scholarly journals Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3

Biomolecules ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 62
Author(s):  
Chad S. Hewitt ◽  
Chittaranjan Das ◽  
Daniel P. Flaherty
Keyword(s):  
Rational Design ◽  
Deubiquitinating Enzyme ◽  
Triple Mutant ◽  
Cellular Assays ◽  
Rational Development ◽  
Alternative Approach ◽  
Covalent Adducts ◽  
Reliable Methods ◽  
Insight Into

There is currently a lack of reliable methods and strategies to probe the deubiquitinating enzyme UCHL3. Current small molecules reported for this purpose display reduced potency and selectivity in cellular assays. To bridge this gap and provide an alternative approach to probe UCHL3, our group has carried out the rational design of ubiquitin-variant activity-based probes with selectivity for UCHL3 over the closely related UCHL1 and other DUBs. The approach successfully produced a triple-mutant ubiquitin variant activity-based probe, UbVQ40V/T66K/V70F-PRG, that was ultimately 20,000-fold more selective for UCHL3 over UCHL1 when assessed by rate of inactivation assays. This same variant was shown to selectively form covalent adducts with UCHL3 in MDA-MB-231 breast cancer cells and no reactivity toward other DUBs expressed. Overall, this study demonstrates the feasibility of the approach and also provides insight into how this approach may be applied to other DUB targets.

scholarly journals Rational design of adjuvants targeting the C-type lectin Mincle

2017 ◽  
Vol 114 (10) ◽  
pp. 2675-2680 ◽  
Author(s):  
Alexiane Decout ◽  
Sandro Silva-Gomes ◽  
Daniel Drocourt ◽  
Sophie Barbe ◽  
Isabelle André ◽  
...  
Keyword(s):  
Immune Responses ◽  
Rational Design ◽  
Biological Evaluation ◽  
Subunit Vaccines ◽  
Rational Development ◽  
Dynamics Simulations ◽  
Mycobacterial Cell Wall ◽  
Molecular Bases ◽  
Insight Into

The advances in subunit vaccines development have intensified the search for potent adjuvants, particularly adjuvants inducing cell-mediated immune responses. Identification of the C-type lectin Mincle as one of the receptors underlying the remarkable immunogenicity of the mycobacterial cell wall, via recognition of trehalose-6,6′-dimycolate (TDM), has opened avenues for the rational design of such molecules. Using a combination of chemical synthesis, biological evaluation, molecular dynamics simulations, and protein mutagenesis, we gained insight into the molecular bases of glycolipid recognition by Mincle. Unexpectedly, the fine structure of the fatty acids was found to play a key role in the binding of a glycolipid to the carbohydrate recognition domain of the lectin. Glucose and mannose esterified atO-6 by a synthetic α-ramified 32-carbon fatty acid showed agonist activity similar to that of TDM, despite their much simpler structure. Moreover, they were seen to stimulate proinflammatory cytokine production in primary human and murine cells in a Mincle-dependent fashion. Finally, they were found to induce strong Th1 and Th17 immune responses in vivo in immunization experiments in mice and conferred protection in a murine model ofMycobacterium tuberculosisinfection. Here we describe the rational development of new molecules with powerful adjuvant properties.

Skeletal elements of crinoid echinoderms: High-resolution structural and microanalytical results

1983 ◽  
Vol 41 ◽  
pp. 194-195
Author(s):  
D. F. Blake ◽  
L. F. Allard ◽  
D. R. Peacor
Keyword(s):  
High Resolution ◽  
Marine Invertebrates ◽  
Sea Urchins ◽  
Structural Features ◽  
Sea Stars ◽  
High Resolution Tem ◽  
Relationship Of ◽  
The Relationship ◽  
Insight Into

Echinodermata is a phylum of marine invertebrates which has been extant since Cambrian time (c.a. 500 m.y. before the present). Modern examples of echinoderms include sea urchins, sea stars, and sea lilies (crinoids). The endoskeletons of echinoderms are composed of plates or ossicles (Fig. 1) which are with few exceptions, porous, single crystals of high-magnesian calcite. Despite their single crystal nature, fracture surfaces do not exhibit the near-perfect {10.4} cleavage characteristic of inorganic calcite. This paradoxical mix of biogenic and inorganic features has prompted much recent work on echinoderm skeletal crystallography. Furthermore, fossil echinoderm hard parts comprise a volumetrically significant portion of some marine limestones sequences. The ultrastructural and microchemical characterization of modern skeletal material should lend insight into: 1). The nature of the biogenic processes involved, for example, the relationship of Mg heterogeneity to morphological and structural features in modern echinoderm material, and 2). The nature of the diagenetic changes undergone by their ancient, fossilized counterparts. In this study, high resolution TEM (HRTEM), high voltage TEM (HVTEM), and STEM microanalysis are used to characterize tha ultrastructural and microchemical composition of skeletal elements of the modern crinoid Neocrinus blakei.

Speed vs. comfort: Testing the efficacy of a single question for predicting transit users’ route choices

2019 ◽  
Author(s):  
Joseph John Pyne Simons ◽  
Ilya Farber
Keyword(s):  
Data Collection ◽  
Efficient Method ◽  
Choice Task ◽  
The Self ◽  
Self Report ◽  
Model Parameters ◽  
Single Item ◽  
Alternative Approach ◽  
Single Question ◽  
Insight Into

Not all transit users have the same preferences when making route decisions. Understanding the factors driving this heterogeneity enables better tailoring of policies, interventions, and messaging. However, existing methods for assessing these factors require extensive data collection. Here we present an alternative approach - an easily-administered single item measure of overall preference for speed versus comfort. Scores on the self-report item predict decisions in a choice task and account for a proportion of the differences in model parameters between people (n=298). This single item can easily be included on existing travel surveys, and provides an efficient method to both anticipate the choices of users and gain more general insight into their preferences.

scholarly journals Preclinical characterization of dostarlimab, a therapeutic anti-PD-1 antibody with potent activity to enhance immune function in in vitro cellular assays and in vivo animal models

mAbs ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 1954136
Author(s):  
Sujatha Kumar ◽  
Srimoyee Ghosh ◽  
Geeta Sharma ◽  
Zebin Wang ◽  
Marilyn R. Kehry ◽  
...  
Keyword(s):  
Animal Models ◽  
Immune Function ◽  
Cellular Assays ◽  
In Vivo Animal Models

scholarly journals First Insight into Nutraceutical Properties of Local Salento Cichorium intybus Varieties: NMR-Based Metabolomic Approach

2021 ◽  
Vol 18 (8) ◽  
pp. 4057
Author(s):  
Chiara Roberta Girelli ◽  
Francesca Serio ◽  
Rita Accogli ◽  
Federica Angilè ◽  
Antonella De Donno ◽  
...  
Keyword(s):  
1H Nmr ◽  
Cichorium Intybus ◽  
Resonance Spectroscopy ◽  
Metabolic Profiles ◽  
Whole Plant ◽  
Different Content ◽  
Insight Into ◽  
Nutraceutical Properties ◽  
Metabolomic Approach

Background: Plants of genus Cichorium are known for their therapeutic and nutraceutical properties determined by a wealth of phytochemical substances contained in the whole plant. The aim of this paper was to characterize the metabolic profiles of local Salento chicory (Cichorium intybus L.) varieties (“Bianca”, “Galatina”, “Leccese”, and “Otranto”) in order to describe their metabolites composition together with possible bioactivity and health beneficial properties. Methods: The investigation was performed by 1H-NMR spectroscopy and Multivariate Analysis (MVA), by which the metabolic profiles of the samples were easily obtained and compared. Results: The supervised Partial Least Squares Discriminant Analysis (PLS-DA) analysis showed as “Bianca” and “Galatina” samples grouped together separated by “Leccese” and “Otranto” varieties. A different content of free amino acids and organic acids was observed among the varieties. In particular a high content of cichoric and monocaffeoyl tartaric acid was observed for the “Leccese” variety. The presence of secondary metabolites adds significant interest in the investigation of Cichorium inthybus, as this vegetable may benefit human health when incorporated into the diet. Conclusions: The 1H-NMR (Nuclear Magnetic Resonance Spectroscopy) based characterization of Salento chicory varieties allowed us to determine the potential usefulness and nutraceutical properties of the product, also providing a method to guarantee its authenticity on a molecular scale.

“Real-World” Microscopy: Understanding Environment-Sensitive Behavior of Ni-Base Welds

1998 ◽  
Vol 4 (S2) ◽  
pp. 528-529
Author(s):  
M. G. Burke ◽  
R. J. Wehrer ◽  
C.M. Brown
Keyword(s):  
Nuclear Power ◽  
Base Alloy ◽  
Analytical Electron Microscopy ◽  
Furnace Cool ◽  
Environmental Embrittlement ◽  
Reported Study ◽  
Compositional Changes ◽  
Insight Into ◽  
Sensitive Behavior

Ni-base alloy welds such as EN82H weld metal are frequently employed in nuclear power applications where resistance to corrosion is required. Results of a recently reported study of the mechanical properties of EN82H welds show that this alloy is susceptible to low-temperature (∼100°C) environmental embrittlement (LTEE) in hydrogenated water. LTEE is a manifestation of hydrogen embrittlement in these alloys.1 Recent LTEE tests have demonstrated a beneficial effect of a high-temperature (∼1100°C) anneal and furnace-cool in alleviating the material's susceptibility to LTEE. Understanding the reason for the reduction in LTEE susceptibility requires detailed characterization of the microstructure so that the specific structural and compositional changes that have been induced by the solution-anneal can be identified. This study reports the results of light optical and analytical electron microscopy (AEM) characterization of the microstructures of as-fabricated and as-solution-annealed EN82H welds with the objective of providing insight into the observed LTEE behavior.

scholarly journals Enhancing the thermostability and activity of uronate dehydrogenase from Agrobacterium tumefaciens LBA4404 by semi-rational engineering

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Hui-Hui Su ◽  
Fei Peng ◽  
Pei Xu ◽  
Xiao-Ling Wu ◽  
Min-Hua Zong ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Rational Design ◽  
Glucuronic Acid ◽  
Specific Activity ◽  
Value Added ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Glucaric Acid ◽  
Triple Mutant ◽  
Pharmaceutical Industries

Abstract Background Glucaric acid, one of the aldaric acids, has been declared a “top value-added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efficient and economical than chemical synthesis. Uronate dehydrogenases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi-rational design, is a major research goal. Results In the present work, three UDHs were obtained from different Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tumefaciens (At) LBA4404 had a better catalytic efficiency than the other two UDHs (800 vs 600 and 530 s−1 mM−1). To further boost the catalytic performance of the UDH from AtLBA4404, site-directed mutagenesis based on semi-rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400-fold improvement in half-life at 59 °C, a 5 °C improvement in $$ {\text{T}}_{ 5 0}^{ 1 0} $$ T 50 10 value and a 2.5-fold improvement in specific activity at 30 °C compared to wild-type UDH. Conclusions In this study, we successfully obtained a triple mutant (A39P/H99Y/H234K) with simultaneously enhanced activity and thermostability, which provides a novel alternative for the industrial production of glucaric acid from glucuronic acid.

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