Journal articles on the topic 'Iterative Simultaneous Yeast Display'
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1
Wen, Fei, Jie Sun, and Huimin Zhao. "Yeast Surface Display of Trifunctional Minicellulosomes for Simultaneous Saccharification and Fermentation of Cellulose to Ethanol." Applied and Environmental Microbiology 76, no. 4 (December 18, 2009): 1251–60. http://dx.doi.org/10.1128/aem.01687-09.
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ABSTRACT By combining cellulase production, cellulose hydrolysis, and sugar fermentation into a single step, consolidated bioprocessing (CBP) represents a promising technology for biofuel production. Here we report engineering of Saccharomyces cerevisiae strains displaying a series of uni-, bi-, and trifunctional minicellulosomes. These minicellulosomes consist of (i) a miniscaffoldin containing a cellulose-binding domain and three cohesin modules, which was tethered to the cell surface through the yeast a-agglutinin adhesion receptor, and (ii) up to three types of cellulases, an endoglucanase, a cellobiohydrolase, and a β-glucosidase, each bearing a C-terminal dockerin. Cell surface assembly of the minicellulosomes was dependent on expression of the miniscaffoldin, indicating that formation of the complex was dictated by the high-affinity interactions between cohesins and dockerins. Compared to the unifunctional and bifunctional minicellulosomes, the quaternary trifunctional complexes showed enhanced enzyme-enzyme synergy and enzyme proximity synergy. More importantly, surface display of the trifunctional minicellulosomes gave yeast cells the ability to simultaneously break down and ferment phosphoric acid-swollen cellulose to ethanol with a titer of ∼1.8 g/liter. To our knowledge, this is the first report of a recombinant yeast strain capable of producing cell-associated trifunctional minicellulosomes. The strain reported here represents a useful engineering platform for developing CBP-enabling microorganisms and elucidating principles of cellulosome construction and mode of action.
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Ito, Junji, Akihiko Kosugi, Tsutomu Tanaka, Kouichi Kuroda, Seiji Shibasaki, Chiaki Ogino, Mitsuyoshi Ueda, Hideki Fukuda, Roy H. Doi, and Akihiko Kondo. "Regulation of the Display Ratio of Enzymes on the Saccharomyces cerevisiae Cell Surface by the Immunoglobulin G and Cellulosomal Enzyme Binding Domains." Applied and Environmental Microbiology 75, no. 12 (May 1, 2009): 4149–54. http://dx.doi.org/10.1128/aem.00318-09.
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ABSTRACT We constructed a novel cell surface display system to control the ratio of target proteins on the Saccharomyces cerevisiae cell surface, using two pairs of protein-protein interactions. One protein pair is the Z domain of protein A derived from Staphylococcus aureus and the Fc domain of human immunoglobulin G. The other is the cohesin (Coh) and dockerin (Dock) from the cellulosome of Clostridium cellulovorans. In this proposed displaying system, the scaffolding proteins (fusion proteins of Z and Coh) were displayed on the cell surface by fusing with the 3′ half of α-agglutinin, and the target proteins fused with Fc or Dock were secreted. As a target protein, a recombinant Trichoderma reesei endoglucanase II (EGII) was secreted into the medium and immediately displayed on the yeast cell surface via the Z and Fc domains. Display of EGII on the cell surface was confirmed by hydrolysis of β-glucan as a substrate, and EGII activity was detected in the cell pellet fraction. Finally, two enzymes, EGII and Aspergillus aculeatus β-glucosidase 1, were codisplayed on the cell surface via Z-Fc and Dock-Coh interactions, respectively. As a result, the yeast displaying two enzymes hydrolyzed β-glucan to glucose very well. These results strongly indicated that the proposed strategy, the simultaneous display of two enzymes on the yeast cell surface, was accomplished by quantitatively controlling the display system using affinity binding.
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Luo, Ruiqi, Baole Qu, Lili An, Yun Zhao, Yang Cao, Peng Ren, and Haiying Hang. "Simultaneous Maturation of Single Chain Antibody Stability and Affinity by CHO Cell Display." Bioengineering 9, no. 8 (August 2, 2022): 360. http://dx.doi.org/10.3390/bioengineering9080360.
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Antibody stability and affinity are two important features of its applications in therapy and diagnosis. Antibody display technologies such as yeast and bacterial displays have been successfully used for improving both affinity and stability. Although mammalian cell display has also been utilized for maturing antibody affinity, it has not been applied for improving antibody stability. Previously, we developed a Chinese hamster ovary (CHO) cell display platform in which activation-induced cytidine deaminase (AID) was used to induce antibody mutation, and antibody affinity was successfully matured using the platform. In the current study, we developed thermo-resistant (TR) CHO cells for the purpose of maturing both antibody stability and affinity. We cultured TR CHO cells displaying an antibody mutant library and labeled them at temperatures above 41 °C, enriching cells that displayed antibody mutants with both the highest affinities and the highest display levels. To evaluate our system, we chose three antibodies to improve their affinities and stabilities. We succeeded in simultaneously improving both affinities and stabilities of all three antibodies. Of note, we obtained an anti-TNFα antibody mutant with a Tm (dissolution temperature) value 12 °C higher and affinity 160-fold greater than the parent antibody after two rounds of cell proliferation and flow cytometric sorting. By using CHO cells with its advantages in protein folding, post-translational modifications, and code usage, this procedure is likely to be widely used in maturing antibodies and other proteins in the future.
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Chen, Hongling, Siyuan Cao, Shaohuan Zu, Bo Yang, Shian Shen, and Xiaoming Sun. "Iterative deblending using the POCS algorithm in the approximate flattened domain." Journal of Geophysics and Engineering 15, no. 4 (April 13, 2018): 1104–19. http://dx.doi.org/10.1093/jge/aaa981.
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Abstract We proposed an improved method to eliminate the interference generated by simultaneous-source acquisition, which can help shorten the acquisition period and improve the quality of seismic data. An iterative mathematical framework is devised, which uses the projection onto convex sets algorithm to estimate the blending noise subtracted from the pseudo-deblended data to separate the blended data in an iterative way. Differently to the conventional method using the coherent-promoting operator only based on the curvelet transform, we combine the curvelet transform and the approximate flattened operator (AFO) to improve the deblended result, which can flatten seismic events approximately to preserve the details of useful signals. This is the first time that the AFO and the curvelet transform are combined to enhance the effect of the coherent-promoting operator and improve the performance of deblending. To display the advantages of the improved method, we use both simulated synthetic data and field data examples to compare and analyse the deblended results using our method and the conventional method, and confirm that the improved method can perform better.
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Albakri, Maram B., Yuwei Jiang, and Patrick Lajoie. "Polyglutamine toxicity assays highlight the advantages of mScarlet for imaging in Saccharomyces cerevisiae." F1000Research 7 (August 10, 2018): 1242. http://dx.doi.org/10.12688/f1000research.15829.1.
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Development of fluorescent proteins (FPs) enabled researchers to visualize protein localization and trafficking in living cells and organisms. The extended palette of available FPs allows simultaneous detection of multiples fluorescent fusion proteins. Importantly, FPs are originally derived from different organisms from jelly fish to corals and each FP display its own biophysical properties. Among these properties, the tendency of FPs to oligomerize inherently affects the behavior of its fusion partner. Here we employed the budding yeast Saccharomyces cerevisiae to determine the impact of the latest generation of red FPs on their binding partner. We used a yeast assay based on the aggregation and toxicity of misfolded polyQ expansion proteins linked to Huntington’s disease. Since polyQ aggregation and toxicity are highly dependent on the sequences flanking the polyQ region, polyQ expansions provide an ideal tool to assess the impact of FPs on their fusion partners. We found that unlike yemRFP and yFusionRed, the synthetically engineered ymScarlet displayed severe polyQ toxicity and aggregation similar to what is observed for green FP variants. Our data indicate that ymScarlet might have significant advantages over the previous generation of red FPs for use in fluorescent fusions in yeast.
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Fujita, Yasuya, Junji Ito, Mitsuyoshi Ueda, Hideki Fukuda, and Akihiko Kondo. "Synergistic Saccharification, and Direct Fermentation to Ethanol, of Amorphous Cellulose by Use of an Engineered Yeast Strain Codisplaying Three Types of Cellulolytic Enzyme." Applied and Environmental Microbiology 70, no. 2 (February 2004): 1207–12. http://dx.doi.org/10.1128/aem.70.2.1207-1212.2004.
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ABSTRACT A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on α-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus β-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of α-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of β-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying β-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.
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Yin, Yiming, Brian D. Quinlan, Tianling Ou, Yan Guo, Wenhui He, and Michael Farzan. "In vitro affinity maturation of broader and more-potent variants of the HIV-1–neutralizing antibody CAP256-VRC26.25." Proceedings of the National Academy of Sciences 118, no. 29 (July 14, 2021): e2106203118. http://dx.doi.org/10.1073/pnas.2106203118.
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Three variable 2 (V2) loops of HIV-1 envelope glycoprotein (Env) trimer converge at the Env apex to form the epitope of an important classes of HIV-1 broadly neutralizing antibodies (bNAbs). These V2-glycan/apex antibodies are exceptionally potent but less broad (∼60 to 75%) than many other bNAbs. Their CDRH3 regions are typically long, acidic, and tyrosine sulfated. Tyrosine sulfation complicates efforts to improve these antibodies through techniques such as phage or yeast display. To improve the breadth of CAP256-VRC26.25 (VRC26.25), a very potent apex antibody, we adapted and extended a B cell display approach. Specifically, we used CRISPR/Cas12a to introduce VRC26.25 heavy- and light-chain genes into their respective loci in a B cell line, ensuring that each cell expresses a single VRC26.25 variant. We then diversified these loci through activation-induced cytidine deaminase–mediated hypermutation and homology-directed repair using randomized CDRH3 sequences as templates. Iterative sorting with soluble Env trimers and further randomization selected VRC26.25 variants with successively improving affinities. Three mutations in the CDRH3 region largely accounted for this improved affinity, and VRC26.25 modified with these mutations exhibited greater breadth and potency than the original antibody. Our data describe a broader and more-potent form of VRC26.25 as well as an approach useful for improving the breadth and potency of antibodies with functionally important posttranslational modifications.
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Goehring, April S., David M. Rivers, and George F. Sprague. "Urmylation: A Ubiquitin-like Pathway that Functions during Invasive Growth and Budding in Yeast." Molecular Biology of the Cell 14, no. 11 (November 2003): 4329–41. http://dx.doi.org/10.1091/mbc.e03-02-0079.
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Ubiquitin is a small modifier protein that is conjugated to substrates to target them for degradation. Recently, a surprising number of ubiquitin-like proteins have been identified that also can be attached to proteins. Herein, we identify two molecular functions for the posttranslational protein modifier from Saccharomyces cerevisiae, Urm1p. Simultaneous loss of Urm1p and Cla4p, a p21-activated kinase that functions in budding, is lethal. This result suggests a role for the urmylation pathway in budding. Furthermore, loss of the urmylation pathway causes defects in invasive growth and confers sensitivity to rapamycin. Our results indicate that the sensitivity to rapamycin is due to a genetic interaction with the TOR pathway, which is important for regulation of cell growth in response to nutrients. We have found that Urm1p can be attached to a number of proteins. Loss of five genes that are also essential in a cla4Δ strain, NCS2, NCS6, ELP2, ELP6, and URE2, affect the level of at least one Urm1p conjugate. Moreover, these five genes have a role in invasive growth and display genetic interactions with the TOR pathway. In summary, our results suggest the urmylation pathway is involved in nutrient sensing and budding.
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Schmieder, Felix, Lars Büttner, Tony Hanitzsch, Volker Busskamp, and Jürgen W. Czarske. "Two-Wavelength Computational Holography for Aberration-Corrected Simultaneous Optogenetic Stimulation and Inhibition of In Vitro Biological Samples." Applied Sciences 12, no. 5 (February 22, 2022): 2283. http://dx.doi.org/10.3390/app12052283.
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Optogenetics is a versatile toolset for the functional investigation of excitable cells such as neurons and cardiomyocytes in vivo and in vitro. While monochromatic illumination of these cells for either stimulation or inhibition already enables a wide range of studies, the combination of activation and silencing in one setup facilitates new experimental interrogation protocols. In this work, we present a setup for the simultaneous holographic stimulation and inhibition of multiple cells in vitro. The system is based on two fast ferroelectric liquid crystal spatial light modulators with frame rates of up to 1.7 kHz. Thereby, we are able to illuminate up to about 50 single spots with better than cellular resolution and without crosstalk, perfectly suited for refined network analysis schemes. System-inherent aberrations are corrected by applying an iterative optimization scheme based on Zernike polynomials. These are superposed on the same spatial light modulators that display the pattern-generating holograms, hence no further adaptive optical elements are needed for aberration correction. A near-diffraction-limited spatial resolution is achieved over the whole field of view, enabling subcellular optogenetic experiments by just choosing an appropriate microscope objective. The setup can pave the way for a multitude of optogenetic experiments, in particular with cardiomyocytes and neural networks.
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Tsai, Shen-Long, Jeongseok Oh, Shailendra Singh, Ruizhen Chen, and Wilfred Chen. "Functional Assembly of Minicellulosomes on the Saccharomyces cerevisiae Cell Surface for Cellulose Hydrolysis and Ethanol Production." Applied and Environmental Microbiology 75, no. 19 (August 14, 2009): 6087–93. http://dx.doi.org/10.1128/aem.01538-09.
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ABSTRACT We demonstrated the functional display of a miniscaffoldin on the Saccharomyces cerevisiae cell surface consisting of three divergent cohesin domains from Clostridium thermocellum (t), Clostridium cellulolyticum (c), and Ruminococcus flavefaciens (f). Incubation with Escherichia coli lysates containing an endoglucanase (CelA) fused with a dockerin domain from C. thermocellum (At), an exoglucanase (CelE) from C. cellulolyticum fused with a dockerin domain from the same species (Ec), and an endoglucanase (CelG) from C. cellulolyticum fused with a dockerin domain from R. flavefaciens (Gf) resulted in the assembly of a functional minicellulosome on the yeast cell surface. The displayed minicellulosome retained the synergistic effect for cellulose hydrolysis. When a β-glucosidase (BglA) from C. thermocellum tagged with the dockerin from R. flavefaciens was used in place of Gf, cells displaying the new minicellulosome exhibited significantly enhanced glucose liberation and produced ethanol directly from phosphoric acid-swollen cellulose. The final ethanol concentration of 3.5 g/liter was 2.6-fold higher than that obtained by using the same amounts of added purified cellulases. The overall yield was 0.49 g of ethanol produced per g of carbohydrate consumed, which corresponds to 95% of the theoretical value. This result confirms that simultaneous and synergistic saccharification and fermentation of cellulose to ethanol can be efficiently accomplished with a yeast strain displaying a functional minicellulosome containing all three required cellulolytic enzymes.
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Yosef, Gal, Hezi Hayun, and Niv Papo. "Simultaneous targeting of CD44 and MMP9 catalytic and hemopexin domains as a therapeutic strategy." Biochemical Journal 478, no. 5 (March 12, 2021): 1139–57. http://dx.doi.org/10.1042/bcj20200628.
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Crosstalk of the oncogenic matrix metalloproteinase-9 (MMP9) and one of its ligands, CD44, involves cleavage of CD44 by the MMP9 catalytic domain, with the CD44–MMP9 interaction on the cell surface taking place through the MMP9 hemopexin domain (PEX). This interaction promotes cancer cell migration and invasiveness. In concert, MMP9-processed CD44 induces the expression of MMP9, which degrades ECM components and facilitates growth factor release and activation, cancer cell invasiveness, and metastasis. Since both MMP9 and CD44 contribute to cancer progression, we have developed a new strategy to fully block this neoplastic process by engineering a multi-specific inhibitor that simultaneously targets CD44 and both the catalytic and PEX domains of MMP9. Using a yeast surface display technology, we first obtained a high-affinity inhibitor for the MMP9 catalytic domain, which we termed C9, by modifying a natural non-specific MMP inhibitor, N-TIMP2. We then conjugated C9 via a flexible linker to PEX, thereby creating a multi-specific inhibitor (C9-PEX) that simultaneously targets the MMP9 catalytic and PEX domains and CD44. It is likely that, via its co-localization with CD44, C9-PEX may compete with MMP9 localization on the cell surface, thereby inhibiting MMP9 catalytic activity, reducing MMP9 cellular levels, interfering with MMP9 homodimerization, and reducing the activation of downstream MAPK/ERK pathway signaling. The developed platform could be extended to other oncogenic MMPs as well as to other important target proteins, thereby offering great promise for creating novel multi-specific therapeutics for cancer and other diseases.
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Yukawa, Masashi, Tomoki Kawakami, Corinne Pinder, and Takashi Toda. "Two XMAP215/TOG Microtubule Polymerases, Alp14 and Dis1, Play Non-Exchangeable, Distinct Roles in Microtubule Organisation in Fission Yeast." International Journal of Molecular Sciences 20, no. 20 (October 15, 2019): 5108. http://dx.doi.org/10.3390/ijms20205108.
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Proper bipolar spindle assembly underlies accurate chromosome segregation. A cohort of microtubule-associated proteins orchestrates spindle microtubule formation in a spatiotemporally coordinated manner. Among them, the conserved XMAP215/TOG family of microtubule polymerase plays a central role in spindle assembly. In fission yeast, two XMAP215/TOG members, Alp14 and Dis1, share essential roles in cell viability; however how these two proteins functionally collaborate remains undetermined. Here we show the functional interplay and specification of Alp14 and Dis1. Creation of new mutant alleles of alp14, which display temperature sensitivity in the absence of Dis1, enabled us to conduct detailed analyses of a double mutant. We have found that simultaneous inactivation of Alp14 and Dis1 results in early mitotic arrest with very short, fragile spindles. Intriguingly, these cells often undergo spindle collapse, leading to a lethal “cut” phenotype. By implementing an artificial targeting system, we have shown that Alp14 and Dis1 are not functionally exchangeable and as such are not merely redundant paralogues. Interestingly, while Alp14 promotes microtubule nucleation, Dis1 does not. Our results uncover that the intrinsic specification, not the spatial regulation, between Alp14 and Dis1 underlies the collaborative actions of these two XMAP215/TOG members in mitotic progression, spindle integrity and genome stability.
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DE SMET, HENDRIK, and LIESBET HEYVAERT. "The meaning of the English present participle." English Language and Linguistics 15, no. 3 (October 4, 2011): 473–98. http://dx.doi.org/10.1017/s136067431100013x.
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While earlier descriptions of the English present participle have tended to be too general or too exclusively focused on its progressive meaning, this article aims to present an account of the meanings of the English present participle that captures their full richness. It starts from the observation that many (though not all) present participle clauses/phrases are paradigmatically related to adjectival phrases, as manifested in their distributional properties (e.g. a challenging year, those living alone). The article analyses the semantic effects that arise from the tension between the verbal semantics of the participial stem and the adjectival semantics of the syntactic slot. These effects involve accommodation of the verbal situation to the requirement that a situation is represented as time-stable and as simultaneous to some contextually given reference time. The progressive meaning is one such semantic effect, but participles may also assume iterative, habitual or gnomic readings. Some construction-specific semantic extensions of this adjectival template are identified and a tentative explanation is offered for them. Those constructions where the present participle has lost its semantic association with adjective phrases, such as the progressive construction and integrated participle clauses, are shown to display loosening or specialization of semantic constraints.
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Wang, Yupeng, Jordan Jarjour, Jim Havranek, David Baker, Andrew M. Scharenberg, and David J. Rawlings. "Engineering XID(Btk) site specific Homing Endonucleases for gene repair in hematopoietic stem cells (42.19)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 42.19. http://dx.doi.org/10.4049/jimmunol.182.supp.42.19.
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Abstract X-linked agammaglobulinemia (XLA), a human immunodeficiency caused by mutations in Btk, represents an ideal candidate target for HSC gene therapy. However, current gene replacement methods carry risks of insertional mutagenesis as well as gene silencing. In contrast, gene repair induced by site specific DNA double strand breaks (DSB) offers a novel means to treat genetic disease with reduced risk and the potential to utilize endogenous gene expression control elements. LAGLIDADG Homing Endonucleases (LHEs) have been shown to efficiently induce site specific DSB and markerless modification of genes without toxicity. Due to the limited number of LHEs currently available, engineering site specific LHEs represents a critical step for enabling technology for DSB-induced gene modification. In the current study we have begun to generate a site specific variant of the LHE I-AniI, targeted to recognize a unique mutation in the mouse BTK gene in the XID model of human XLA. Our approaches combine: protein computational design with random mutation and selection using yeast library surface display; or alternatively, iterative somatic hyper-mutation and selection within DT40 B cell library targeted to the light chain locus. Using these combinatorial methods we have generated a panel of I-AniI variants with novel DNA binding and cleavage specificity towards the XID site. Preliminary characterization of a subset of these I-AniI variants will be presented. Ultimately, new I-Anil variants and donor DNA repair template will be delivered into HSC derived from XID/Tec-/- mice; and evaluated for capacity to re-engraft and reconstitute B lineage development and function in vivo.
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van Rooij, M. P. C., T. Q. Dang, and L. M. Larosiliere. "Improving Aerodynamic Matching of Axial Compressor Blading Using a Three-Dimensional Multistage Inverse Design Method." Journal of Turbomachinery 129, no. 1 (February 1, 2005): 108–18. http://dx.doi.org/10.1115/1.2372773.
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Current turbomachinery design systems increasingly rely on multistage CFD as a means to diagnose designs and assess performance potential. However, design weaknesses attributed to improper stage matching are addressed using often ineffective strategies involving a costly iterative loop between blading modification, revision of design intent, and further evaluation of aerodynamic performance. A scheme is proposed herein which greatly simplifies the design point blade row matching process. It is based on a three-dimensional viscous inverse method that has been extended to allow blading analysis and design in a multi-blade row environment. For computational expediency, blade row coupling is achieved through an averaging-plane approximation. To limit computational time, the inverse method was parallelized. The proposed method allows improvement of design point blade row matching by direct regulation of the circulation capacity of the blading within a multistage environment. During the design calculation, blade shapes are adjusted to account for inflow and outflow conditions while producing a prescribed pressure loading. Thus, it is computationally ensured that the intended pressure-loading distribution is consistent with the derived blading geometry operating in a multiblade row environment that accounts for certain blade row interactions. The viability of the method is demonstrated in design exercises involving the rotors of a 2.5 stage, highly loaded compressor. Individually redesigned rotors display mismatching when run in the 2.5 stage, evident as a deviation from design intent. However, simultaneous redesign of the rotors in their multistage environment produces the design intent, indicating that aerodynamic matching has been achieved.
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Banach, Bailey B., Prabhanshu Tripathi, Lais Da Silva Pereira, Jason Gorman, Thuy Duong Nguyen, Marlon Dillon, Ahmed S. Fahad, et al. "Highly protective antimalarial antibodies via precision library generation and yeast display screening." Journal of Experimental Medicine 219, no. 8 (June 23, 2022). http://dx.doi.org/10.1084/jem.20220323.
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The monoclonal antibody CIS43 targets the Plasmodium falciparum circumsporozoite protein (PfCSP) and prevents malaria infection in humans for up to 9 mo following a single intravenous administration. To enhance the potency and clinical utility of CIS43, we used iterative site-saturation mutagenesis and DNA shuffling to screen precise gene-variant yeast display libraries for improved PfCSP antigen recognition. We identified several mutations that improved recognition, predominately in framework regions, and combined these to produce a panel of antibody variants. The most improved antibody, CIS43_Var10, had three mutations and showed approximately sixfold enhanced protective potency in vivo compared to CIS43. Co-crystal and cryo-electron microscopy structures of CIS43_Var10 with the peptide epitope or with PfCSP, respectively, revealed functional roles for each of these mutations. The unbiased site-directed mutagenesis and screening pipeline described here represent a powerful approach to enhance protective potency and to enable broader clinical use of antimalarial antibodies.
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Kajiwara, Kaho, Wataru Aoki, Naoki Koike, and Mitsuyoshi Ueda. "Development of a yeast cell surface display method using the SpyTag/SpyCatcher system." Scientific Reports 11, no. 1 (May 26, 2021). http://dx.doi.org/10.1038/s41598-021-90593-w.
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AbstractYeast cell surface display (YSD) has been used to engineer various proteins, including antibodies. Directed evolution, which subjects a gene to iterative rounds of mutagenesis, selection and amplification, is useful for protein engineering. In vivo continuous mutagenesis, which continuously diversifies target genes in the host cell, is a promising tool for accelerating directed evolution. However, combining in vivo continuous evolution and YSD is difficult because mutations in the gene encoding the anchor proteins may inhibit the display of target proteins on the cell surface. In this study, we have developed a modified YSD method that utilises SpyTag/SpyCatcher-based in vivo protein ligation. A nanobody fused with a SpyTag of 16 amino acids and an anchor protein fused with a SpyCatcher of 113 amino acids are encoded by separate gene cassettes and then assembled via isopeptide bond formation. This system achieved a high display efficiency of more than 90%, no intercellular protein ligation events, and the enrichment of target cells by cell sorting. These results suggested that our system demonstrates comparable performance with conventional YSD methods; therefore, it can be an appropriate platform to be integrated with in vivo continuous evolution.
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"Directed Evolution with Fast and Efficient Selection Technologies." CHIMIA 55, no. 4 (April 25, 2001): 325. http://dx.doi.org/10.2533/chimia.2001.325.
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Directed molecular evolution has proven to be a very powerful concept for the generation of proteins with improved properties, such as increased activity, binding affinity, folding efficiency or enhanced chemical and/or thermodynamic stability. We review here advances in the selection of proteins carrying desired mutations from pools of proteins that mostly carry unfavourable alterations. A short overview of the concept of directed evolution with a discussion of randomisation strategies is given first. Two technologies for the selection of proteins, each with its own advantages, are then discussed: In Ribosome Display, all steps are carried out in a cell-free system, which allows one to create very large libraries (diversity > 1011), rapidly introduce mutations and thus obtain an iterative evolution. Examples with antibodies evolved for affinity or stability are discussed. In the Protein Fragment Complementation Assay, a library-versus-library selection is possible, that is, a simultaneous selection of binders against many targets. Examples with peptide and antibody libraries are discussed.
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Szent-Gyorgyi, Christopher, Lydia A. Perkins, Brigitte F. Schmidt, Zhen Liu, Marcel P. Bruchez, and Robert van de Weerd. "Bottom-Up Design: A Modular Golden Gate Assembly Platform of Yeast Plasmids for Simultaneous Secretion and Surface Display of Distinct FAP Fusion Proteins." ACS Synthetic Biology, October 19, 2022. http://dx.doi.org/10.1021/acssynbio.2c00283.
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Zhong, Zhenhui, Yafei Wang, Ming Wang, Fan Yang, Quentin Angelo Thomas, Yan Xue, Yaxin Zhang, et al. "Histone chaperone ASF1 mediates H3.3-H4 deposition in Arabidopsis." Nature Communications 13, no. 1 (November 15, 2022). http://dx.doi.org/10.1038/s41467-022-34648-0.
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AbstractHistone chaperones and chromatin remodelers control nucleosome dynamics, which are essential for transcription, replication, and DNA repair. The histone chaperone Anti-Silencing Factor 1 (ASF1) plays a central role in facilitating CAF-1-mediated replication-dependent H3.1 deposition and HIRA-mediated replication-independent H3.3 deposition in yeast and metazoans. Whether ASF1 function is evolutionarily conserved in plants is unknown. Here, we show that Arabidopsis ASF1 proteins display a preference for the HIRA complex. Simultaneous mutation of both Arabidopsis ASF1 genes caused a decrease in chromatin density and ectopic H3.1 occupancy at loci typically enriched with H3.3. Genetic, transcriptomic, and proteomic data indicate that ASF1 proteins strongly prefers the HIRA complex over CAF-1. asf1 mutants also displayed an increase in spurious Pol II transcriptional initiation and showed defects in the maintenance of gene body CG DNA methylation and in the distribution of histone modifications. Furthermore, ectopic targeting of ASF1 caused excessive histone deposition, less accessible chromatin, and gene silencing. These findings reveal the importance of ASF1-mediated histone deposition for proper epigenetic regulation of the genome.
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Chen, Li-Qun, Shweta Chhajed, Tong Zhang, Joseph M. Collins, Qiuying Pang, Wenyuan Song, Yan He, and Sixue Chen. "Protein complex formation in methionine chain-elongation and leucine biosynthesis." Scientific Reports 11, no. 1 (February 10, 2021). http://dx.doi.org/10.1038/s41598-021-82790-4.
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AbstractDuring the past two decades, glucosinolate (GLS) metabolic pathways have been under extensive studies because of the importance of the specialized metabolites in plant defense against herbivores and pathogens. The studies have led to a nearly complete characterization of biosynthetic genes in the reference plant Arabidopsis thaliana. Before methionine incorporation into the core structure of aliphatic GLS, it undergoes chain-elongation through an iterative three-step process recruited from leucine biosynthesis. Although enzymes catalyzing each step of the reaction have been characterized, the regulatory mode is largely unknown. In this study, using three independent approaches, yeast two-hybrid (Y2H), coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC), we uncovered the presence of protein complexes consisting of isopropylmalate isomerase (IPMI) and isopropylmalate dehydrogenase (IPMDH). In addition, simultaneous decreases in both IPMI and IPMDH activities in a leuc:ipmdh1 double mutants resulted in aggregated changes of GLS profiles compared to either leuc or ipmdh1 single mutants. Although the biological importance of the formation of IPMI and IPMDH protein complexes has not been documented in any organisms, these complexes may represent a new regulatory mechanism of substrate channeling in GLS and/or leucine biosynthesis. Since genes encoding the two enzymes are widely distributed in eukaryotic and prokaryotic genomes, such complexes may have universal significance in the regulation of leucine biosynthesis.
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Klewinghaus, Daniel, Lukas Pekar, Paul Arras, Simon Krah, Bernhard Valldorf, Harald Kolmar, and Stefan Zielonka. "Grabbing the Bull by Both Horns: Bovine Ultralong CDR-H3 Paratopes Enable Engineering of ‘Almost Natural’ Common Light Chain Bispecific Antibodies Suitable For Effector Cell Redirection." Frontiers in Immunology 12 (January 11, 2022). http://dx.doi.org/10.3389/fimmu.2021.801368.
Full textAbstract:
A subset of antibodies found in cattle comprises ultralong CDR-H3 regions of up to 70 amino acids. Interestingly, this type of immunoglobulin usually pairs with the single germline VL gene, V30 that is typically very conserved in sequence. In this work, we have engineered ultralong CDR-H3 common light chain bispecific antibodies targeting Epidermal Growth Factor Receptor (EGFR) on tumor cells as well as Natural Cytotoxicity Receptor NKp30 on Natural Killer (NK) cells. Antigen-specific common light chain antibodies were isolated by yeast surface display by means of pairing CDR-H3 diversities following immunization with a single V30 light chain. After selection, EGFR-targeting paratopes as well as NKp30-specific binders were combined into common light chain bispecific antibodies by exploiting the strand-exchange engineered domain (SEED) technology for heavy chain heterodimerization. Biochemical characterization of resulting bispecifics revealed highly specific binding to the respective antigens as well as simultaneous binding to both targets. Most importantly, engineered cattle-derived bispecific common light chain molecules elicited potent NK cell redirection and consequently tumor cell lysis of EGFR-overexpressing cells as well as robust release of proinflammatory cytokine interferon-γ. Taken together, this data is giving clear evidence that bovine bispecific ultralong CDR-H3 common light chain antibodies are versatile for biotechnological applications.
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Ullrich, Alexander, Fabian Eckelmann, and Stephane Ghozzi. "Dashboards as strategy to integrate multiple data streams for real time surveillance." Online Journal of Public Health Informatics 11, no. 1 (May 30, 2019). http://dx.doi.org/10.5210/ojphi.v11i1.9701.
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ObjectiveProviding an integrative tool for public health experts to rapidly assess the epidemiological situation based on data streams from different surveillance systems and relevant external factors, e.g. weather or socio-economic conditions. The efficient implementation in a modular architecture of disease- or task-specific visualisations and interactions, their combination in dashboards and integration in a consistent, general web application. The user-oriented development through an iterative process in close collaboration with epidemiologists.IntroductionThe mission of the Infectious-Disease-Epidemiology Department at the Robert Koch Institute is the prevention, detection and control of infections in the German population. For this purpose it has a set of surveillance and outbreak-detection systems in place. Some of these cover a wide range of diseases, e.g. the traditional surveillance of about 80 notifiable diseases, while others are specialised for the timely assessment of only one or a few diseases, e.g. participatory syndromic surveillance of acute respiratory infections. Many different such data sources have to be combined to allow a holistic view of the epidemiological situation. The continuous integration of many heterogeneous data streams into a readily available and accessible product remains a big challenge in infectious-disease epidemiology.MethodsThe first step in the development of visualisation and analysis dashboards was the identification of relevant epidemiological questions. This was done through the review and analysis of existing epidemiological tools and workflows, among others through surveys and interviews. With the help of domain experts we identified the relevant data sources for specific tasks. We then chose data visualisations that are common in the field of infectious-disease epidemiology, e.g. disease maps, epicurves and age pyramids, as well as visualisations that were suggested by experts, e.g. time-series graph with severity thresholds. In an iterative process of propositions and expert feedback, we refined the user experience, adjusting variables, control parameters and the layout.We have used two different technologies for the dashboard development. For tasks that needed extensive data integration and statistical computing we used the Shiny web-framework of the statistical programming language R, which allows for a seamless integration of data-wrangling, statistical methods and web design with interactive visualizations. For tasks where a more flexible and fluid user experience is desired and for the integration in a general web application, we used the more versatile single-page application (SPA) framework AngularJS in combination with ASP.NET. In both approaches we used standard open-source visualisation libraries such as Leaflet or Plotly. The dashboards were designed in a modular way, abstracting data sources and visualisations in order to reuse them and adapt them easily to other data sources. Where applicable, interfaces to live data bases and OLAP cubes where developed and implemented.ResultsWe have developed a set of dashboards that allow the exploration of infectious-disease data, each designed for a specific epidemiological task. While still under active development, the dashboards are accepted and routinely used by epidemiologists of the Robert Koch Institute. The expansion to other user groups (e.g. local health agencies) is planned for the near future. Further dashboards will be developed as new epidemiological tasks are identified.A general dashboard ("Signals Dashboard", see Figure 1 A) is displaying laboratory confirmed cases and their distribution across time, space, age and sex in linked widgets. Additionally it highlights anomalous clusters of cases in all widgets and lists the anomalies in an interactive table. The dashboard is available for all (approx. 80) notifiable diseases. The "Severity Dashboard" (Figure 1 B) integrates influenza-related syndromic data, virological information and laboratory confirmed cases. The indicators transmissibility, seriousness and impact, as defined by the PISA guidelines of the World Health Organization, are displayed in time-series charts (absolute and cumulative) and tables; parameter-adjustable severity assessments are computed on the fly. This dashboard has then been adapted to monitor in real time the severity of rotavirus infections. One further dashboard focusses on vaccine-preventable diseases and allows the simultaneous exploration of incidences and vaccination rates through synchronized maps and histograms. Lastly, a "Context Dashboard" enables the exploration of possible connections between tick-related diseases such as TBE and Lyme disease on the one hand, and weather and environment as external factors on the other. It provides visual comparisons through maps and time-series charts, correlation analysis and statistical modeling. The user can choose a set of (lagged) variables to be included in a linear statistical model, which is immediately trained. The contributions and significance of the chosen factors, as well as the fit and prediction accuracies, are displayed in tables, scatter plots and time series. Both "Signals" and "Severity" dashboards serve the rapid assessment of the epidemiological situation and as such display live data as read from internal databases and cubes. The others are at present rather meant for retrospective analyses but will be connected to live data streams in the future.ConclusionsDashboards can provide a way to integrate different epidemiological data streams and statistical methods, offering experts a useful tool to assess the epidemiological situation. Close collaboration between epidemiologists and data scientists in the design and development is beneficial to the relevance and sustainability of such a tool.