To site-specifically incorporate an unnatural amino acid (UAA) into target proteins in Escherichia coli, we use a suppressor plasmid that provides an engineered suppressor tRNA and an aminoacyl-tRNA ...synthetase (aaRS) specific for the UAA of interest. The continuous drive to further improve UAA incorporation efficiency in E. coli has resulted in several generations of suppressor plasmids. Here we describe a new, highly efficient suppressor plasmid, pUltra, harboring a single copy each of the tRNA and aaRS expression cassettes that exhibits higher suppression activity than its predecessors. This system is able to efficiently incorporate up to three UAAs within the same protein at levels up to 30% of the level of wild-type protein expression. Its unique origin of replication (CloDF13) and antibiotic resistance marker (spectinomycin) allow pUltra to be used in conjunction with the previously reported pEVOL suppressor plasmid, each encoding a distinct tRNA/aaRS pair, to simultaneously insert two different UAAs into the same protein. We demonstrate the utility of this system by efficiently incorporating two bio-orthogonal UAAs containing keto and azido side chains into ketosteroid isomerase and subsequently derivatizing these amino acid residues with two distinct fluorophores, capable of Förster resonance energy transfer interaction. Finally, because of its minimal composition, two different tRNA/aaRS pairs were encoded in pUltra, allowing the generation of a single plasmid capable of dual suppression. The high suppression efficiency and the ability to harbor multiple tRNA/aaRS pairs make pUltra a useful system for conducting single- and multiple-UAA mutagenesis in E. coli.
The adoptive transfer of autologous T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a promising cancer therapy. Despite impressive clinical efficacy, the general ...application of current CAR–T-cell therapy is limited by serious treatment-related toxicities. One approach to improve the safety of CAR-T cells involves making their activation and proliferation dependent upon adaptor molecules that mediate formation of the immunological synapse between the target cancer cell and T-cell. Here, we describe the design and synthesis of structurally defined semisynthetic adaptors we refer to as “switch” molecules, in which anti-CD19 and anti-CD22 antibody fragments are site-specifically modified with FITC using genetically encoded noncanonical amino acids. This approach allows the precise control over the geometry and stoichiometry of complex formation between CD19- or CD22-expressing cancer cells and a “universal” anti-FITC–directed CAR-T cell. Optimization of this CAR–switch combination results in potent, dose-dependent in vivo antitumor activity in xenograft models. The advantage of being able to titrate CAR–T-cell in vivo activity was further evidenced by reduced in vivo toxicity and the elimination of persistent B-cell aplasia in immune-competent mice. The ability to control CAR-T cell and cancer cell interactions using intermediate switch molecules may expand the scope of engineered T-cell therapy to solid tumors, as well as indications beyond cancer therapy.
Hallmarks of Aging in the Liver Hunt, Nicholas J.; Kang, Sun Woo (Sophie); Lockwood, Glen P. ...
Computational and Structural Biotechnology Journal,
01/2019, Volume:
17
Journal Article
Peer reviewed
Open access
While the liver demonstrates remarkable resilience during aging, there is growing evidence that it undergoes all the cellular hallmarks of aging, which increases the risk of liver and systemic ...disease. The aging process in the liver is driven by alterations of the genome and epigenome that contribute to dysregulation of mitochondrial function and nutrient sensing pathways, leading to cellular senescence and low-grade inflammation. These changes promote multiple phenotypic changes in all liver cells (hepatocytes, liver sinusoidal endothelial, hepatic stellate and Küpffer cells) and impairment of hepatic function. In particular, age-related changes in the liver sinusoidal endothelial cells are a significant but under-recognized risk factor for the development of age-related cardiometabolic disease.
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•Liver aging is driven by transcription and metabolic epigenome alterations.•This leads to cellular senescence and low-grade inflammation.•Hepatocyte, sinusoidal endothelial, stellate and Küpffer cells undergoes the hallmarks of aging.•Each cell type demonstrates phenotypical cellular changes with age.
Upper bounds of dual flagged Weyl characters Peng, Simon C.Y.; Lin, Zhuowei; Sun, Sophie C.C.
Advances in applied mathematics,
September 2024, 2024-09-00, Volume:
160
Journal Article
Peer reviewed
Open access
For a subset D of boxes in an n×n square grid, let χD(x) denote the dual character of the flagged Weyl module associated to D. It is known that χD(x) specifies to a Schubert polynomial (resp., a key ...polynomial) in the case when D is the Rothe diagram of a permutation (resp., the skyline diagram of a composition). One can naturally define a lower and an upper bound of χD(x). Mészáros, St. Dizier and Tanjaya conjectured that χD(x) attains the upper bound if and only if D avoids a certain single subdiagram. We provide a proof of this conjecture.
Selective covalent bond formation at a protein–protein interface potentially can be achieved by genetically introducing into a protein an appropriately “tuned” electrophilic unnatural amino acid that ...reacts with a native nucleophilic residue in its cognate receptor upon complex formation. We have evolved orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs that genetically encode three aza-Michael acceptor amino acids, N ε-acryloyl-(S)-lysine (AcrK, 1), p-acrylamido-(S)-phenylalanine (AcrF, 2), and p-vinylsulfonamido-(S)-phenylalanine (VSF, 3), in response to the amber stop codon in Escherichia coli. Using an αErbB2 Fab-ErbB2 antibody-receptor pair as an example, we demonstrate covalent bond formation between an αErbB2-VSF mutant and a specific surface lysine ε-amino group of ErbB2, leading to near quantitative cross-linking to either purified ErbB2 in vitro or to native cellular ErbB2 at physiological pH. This efficient biocompatible reaction may be useful for creating novel cell biological probes, diagnostics, or therapeutics that selectively and irreversibly bind a target protein in vitro or in living cells.
Gustafson and Milne proved an identity which can be used to express a Schur function sμ(x1,x2,…,xn) with μ=(μ1,μ2,…,μk) in terms of the Schur function sλ(x1,x2,…,xn), where λ=(λ1,λ2,…,λk) is a ...partition such that λi=μi+n−k for 1≤i≤k. On the other hand, Fehér, Némethi and Rimányi found an identity which relates sμ(x1,x2,…,xn) to the Schur function sλ(x1,x2,…,xℓ), where λ=(λ1,λ2,…,λℓ) is a partition obtained from μ by removing some of the largest parts of μ. Fehér, Némethi and Rimányi gave a geometric explanation of their identity, and they raised the question of finding a combinatorial proof. In this paper, we establish a Gustafson-Milne type identity as well as a Fehér-Némethi-Rimányi type identity for factorial Grothendieck polynomials. Specializing a factorial Grothendieck polynomial to a Schur function, we obtain a combinatorial proof of the Fehér-Némethi-Rimányi identity.
Lung cancer is the leading cause of cancer deaths worldwide. Despite never smokers comprising between 10 and 25% of all cases, lung cancer in never smokers (LCNS) is relatively under characterized ...from an etiological and biological perspective. The application of multi-omics techniques on large patient cohorts has significantly advanced the current understanding of LCNS tumor biology. By synthesizing the findings of multi-omics studies on LCNS from a clinical perspective, we can directly translate knowledge regarding tumor biology into implications for patient care. Primarily focused on never smokers with lung adenocarcinoma, this review details the predominance of driver mutations, particularly in East Asian patients, as well as the frequency and importance of germline variants in LCNS. The mutational patterns present in LCNS tumors are thoroughly explored, highlighting the high abundance of the APOBEC signature. Moreover, this review recognizes the spectrum of immune profiles present in LCNS tumors and posits how it can be translated to treatment selection. The recurring and novel insights from multi-omics studies on LCNS tumor biology have a wide range of clinical implications. Risk factors such as exposure to outdoor air pollution, second hand smoke, and potentially diet have a genomic imprint in LCNS at varying degrees, and although they do not encompass all LCNS cases, they can be leveraged to stratify risk. Germline variants similarly contribute to a notable proportion of LCNS, which warrants detailed documentation of family history of lung cancer among never smokers and demonstrates value in developing testing for pathogenic variants in never smokers for early detection in the future. Molecular driver subtypes and specific co-mutations and mutational signatures have prognostic value in LCNS and can guide treatment selection. LCNS tumors with no known driver alterations tend to be stem-like and genes contributing to this state may serve as potential therapeutic targets. Overall, the comprehensive findings of multi-omics studies exert a wide influence on clinical management and future research directions in the realm of LCNS.
Abstract
Age-related changes in the liver sinusoidal endothelium, particularly the reduction in fenestrations, contribute to insulin resistance in old age. Metformin impacts on the aging process and ...improves insulin resistance. Therefore, the effects of metformin on the liver sinusoidal endothelium were studied. Metformin increased fenestrations in liver sinusoidal endothelial cells isolated from both young and old mice. Mice administered metformin in the diet for 12 months had increased fenestrations and this was associated with lower insulin levels. The effect of metformin on fenestrations was blocked by inhibitors of AMP-activated protein kinase (AMPK), endothelial nitric oxide synthase, and myosin light chain kinase phosphorylation. Metformin led to increased transgelin expression and structural changes in the actin cytoskeleton but had no effect on lactate production. Metformin also generated fenestration-like structures in SK-Hep1 cells, a liver endothelial cell line, and this was associated with increased ATP, cGMP, and mitochondrial activity. In conclusion, metformin ameliorates age-related changes in the liver sinusoidal endothelial cell via AMPK and endothelial nitric oxide pathways, which might promote insulin sensitivity in the liver, particularly in old age.
Lung cancer is the leading cause of cancer death worldwide. The disease is particularly difficult to detect, and patients often present at an advanced stage. Current treatments have limited ...effectiveness, and unfortunately, the prognosis remains poor. Recent insights into the molecular pathogenesis and biologic behavior of lung cancer have led to the development of rationally designed methods of early detection, prevention, and treatment of this disease. This article will review the important clinical implications of these advances, with a focus on new molecularly targeted therapies currently in development.
To date, over 100 noncanonical amino acids (ncAAs) have been genetically encoded in living cells in order to expand the functional repertoire of the canonical 20 amino acids. More recently, this ...technology has been expanded to the field of protein therapeutics, where traditional chemical methods typically result in heterogeneous mixtures of proteins. The site‐specific incorporation of ncAAs with orthogonal chemical groups allows unprecedented control over the site of conjugation and the stoichiometry, thus facilitating the rational optimization of the biological functions and/or pharmacokinetics of biologics. Herein, we discuss the recent contribution of ncAA technology in enhancing the pharmacological properties of current protein therapeutics as well as developing novel therapeutic modalities.
Expanding the code: The genetic encoding of noncanonical amino acids allows for the site‐specific introduction of reactive moieties that are typically unavailable to native proteins. In this review, we discuss how this technology has been adopted to overcome existing challenges in current protein therapeutics and to develop novel therapeutic modalities.
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