various industrial applications. Chlamydomonas is an emerging model for microalgae to be manipulated by multiple biotechnological tools in order to produce high-value bioproducts such as biofuels, bioactive peptides, pigments, nutraceuticals, and medicines. Specifically, Chlamydomonas reinhardtii has become a subject of different genetic-editing techniques adapted to modulate the production of microalgal metabolites. The main nuclear genome-editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and more recently discovered the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas) nuclease system. The latter, shown to have an interesting editing capacity, has become an essential tool for genome editing. In this review, we highlight the available literature on the methods and the applications of CRISPR-Cas for C. reinhardtii genetic engineering, including recent transformation methods, most used bioinformatic tools, best strategies for the expression of Cas protein and sgRNA, the CRISPR-Cas mediated gene knock-in/knock-out strategies, and finally the literature related to CRISPR expression and modification approaches.
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Cary, Daniele C., and B. Matija Peterlin. "Targeting the latent reservoir to achieve functional HIV cure." F1000Research 5 (May 26, 2016): 1009. http://dx.doi.org/10.12688/f1000research.8109.1.
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While highly active anti-retroviral therapy has greatly improved the lives of HIV-infected individuals, current treatments are unable to completely eradicate the virus. This is due to the presence of HIV latently infected cells which harbor transcriptionally silent HIV. Latent HIV does not replicate or produce viral proteins, thereby preventing efficient targeting by anti-retroviral drugs. Strategies to target the HIV latent reservoir include viral reactivation, enhancing host defense mechanisms, keeping latent HIV silent, and using gene therapy techniques to knock out or reactivate latent HIV. While research into each of these areas has yielded promising results, currently no one mechanism eradicates latent HIV. Instead, combinations of these approaches should be considered for a potential HIV functional cure.
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Lewis, Matthieu, Valerie Prouzet-Mauleon, Elodie Richard, Beatrice Turcq, Richard Iggo, and Francois-xavier Mahon. "Identification of Imatinib-Sensitizing Genes in Chronic Myeloid Leukemia with a Genome-Scale Crispr Knock-out Screen." Blood 128, no. 22 (December 2, 2016): 4233. http://dx.doi.org/10.1182/blood.v128.22.4233.4233.
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Abstract Background: Resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) can either originate from mutations in the BCR-ABL1 gene, which are mostly well characterized, or emerge from unknown alternative mutations elsewhere in the genome. Small hairpin (sh)RNA screens have been used to discover such genes but are becoming limited due to sup-optimal protein depletion and non-reliable off-target effects. More efficient screening techniques in human cells are now available as a result of the increasing understanding of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) /Cas9 system. Aims: Our goal is to uncover imatinib (IM)-sensitizing genes that cause IM resistance when knocked-out. Characterizing these genes may help understand the mechanisms of IM uptake, metabolism, degradation and/or activity in CML cells. Additionally, we also expect to unveil alternative, BCR-ABL1 independent, oncogenic pathways in CML cells. Methods: In order to find other genes involved in IM resistance, we performed a genome scale CRISPR knock-out (GeCKO) screen, which contains 121,413 sgRNAs that target 20,914 protein coding genes and miRNAs. We transduced one sgRNA per cell and challenged the K562-GeCKO cell pool to IM selection. We compared the abundance of sgRNAs between pre/post-IM treatment by next generation sequencing (NGS). Results: After IM selection, the sgRNAs from surviving cells were identified by NGS and unveiled potential IM-sensitizing genes. The most enriched sgRNAs (FDR < 0.01) targeted genes involved in transcriptional (KLF1, MED24) and translational (EIF2AK1, UBE2M) regulation, apoptosis (BAX, BCL2L11) and cell cycle regulation (BAP1, SPRED2). Subsequent screens on LAMA84 cells are currently underway in order to validate our findings. Additionally, the establishment of individual gene knock-out cell lines are in progress in order to fully understand the role of each gene in IM resistance. Summary/Conclusion: Using a CRISPR knock-out screen, we produced a list of 19 genes (FDR < 0.05) that may play a role in IM resistance. Encouragingly, a subset of these genes (BAX, BAP1, BCL2L11 and SPRED2) have already been correlated to CML progression and/or TKI resistance in the past. We aim to bolster our findings by establishing individual gene KO cell lines and study resistance in LAMA84 cells. The utilization of CRISPR libraries may not only help understand TKI resistance in CML, but also help identify numerous novel genes involved in drug resistances for a myriad of different diseases. Disclosures Mahon: ARIAD: Honoraria; PFIZER: Honoraria; BMS: Consultancy, Honoraria; NOVARTIS PHARMA: Consultancy, Honoraria, Research Funding.
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Indriani, Mutiya, and Nurhafizah Nurhafizah. "Pengembangan Kecerdasan Musikal Anak di Taman Kanak-Kanak Pertiwi 3 Kantor Gubernur Padang." Jurnal Family Education 4, no. 1 (January 24, 2024): 1–7. http://dx.doi.org/10.24036/jfe.v4i1.150.
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This research is carried out every day the implementation of activities carried out in Kindergarten Pertiwi 3 Of the Padang Governor's Office held knock play activities in the classroom so that every day children feel interesting activities and do not get bored for children for the development of children's musical intelligence. This research aims to get an overview of the development of early childhood musical intelligence in Kindergarten Pertiwi 3 Padang Governor's Office. This type of research is qualitative research using a descriptive approach. To see how the development of early childhood musical intelligence in Kindergarten Pertiwi 3 Padang Governor's Office. The subjects of the study were B3-graders at Kindergarten 3 of the Padang Governor's Office. The informant of this study is the principal and teacher in Kindergarten Pertiwi 3 Of the Padang Governor's Office.
Data collection techniques are used in the form of observations, interviews, and documentation. Data analysis techniques are: 1) data collection, 2) data reduction, 3) presentation of data, 4) verification. While the data absorbing technique used in the form of triangulation techniques. The results of the study in general showed that in the development of early childhood musical intelligence in Kindergarten 3 the Governor's Office of Padang teachers has prepared a Daily Learning Implementation Plan (RPPH). Activities carried out every day have been done in accordance with what has been planned, activities carried out every day using various activities such as, playing beats, singing and others. Furthermore, after the child conducts teacher activities to conduct evaluations by means of observation and daily assessment.
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Reddy, D. Samba, and Tina Reddy. "Pharmaceutical and Biotechnological Applications of Transgenic Technology." International Journal of Pharmaceutical Sciences and Nanotechnology 11, no. 4 (July 31, 2018): 4145–53. http://dx.doi.org/10.37285/ijpsn.2018.11.4.1.
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A transgenic animal is a genetically modified species in which researchers have modified an existing gene or genes by genetic engineering techniques. Genetic modification involves the mutation, insertion, or deletion of genes. Mouse is the most widely used mammalian species for creating transgenic lines. There are two types of transgenic animals: (i) gene deleted (“knock-out”) and (ii) gene overexpressed (“knock-in”). The loss or gain of gene activity often causes changes in a mouse's phenotype, which includes appearance, behavior and other observable characteristics. Knockout mice are key animal models for studying the role of genes which have been sequenced but whose functions have not been determined. They include constitutive knockouts (gene deleted since birth) and conditional knockout (gene turned off later after birth). The first knockout mouse was created in 1989 by Mario Capecchi, Martin Evans, and Oliver Smithies, for which they were awarded the 2007 Nobel Prize in Physiology or Medicine. Transgenic mouse models have revolutionized the biomedical research and provided a power tool for understanding health and disease. Transgenic animals have been created for bulk production of biotechnology and pharmaceutical products. In 2009, the FDA approved the first human biological drug ATryn, an anticoagulant extracted from the transgenic goat's milk. The recently discovered CRISPER gene editing technology is providing new frontiers in correcting abnormal genes and hopefully provide cures for genetic diseases in the future.
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Rai, Arti K. "PHARMACOGENETIC INTERVENTIONS, ORPHAN DRUGS, AND DISTRIBUTIVE JUSTICE: THE ROLE OF COST-BENEFIT ANALYSIS." Social Philosophy and Policy 19, no. 2 (July 2002): 246–70. http://dx.doi.org/10.1017/s0265052502192107.
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With the human genome mapped, and with the mapping of more than one hundred animal genomes in progress, the amount of genetic data available is increasing exponentially. This exponential increase in data is having an immediate impact on the process of drug development. By using techniques of information technology to manipulate data regarding the genes, proteins, and biochemical pathways associated with various diseases, scientists are beginning to be able to design drugs in a systematic fashion. In the context of any given disease, scientists look to see whether a gene, a protein for which the gene codes, or another protein in the relevant biochemical pathway could be the “target” biological molecule, the “knocking out” of which would halt or slow the disease's progression. Once a target molecule has been identified and characterized structurally, drug therapies that would be likely to knock out this target can be identified and tested systematically. The merger of information technology and genetic technology has changed the process of pharmaceutical development so much that a new term—bioinformatics—has been coined to describe this new approach to such development.
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Kheifets, Anatoli S. "Shake-Off Process in Non-Sequential Single-Photon Double Ionization of Closed-Shell Atomic Targets." Atoms 10, no. 3 (September 7, 2022): 89. http://dx.doi.org/10.3390/atoms10030089.
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Amusia and Kheifets in 1984 introduced a Green’s function formalism to describe the effect of many-electron correlation on the ionization spectra of atoms. Here, we exploit this formalism to model the shake-off (SO) process, leading to the non-sequential single-photon two-electron ionization (double photoionization—DPI) of closed-shell atomic targets. We separate the SO process from another knock-out (KO) mechanism of DPI and show the SO prevalence away from the DPI threshold. We use this kinematic regime to validate our model by making a comparison with more elaborate techniques, such as convergent and time-dependent close coupling. We also use our model to evaluate the attosecond time delay associated with the SO process. Typically, the SO is very fast, taking only a few attoseconds to complete. However, it can take much longer in the DPI of strongly correlated systems, such as the H− ion as well as the subvalent shells of the Ar and Xe atoms and Cl− ion.
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BUFF, ROBERT. "WORST-CASE SCENARIOS FOR AMERICAN OPTIONS." International Journal of Theoretical and Applied Finance 03, no. 01 (January 2000): 25–58. http://dx.doi.org/10.1142/s0219024900000036.
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One approach to cope with uncertain diffusion parameters when pricing options portfolios is to identify the parameters [Formula: see text] in a subset [Formula: see text] of the parameter space which form the worst-case for a particular portfolio. For the sell-side, this leads to a nonlinear algorithm that maximizes the expected liability under the risk-neutral measure. [Formula: see text] depends on the portfolio under consideration. Moreover, the algorithm must take into account that the exposure to [Formula: see text]-risk changes when non-vanilla components such as barrier or American options knock out or are exercised early. In this paper, we describe techniques to price portfolios with American options under worst-case scenarios based on uncertain volatility models. We also present heuristics which reduce the computational complexity that arises from the necessity to consider many early exercise combinations at a time. These heuristics reduce the compute time by almost one half.
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Dabrowska, Magdalena, Agata Ciolak, Emilia Kozlowska, Agnieszka Fiszer, and Marta Olejniczak. "Generation of New Isogenic Models of Huntington’s Disease Using CRISPR-Cas9 Technology." International Journal of Molecular Sciences 21, no. 5 (March 8, 2020): 1854. http://dx.doi.org/10.3390/ijms21051854.
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Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by the expansion of CAG repeats in exon 1 of the huntingtin gene (HTT). Despite its monogenic nature, HD pathogenesis is still not fully understood, and no effective therapy is available to patients. The development of new techniques such as genome engineering has generated new opportunities in the field of disease modeling and enabled the generation of isogenic models with the same genetic background. These models are very valuable for studying the pathogenesis of a disease and for drug screening. Here, we report the generation of a series of homozygous HEK 293T cell lines with different numbers of CAG repeats at the HTT locus and demonstrate their usefulness for testing therapeutic reagents. In addition, using the CRISPR-Cas9 system, we corrected the mutation in HD human induced pluripotent stem cells and generated a knock-out of the HTT gene, thus providing a comprehensive set of isogenic cell lines for HD investigation.
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Lujan, Henry, Eric Romer, Richard Salisbury, Saber Hussain, and Christie Sayes. "Determining the Biological Mechanisms of Action for Environmental Exposures: Applying CRISPR/Cas9 to Toxicological Assessments." Toxicological Sciences 175, no. 1 (February 27, 2020): 5–18. http://dx.doi.org/10.1093/toxsci/kfaa028.
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Abstract Toxicology is a constantly evolving field, especially in the area of developing alternatives to animal testing. Toxicological research must evolve and utilize adaptive technologies in an effort to improve public, environmental, and occupational health. The most commonly cited mechanisms of toxic action after exposure to a chemical or particle test substance is oxidative stress. However, because oxidative stress involves a plethora of genes and proteins, the exact mechanism(s) are not commonly defined. Exact mechanisms of toxicity can be revealed using an emerging laboratory technique referred to as CRISPR (clustered regularly interspaced short palindromic repeats). This article reviews the most common CRISPR techniques utilized today and how each may be applied in Toxicological Sciences. Specifically, the CRISPR/CRISPR-associated protein complex is used for single gene knock-outs, whereas CRISPR interference/activation is used for silencing or activating (respectively) ribonucleic acid. Finally, CRISPR libraries are used for knocking-out entire gene pathways. This review highlights the application of CRISPR in toxicology to elucidate the exact mechanism through which toxicants perturb normal cellular functions.
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Nelson, Nicole C. "A Knockout Experiment: Disciplinary Divides and Experimental Skill in Animal Behaviour Genetics." Medical History 59, no. 3 (June 19, 2015): 465–85. http://dx.doi.org/10.1017/mdh.2015.30.
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In the early 1990s, a set of new techniques for manipulating mouse DNA allowed researchers to ‘knock out’ specific genes and observe the effects of removing them on a live mouse. In animal behaviour genetics, questions about how to deploy these techniques to study the molecular basis of behaviour became quite controversial, with a number of key methodological issues dissecting the interdisciplinary research field along disciplinary lines. This paper examines debates that took place during the 1990s between a predominately North American group of molecular biologists and animal behaviourists around how to design, conduct, and interpret behavioural knockout experiments. Drawing from and extending Harry Collins’s work on how research communities negotiate what counts as a ‘well-done experiment,’ I argue that the positions practitioners took on questions of experimental skill reflected not only the experimental traditions they were trained in but also their differing ontological and epistemological commitments. Different assumptions about the nature of gene action, eg., were tied to different positions in the knockout mouse debates on how to implement experimental controls. I conclude by showing that examining representations of skill in the context of a community’s knowledge commitments sheds light on some of the contradictory ways in which contemporary animal behaviour geneticists talk about their own laboratory work as a highly skilled endeavour that also could be mechanised, as easy to perform and yet difficult to perform well.
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Yu, Zhixiong, Yumeng Dai, Tingting Li, Wu Gu, Yi Yang, Xiang Li, Pai Peng, et al. "A Novel Pathway of Chlorimuron-Ethyl Biodegradation by Chenggangzhangella methanolivorans Strain CHL1 and Its Molecular Mechanisms." International Journal of Molecular Sciences 23, no. 17 (August 31, 2022): 9890. http://dx.doi.org/10.3390/ijms23179890.
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Chlorimuron-ethyl is a widely used herbicide in agriculture. However, uncontrolled chlorimuron-ethyl application causes serious environmental problems. Chlorimuron-ethyl can be effectively degraded by microbes, but the underlying molecular mechanisms are not fully understood. In this study, we identified the possible pathways and key genes involved in chlorimuron-ethyl degradation by the Chenggangzhangella methanolivorans strain CHL1, a Methylocystaceae strain with the ability to degrade sulfonylurea herbicides. Using a metabolomics method, eight intermediate degradation products were identified, and three pathways, including a novel pyrimidine-ring-opening pathway, were found to be involved in chlorimuron-ethyl degradation by strain CHL1. Transcriptome sequencing indicated that three genes (atzF, atzD, and cysJ) are involved in chlorimuron-ethyl degradation by strain CHL1. The gene knock-out and complementation techniques allowed for the functions of the three genes to be identified, and the enzymes involved in the different steps of chlorimuron-ethyl degradation pathways were preliminary predicted. The results reveal a previously unreported pathway and the key genes of chlorimuron-ethyl degradation by strain CHL1, which have implications for attempts to enrich the biodegradation mechanism of sulfonylurea herbicides and to construct engineered bacteria in order to remove sulfonylurea herbicide residues from environmental media.
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Thomson, Michael J., Sudip Biswas, Nikolaos Tsakirpaloglou, and Endang M. Septiningsih. "Functional Allele Validation by Gene Editing to Leverage the Wealth of Genetic Resources for Crop Improvement." International Journal of Molecular Sciences 23, no. 12 (June 12, 2022): 6565. http://dx.doi.org/10.3390/ijms23126565.
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Advances in molecular technologies over the past few decades, such as high-throughput DNA marker genotyping, have provided more powerful plant breeding approaches, including marker-assisted selection and genomic selection. At the same time, massive investments in plant genetics and genomics, led by whole genome sequencing, have led to greater knowledge of genes and genetic pathways across plant genomes. However, there remains a gap between approaches focused on forward genetics, which start with a phenotype to map a mutant locus or QTL with the goal of cloning the causal gene, and approaches using reverse genetics, which start with large-scale sequence data and work back to the gene function. The recent establishment of efficient CRISPR-Cas-based gene editing promises to bridge this gap and provide a rapid method to functionally validate genes and alleles identified through studies of natural variation. CRISPR-Cas techniques can be used to knock out single or multiple genes, precisely modify genes through base and prime editing, and replace alleles. Moreover, technologies such as protoplast isolation, in planta transformation, and the use of developmental regulatory genes promise to enable high-throughput gene editing to accelerate crop improvement.
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Domínguez-Santos, Rebeca, Katarina Kosalková, Isabel-Clara Sánchez-Orejas, Carlos Barreiro, Yolanda Pérez-Pertejo, Rosa M. Reguera, Rafael Balaña-Fouce, and Carlos García-Estrada. "Characterization of the Gene Encoding S-adenosyl-L-methionine (AdoMet) Synthetase in Penicillium chrysogenum; Role in Secondary Metabolism and Penicillin Production." Microorganisms 10, no. 1 (December 30, 2021): 78. http://dx.doi.org/10.3390/microorganisms10010078.
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The filamentous fungus Penicillium chrysogenum (recently reidentified as Penicillium rubens) is used in the industrial production of the β-lactam antibiotic penicillin. There are several mechanisms regulating the production of this antibiotic, acting both at the genetic and epigenetic levels, the latter including the modification of chromatin by methyltransferases. S-adenosyl-L-methionine (AdoMet) is the main donor of methyl groups for methyltransferases. In addition, it also acts as a donor of aminopropyl groups during the biosynthesis of polyamines. AdoMet is synthesized from L-methionine and ATP by AdoMet-synthetase. In silico analysis of the P. chrysogenum genome revealed the presence of a single gene (Pc16g04380) encoding a putative protein with high similarity to well-known AdoMet-synthetases. Due to the essential nature of this gene, functional analysis was carried out using RNAi-mediated silencing techniques. Knock-down transformants exhibited a decrease in AdoMet, S-adenosyl-L-homocysteine (AdoHcy), spermidine and benzylpenicillin levels, whereas they accumulated a yellow-orange pigment in submerged cultures. On the other hand, overexpression led to reduced levels of benzylpenicillin, thereby suggesting that the AdoMet synthetase, in addition to participate in primary metabolism, also controls secondary metabolism in P. chrysogenum.
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Ellrich, Jens. "Electric Low-Frequency Stimulation of the Tongue Induces Long-Term Depression of the Jaw-Opening Reflex in Anesthetized Mice." Journal of Neurophysiology 92, no. 6 (December 2004): 3332–37. http://dx.doi.org/10.1152/jn.00156.2004.
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Long-term depression (LTD) of somatosensory processing has been demonstrated in slice preparations of the spinal dorsal horn. Although LTD could be reliably induced in vitro, inconsistent results were encountered when the same types of experiments were conducted in adult animals in vivo. We addressed the hypothesis that LTD of orofacial sensorimotor processing can be induced in mice under general anesthesia. The effects of electric low- and high-frequency conditioning stimulation of the tongue on the sensorimotor jaw-opening reflex (JOR) elicited by electric tongue stimulation were investigated. Low-frequency stimulation induced a sustained decrease of the reflex integral for ≥1 h after the end of conditioning stimulation. After additional high-frequency stimulation, the reflex partly recovered from LTD. High-frequency stimulation alone induced a transient increase of the JOR integral for <10 min. The LTD of the sensorimotor jaw-opening reflex in anesthetized mice may be an appropriate model to investigate the central mechanisms and the pharmacology of synaptic plasticity in the orofacial region. The application of electrophysiological techniques in mice provides the opportunity to include adequate knock-out models to elucidate the neurobiology of LTD.
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Baylis, Howard A., and Rafael P. Vázquez-Manrique. "Reverse Genetic Strategies inCaenorhabditis elegans: Towards Controlled Manipulation of the Genome." Scientific World JOURNAL 11 (2011): 1394–410. http://dx.doi.org/10.1100/tsw.2011.126.
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Caenorhabditis eleganshas a complete annotated genome sequence that is augmented by increasing quantities of data from high-throughput postgenomic analyses. This has led to an increasing need to identify the biological functions of specific genes using reverse genetics, i.e., moving from gene to phenotype. Fundamental to this aim is the ability to alter the structure of particular genes by means that are not accessible to classical genetic strategies. Thus, one dream ofC. elegansresearchers is to establish a toolkit for the controlled manipulation of anylociwithin the genome. AlthoughC. elegansis amenable to a wide variety of genetic and molecular manipulations, controlled manipulation of endogenous genes by, for example, gene targeting has proved elusive until relatively recently. In this review, we describe and discuss the different methods available for the inactivation and modification of endogenous loci with a focus on strategies that permit some measure of control in this process. We describe methods that use random mutagenesis to isolate mutations in specific genes. We then focus on techniques that allow controlled manipulation of the genome: gene modification by transposon mobilisation, gene knock-out mediated by zinc-finger nucleases, and gene targeting by biolistic transformation.
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Kmita, Angelika, Rafał Dańko, Mariusz Holtzer, Józef Dańko, Dariusz Drożyński, Mateusz Skrzyński, Agnieszka Roczniak, Daniel Robert Gruszka, Jarosław Jakubski, and Sara Tapola. "Eco-Friendly Inorganic Binders: A Key Alternative for Reducing Harmful Emissions in Molding and Core-Making Technologies." International Journal of Molecular Sciences 25, no. 10 (May 17, 2024): 5496. http://dx.doi.org/10.3390/ijms25105496.
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Many years of foundry practice and much more accurate analytical methods have shown that sands with organic binders, in addition to their many technological advantages, pose risks associated with the emission of many compounds, including harmful ones (e.g., formaldehyde, phenol, benzene, polycyclic aromatic hydrocarbons, and sulfur), arising during the pouring of liquid casting alloys into molds, their cooling, and knock-out. The aim of this research is to demonstrate the potential benefits of adopting inorganic binders in European iron foundries. This will improve the environmental and working conditions by introducing cleaner and more ecological production methods, while also ranking the tested binders studied in terms of their harmful content. The article pays special attention to the analysis of seven innovative inorganic binders and one organic binder, acting as a reference for emissions of gases from the BTEX (benzene, toluene, ethylbenzene, and xylenes) and PAHs (polycyclic aromatic hydrocarbons) groups and other compounds such as phenol, formaldehyde, and isocyanates (MDI and TDI) generated during the mold pouring process with liquid metals. The knowledge gained will, for the first time, enrich the database needed to update the Reference Document on The Best Available Techniques for the Smitheries and Foundries Industry (SF BREF).
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Dalla Costa, Lorenza, Daniela Vinciguerra, Lisa Giacomelli, Umberto Salvagnin, Stefano Piazza, Katia Spinella, Mickael Malnoy, Claudio Moser, and Ugo Marchesi. "Integrated approach for the molecular characterization of edited plants obtained via Agrobacterium tumefaciens-mediated gene transfer." European Food Research and Technology 248, no. 1 (November 2, 2021): 289–99. http://dx.doi.org/10.1007/s00217-021-03881-0.
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AbstractAgrobacterium tumefaciens-mediated gene transfer—actually the most used method to engineer plants—may lead to integration of multiple copies of T-DNA in the plant genome, as well as to chimeric tissues composed of modified cells and wild type cells. A molecular characterization of the transformed lines is thus a good practice to select the best ones for further investigation. Nowadays, several quantitative and semi-quantitative techniques are available to estimate the copy number (CN) of the T-DNA in genetically modified plants. In this study, we compared three methods based on (1) real-time polymerase chain reaction (qPCR), (2) droplet digital PCR (ddPCR), and (3) next generation sequencing (NGS), to carry out a molecular characterization of grapevine edited lines. These lines contain a knock-out mutation, obtained via CRISPR/Cas9 technology, in genes involved in plant susceptibility to two important mildew diseases of grapevine. According to our results, qPCR and ddPCR outputs are largely in agreement in terms of accuracy, especially for low CN values, while ddPCR resulted more precise than qPCR. With regard to the NGS analysis, the CNs detected with this method were often not consistent with those calculated by qPCR and ddPCR, and NGS was not able to discriminate the integration points in three out of ten lines. Nevertheless, the NGS method can positively identify T-DNA truncations or the presence of tandem/inverted repeats, providing distinct and relevant information about the transgene integration asset. Moreover, the expression analysis of Cas9 and single guide RNA (sgRNA), and the sequencing of the target site added new information to be related to CN data. This work, by reporting a practical case-study on grapevine edited lines, explores pros and cons of the most advanced diagnostic techniques available for the precocious selection of the proper transgenic material. The results may be of interest both to scientists developing new transgenic lines, and to laboratories in charge of GMO control.
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Fertaki, Stefani, Panagiota Giannoutsou, and Malvina G. Orkoula. "Combining Raman Microspectroscopy and X-ray Microcomputed Tomography for the Study of Bone Quality in Apolipoprotein-Deficient Animal Models." Molecules 28, no. 20 (October 20, 2023): 7196. http://dx.doi.org/10.3390/molecules28207196.
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Raman microspectroscopy and X-ray microcomputed tomography (micro-CT) were used for assessment of the quality of the femur and tibia bones in apolipoprotein-deficient mice compared to control littermates. The cortical and trabecular bone was investigated separately. Raman spectra revealed no differences in the bioapatite-to-collagenous matrix ratio of the cortical bone. The quantities of calcium and collagen, which were measured using atomic absorption spectrometry and thermogravimetric analysis, respectively, were also found to be equal in the two groups. Density and morphometric parameters, which were measured using micro-CT, verified the cortical mineral stability. Bone quality indices were measured using Raman spectra. A decreased collagen crosslink (trivalent-to-divalent) ratio revealed delayed maturation of the collagen network. Such a decrease has been reported in the literature to be connected to decreased bone strength. For the trabecular bone, micro-CT revealed severe osteoporosis in the knock-out group, which was evident from a decreased mineral density, trabecular thickness and increased bone surface/volume ratio. The trabecular bone was not accessible for Raman spectroscopy. According to these results, the cortical and trabecular femur bone is expected to exhibit proneness to fracturing, each for a different reason. A combination of the two techniques was regarded as necessary for an overall assessment of bone quality.
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Martín-Valmaseda, Marina, Sama Rahimi Devin, Germán Ortuño-Hernández, Cristian Pérez-Caselles, Sayyed Mohammad Ehsan Mahdavi, Geza Bujdoso, Juan Alfonso Salazar, Pedro Martínez-Gómez, and Nuria Alburquerque. "CRISPR/Cas as a Genome-Editing Technique in Fruit Tree Breeding." International Journal of Molecular Sciences 24, no. 23 (November 23, 2023): 16656. http://dx.doi.org/10.3390/ijms242316656.
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CRISPR (short for “Clustered Regularly Interspaced Short Palindromic Repeats”) is a technology that research scientists use to selectively modify the DNA of living organisms. CRISPR was adapted for use in the laboratory from the naturally occurring genome-editing systems found in bacteria. In this work, we reviewed the methods used to introduce CRISPR/Cas-mediated genome editing into fruit species, as well as the impacts of the application of this technology to activate and knock out target genes in different fruit tree species, including on tree development, yield, fruit quality, and tolerance to biotic and abiotic stresses. The application of this gene-editing technology could allow the development of new generations of fruit crops with improved traits by targeting different genetic segments or even could facilitate the introduction of traits into elite cultivars without changing other traits. However, currently, the scarcity of efficient regeneration and transformation protocols in some species, the fact that many of those procedures are genotype-dependent, and the convenience of segregating the transgenic parts of the CRISPR system represent the main handicaps limiting the potential of genetic editing techniques for fruit trees. Finally, the latest news on the legislation and regulations about the use of plants modified using CRISPR/Cas systems has been also discussed.
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Shen, Hui, Ying Zhou, Changguang Liao, Qiaoli Xie, Guoping Chen, Zongli Hu, and Ting Wu. "The AlkB Homolog SlALKBH10B Negatively Affects Drought and Salt Tolerance in Solanum lycopersicum." International Journal of Molecular Sciences 25, no. 1 (December 22, 2023): 173. http://dx.doi.org/10.3390/ijms25010173.
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ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a single-protein repair system that safeguards cellular DNA and RNA against the harmful effects of alkylating agents. ALKBH10B, the first discovered N6-methyladenosine (m6A) demethylase in Arabidopsis (Arabidopsis thaliana), has been shown to regulate plant growth, development, and stress responses. However, until now, the functional role of the plant ALKBH10B has solely been reported in arabidopsis, cotton, and poplar, leaving its functional implications in other plant species shrouded in mystery. In this study, we identified the AlkB homolog SlALKBH10B in tomato (Solanum lycopersicum) through phylogenetic and gene expression analyses. SlALKBH10B exhibited a wide range of expression patterns and was induced by exogenous abscisic acid (ABA) and abiotic stresses. By employing CRISPR/Cas9 gene editing techniques to knock out SlALKBH10B, we observed an increased sensitivity of mutants to ABA treatment and upregulation of gene expression related to ABA synthesis and response. Furthermore, the Slalkbh10b mutants displayed an enhanced tolerance to drought and salt stress, characterized by higher water retention, accumulation of photosynthetic products, proline accumulation, and lower levels of reactive oxygen species and cellular damage. Collectively, these findings provide insights into the negative impact of SlALKBH10B on drought and salt tolerance in tomato plant, expanding our understanding of the biological functionality of SlALKBH10B.
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Efendi, Siska Chiko, Vindy Fetricia Amanda, and Yaherwandi Yaherwandi. "KELIMPAHAN POPULASI Helopeltis sp. DAN TINGKAT KERUSAKAN BUAH KAKAO DI KECAMATAN SITIUNG KABUPATEN DHARMASRAYA." Agrika 14, no. 1 (May 30, 2020): 33. http://dx.doi.org/10.31328/ja.v14i1.1275.
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ABSTRAKDharmasraya merupakan kabupaten yang berpotensi untuk pengembangan kakao, terbukti dengan meningkatnya luas areal perkebunan setiap tahun. Pengembangan kakao di Dharmasraya dihadapkan pada beberapa kendala yang mengakibatkan produksi kakao rendah, salah satunya adalah serangan kepik penghisap buah kakao (Helopeltis sp.) Tujuan penelitian ini adalah mempelajari kelimpahan populasi dan tingkat kerusakan kepik penghisap buah kakao. Penelitian dilaksanakan di Kecamatan Sitiung Kabupaten Dharmasraya yang terdiri dari tiga nagari yaitu Siguntur, Sitiung dan Gunung Medan. Penelitian dilaksanakan pada bulan November 2018 sampai dengan Januari 2019. Penelitian ini berbentuk survei menggunakan metode Purposive Random Sampling. Penentuan tanaman sampel dilakukan secara sistematis, sehingga terdapat 30 batang tanaman sampel pada satu lahan. Serangga contoh dikoleksi dengan cara hand collecting dan teknik chemical knock down. Kelimpahan kepik penghisap buah kakao yang diperoleh pada penelitian tergolong rendah yaitu 79 individu dengan rata-rata 0,23-0,36 individu/batang. Persentase kerusakan tergolong tinggi terdapat di Nagari Siguntur yakni 81,43% dan terendah di Nagari Gunung Medan yakni 70,36%. Intensitas kerusakan yang tertinggi terdapat di Nagari Siguntur yakni 73,12% dan terendah di Nagari Gunung Medan yakni 68,15%.ABSTRACTDharmasraya is a district that has the potential for cacao development, proven by increasing the cacao plantation area every year. Cacao development in Dharmasraya was faced with several obstacleswhich resulted in low cacao production. One of the obstacles in the cultivation process is (Helopeltis sp.) Therefore,the purpose of this research was to study the population abundance and the damage levels of to cacao fruit sucking bugs. Research was carried out in SitiungSubdistrict, Dharmasraya which consisted of three nagari namely Siguntur, Sitiung and Gunung Medan. The research began in November 2018 to January 2019. This research was a survey using the Purposive Random Sampling method. The sample plants were determine systematically, so there were 30 plant samples in each nagari. Insect samples were collected by hand collecting and chemical knock down techniques. The abundance of cacao fruit sucking bugs was 79 individuals, with averages were 0.23 – 0.36 individual / plant. The percentage of damage was high, where the highest was obtained in Siguntur 81.43% and the lowest was in Gunung Medan 70.36%. In addition, the highest damage intensity was found in Sigunturthat was 73.12% and the lowest in Gunung Medan that was 68.15%.
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Plenker, T. "Computer aided decision support on choosing the right technology for sewer rehabilitation." Water Science and Technology 46, no. 6-7 (September 1, 2002): 403–10. http://dx.doi.org/10.2166/wst.2002.0706.
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The decision, whether and how to rehabilitate a damaged sewer is a complex one. Detailed information available from sewer inspection can be used for evaluating its condition and determining the urgency of rehabilitation. There are various rehabilitation techniques, from conventional open trenching to newer trenchless construction. Municipal waste water departments and companies must use their tight budgets in the most efficient and responsible way. Therefore, a decision support system for choosing the right/best sewer rehab technology is urgently needed. For a computer aided selection of the right technology for sewer rehabilitation a formalised weighing and ranking procedure should be used. That procedure should be based on a detailed description of the rehabilitation task. So the state of the existing sewer has to be identified, future technical requirements have to be formulated and specific local restraints taken into account. First, rehab technologies not capable of solving the problem under given constraints, are eliminated by “knock-out” criteria. Thus the number of potential solutions is reduced to those which are subsequently compared (computer aided) in pairs. There is no scoring hocus-pocus and criteria weighing in this decision system. The result is found just by comparing pairs of options on as many criteria as needed, and these criteria may be measured on different scales and need not be independent from each other.
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Seibel-Ehlert, Ulla, Nicole Plank, Asuka Inoue, Guenther Bernhardt, and Andrea Strasser. "Label-Free Investigations on the G Protein Dependent Signaling Pathways of Histamine Receptors." International Journal of Molecular Sciences 22, no. 18 (September 9, 2021): 9739. http://dx.doi.org/10.3390/ijms22189739.
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G protein activation represents an early key event in the complex GPCR signal transduction process and is usually studied by label-dependent methods targeting specific molecular events. However, the constrained environment of such “invasive” techniques could interfere with biological processes. Although histamine receptors (HRs) represent (evolving) drug targets, their signal transduction is not fully understood. To address this issue, we established a non-invasive dynamic mass redistribution (DMR) assay for the human H1–4Rs expressed in HEK cells, showing excellent signal-to-background ratios above 100 for histamine (HIS) and higher than 24 for inverse agonists with pEC50 values consistent with literature. Taking advantage of the integrative nature of the DMR assay, the involvement of endogenous Gαq/11, Gαs, Gα12/13 and Gβγ proteins was explored, pursuing a two-pronged approach, namely that of classical pharmacology (G protein modulators) and that of molecular biology (Gα knock-out HEK cells). We showed that signal transduction of hH1–4Rs occurred mainly, but not exclusively, via their canonical Gα proteins. For example, in addition to Gαi/o, the Gαq/11 protein was proven to contribute to the DMR response of hH3,4Rs. Moreover, the Gα12/13 was identified to be involved in the hH2R mediated signaling pathway. These results are considered as a basis for future investigations on the (patho)physiological role and the pharmacological potential of H1–4Rs.
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Bonin, Robert P., Loren J. Martin, John F. MacDonald, and Beverley A. Orser. "α5GABAA Receptors Regulate the Intrinsic Excitability of Mouse Hippocampal Pyramidal Neurons." Journal of Neurophysiology 98, no. 4 (October 2007): 2244–54. http://dx.doi.org/10.1152/jn.00482.2007.
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GABAA receptors generate both phasic and tonic forms of inhibition. In hippocampal pyramidal neurons, GABAA receptors that contain the α5 subunit generate a tonic inhibitory conductance. The physiological role of this tonic inhibition is uncertain, although α5GABAA receptors are known to influence hippocampal-dependent learning and memory processes. Here we provide evidence that α5GABAA receptors regulate the strength of the depolarizing stimulus that is required to generate an action potential in pyramidal neurons. Neurons from α5 knock-out (α5−/−) and wild-type (WT) mice were studied in brain slices and cell cultures using whole cell and perforated-patch-clamp techniques. Membrane resistance was 1.6-fold greater in α5−/− than in WT neurons, but the resting membrane potential and chloride equilibrium potential were similar. Membrane hyperpolarization evoked by an application of exogenous GABA was greater in WT neurons. Inhibiting the function of α5GABAA receptor with nonselective (picrotoxin) or α5 subunit-selective (L-655,708) compounds depolarized WT neurons by ∼3 mV, whereas no change was detected in α5−/− neurons. The depolarizing current required to generate an action potential was twofold greater in WT than in α5−/− neurons, whereas the slope of the input-output relationship for action potential firing was similar. We conclude that shunting inhibition mediated by α5GABAA receptors regulates the firing of action potentials and may synchronize network activity that underlies hippocampal-dependent behavior.
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Simpson, Evan, and Richard J. Santen. "Celebrating 75 years of oestradiol." Journal of Molecular Endocrinology 55, no. 3 (October 5, 2015): T1—T20. http://dx.doi.org/10.1530/jme-15-0128.
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Oestrogens exert important effects on the reproductive as well as many other organ systems in both men and women. The history of the discovery of oestrogens, the mechanisms of their synthesis, and their therapeutic applications are very important components of the fabric of endocrinology. These aspects provide the rationale for highlighting several key components of this story. Two investigators, Edward Doisy and Alfred Butenandt, purified and crystalized oestrone nearly simultaneously in 1929, and Doisy later discovered oestriol and oestradiol. Butenandt won the Nobel Prize for this work and Doisy's had to await his purification of vitamin K. Early investigators quickly recognized that oestrogens must be synthesized from androgens and later investigators called this process aromatization. The aromatase enzyme was then characterized, its mechanism determined, and its structure identified after successful crystallization. With the development of knock-out methodology, the precise effects of oestrogen in males and females were defined and clinical syndromes of deficiency and excess described. Their discovery ultimately led to the development of oral contraceptives, treatment of menopausal symptoms, therapies for breast cancer, and induction of fertility, among others. The history of the use of oestrogens for postmenopausal women to relieve symptoms has been characterized by cyclic periods of enthusiasm and concern. The individuals involved in these studies, the innovative thinking required, and the detailed understanding made possible by evolving biologic and molecular techniques provide many lessons for current endocrinologists.