QTY code designed thermostable and water-soluble chimeric chemokine receptors with tunable ligand affinity

Chemokine receptors are of great interest as they play a critical role in many immunological and pathological processes. The ability to study chemokine receptors in aqueous solution without detergent would be significant because natural receptors require detergents to become soluble. We previously reported using the QTY code to design detergent-free chemokine receptors. We here report the design of 2 detergent-free chimeric chemokine receptors that were experimentally unattainable in detergent solution. We designed chimeric receptors by switching the N terminus and 3 extracellular (EC) loops between different receptors. Specifically, we replaced the N terminus and 3 EC loops of CCR5QTYwith the N terminus and 3 EC loops of CXCR4. The ligand for CXCR4; namely CXCL12, binds to the chimeric receptor CCR5QTY(7TM)-CXCR4 (N terminus+3 EC loops), but with lower affinity compared to CXCR4; the CCL5 ligand of CCR5 binds the chimeric receptor with ∼20× lower affinity. The chimeric design helps to elucidate the mechanism of native receptor-ligand interaction. We also show that all detergent-free QTY-designed chemokine receptors, expressed inEscherichia coli, bind to their respective chemokines with affinities in the nanomolar (nM) range, similar to the affinities of native receptors and SF9-produced QTY variants. These QTY-designed receptors exhibit remarkable thermostability in the presence of arginine and retain ligand-binding activity after heat treatment at 60 °C for 4 h and 24 h, and at 100 °C for 10 min. Our design approach enables affordable scale-up production of detergent-free QTY variant chemokine receptors with tunable functionality for various uses.

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QTY Code-designed Water-soluble Fc-fusion Cytokine Receptors Bind to their Respective Ligands

QRB Discovery ◽

10.1017/qrd.2020.4 ◽

2020 ◽

Vol 1 ◽

Cited By ~ 2

Author(s):

Shilei Hao ◽

David Jin ◽

Shuguang Zhang ◽

Rui Qing

Keyword(s):

Chemokine Receptors ◽

Protein Modification ◽

Viral Infections ◽

Critical Role ◽

Water Soluble ◽

Cytokine Receptors ◽

Interleukin Receptors ◽

Physiological Properties ◽

Life Threatening ◽

Hydrophobic Amino Acids

AbstractCytokine release syndrome (CRS), or ‘cytokine storm’, is the leading side effect during chimeric antigen receptor (CAR)-T therapy that is potentially life-threatening. It also plays a critical role in viral infections such as Coronavirus Disease 2019 (COVID-19). Therefore, efficient removal of excessive cytokines is essential for treatment. We previously reported a novel protein modification tool called the QTY code, through which hydrophobic amino acids Leu, Ile, Val and Phe are replaced by Gln (Q), Thr (T) and Tyr (Y). Thus, the functional detergent-free equivalents of membrane proteins can be designed. Here, we report the application of the QTY code on six variants of cytokine receptors, including interleukin receptors IL4Rα and IL10Rα, chemokine receptors CCR9 and CXCR2, as well as interferon receptors IFNγR1 and IFNλR1. QTY-variant cytokine receptors exhibit physiological properties similar to those of native receptors without the presence of hydrophobic segments. The receptors were fused to the Fc region of immunoglobulin G (IgG) protein to form an antibody-like structure. These QTY code-designed Fc-fusion receptors were expressed in Escherichia coli and purified. The resulting water-soluble fusion receptors bind to their respective ligands with Kd values affinity similar to isolated native receptors. Our cytokine receptor–Fc-fusion proteins potentially serve as an antibody-like decoy to dampen the excessive cytokine levels associated with CRS and COVID-19 infection.

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CXCR3 Requires Tyrosine Sulfation for Ligand Binding and a Second Extracellular Loop Arginine Residue for Ligand-Induced Chemotaxis

Molecular and Cellular Biology ◽

10.1128/mcb.00556-06 ◽

2006 ◽

Vol 26 (15) ◽

pp. 5838-5849 ◽

Cited By ~ 85

Author(s):

Richard A. Colvin ◽

Gabriele S. V. Campanella ◽

Lindsay A. Manice ◽

Andrew D. Luster

Keyword(s):

Ligand Binding ◽

Transmembrane Domain ◽

Nk Cell ◽

Cell Trafficking ◽

Extracellular Loop ◽

Ligand Interaction ◽

Lower Affinity ◽

Extracellular Domains ◽

N Terminus ◽

Charged Residues

ABSTRACT CXCR3 is a G-protein-coupled seven-transmembrane domain chemokine receptor that plays an important role in effector T-cell and NK cell trafficking. Three gamma interferon-inducible chemokines activate CXCR3: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). Here, we identify extracellular domains of CXCR3 that are required for ligand binding and activation. We found that CXCR3 is sulfated on its N terminus and that sulfation is required for binding and activation by all three ligands. We also found that the proximal 16 amino acid residues of the N terminus are required for CXCL10 and CXCL11 binding and activation but not CXCL9 activation. In addition, we found that residue R216 in the second extracellular loop is required for CXCR3-mediated chemotaxis and calcium mobilization but is not required for ligand binding or ligand-induced CXCR3 internalization. Finally, charged residues in the extracellular loops contribute to the receptor-ligand interaction. These findings demonstrate that chemokine activation of CXCR3 involves both high-affinity ligand-binding interactions with negatively charged residues in the extracellular domains of CXCR3 and a lower-affinity receptor-activating interaction in the second extracellular loop. This lower-affinity interaction is necessary to induce chemotaxis but not ligand-induced CXCR3 internalization, further suggesting that different domains of CXCR3 mediate distinct functions.

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Mesoporous Silica Molecular Sieve based Nanocarriers: Transpiring Drug Dissolution Research

Current Pharmaceutical Design ◽

10.2174/1381612822666161026162005 ◽

2017 ◽

Vol 23 (3) ◽

pp. 467-480 ◽

Cited By ~ 8

Author(s):

Satyanarayan Pattnaik ◽

Kamla Pathak

Keyword(s):

Drug Release ◽

Mesoporous Silica ◽

Molecular Sieves ◽

Release Kinetics ◽

Drug Loading ◽

Scale Up ◽

Water Soluble ◽

Drug Dissolution ◽

Water Soluble Drugs ◽

Loading Methods

Background: Improvement of oral bioavailability through enhancement of dissolution for poorly soluble drugs has been a very promising approach. Recently, mesoporous silica based molecular sieves have demonstrated excellent properties to enhance the dissolution velocity of poorly water-soluble drugs. Description: Current research in this area is focused on investigating the factors influencing the drug release from these carriers, the kinetics of drug release and manufacturing approaches to scale-up production for commercial manufacture. Conclusion: This comprehensive review provides an overview of different methods adopted for synthesis of mesoporous materials, influence of processing factors on properties of these materials and drug loading methods. The drug release kinetics from mesoporous silica systems, the manufacturability and stability of these formulations are reviewed. Finally, the safety and biocompatibility issues related to these silica based materials are discussed.

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Compritol®888 ATO a Lipid Excipient for Sustained Release of Highly Water Soluble Active: Formulation, Scale-up and IVIVC Study

Current Drug Delivery ◽

10.2174/1567201811310050006 ◽

2013 ◽

Vol 10 (5) ◽

pp. 548-556 ◽

Cited By ~ 15

Author(s):

Shilpa Patere ◽

Neha Desai ◽

Ankitkumar Jain ◽

Prashant Kadam ◽

Urmila Thatte ◽

...

Keyword(s):

Sustained Release ◽

Scale Up ◽

Water Soluble ◽

Active Formulation

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Unique structural solution from a VH3-30 antibody targeting the hemagglutinin stem of influenza A viruses

Nature Communications ◽

10.1038/s41467-020-20879-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Wayne D. Harshbarger ◽

Derrick Deming ◽

Gordon J. Lockbaum ◽

Nattapol Attatippaholkun ◽

Maliwan Kamkaew ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Influenza A ◽

Critical Role ◽

Binding Activity ◽

Human Antibody ◽

Broadly Neutralizing Antibodies ◽

Influenza A Viruses ◽

Universal Vaccine ◽

Structural Details ◽

Structural Solution

AbstractBroadly neutralizing antibodies (bnAbs) targeting conserved influenza A virus (IAV) hemagglutinin (HA) epitopes can provide valuable information for accelerating universal vaccine designs. Here, we report structural details for heterosubtypic recognition of HA from circulating and emerging IAVs by the human antibody 3I14. Somatic hypermutations play a critical role in shaping the HCDR3, which alone and uniquely among VH3-30 derived antibodies, forms contacts with five sub-pockets within the HA-stem hydrophobic groove. 3I14 light-chain interactions are also key for binding HA and contribute a large buried surface area spanning two HA protomers. Comparison of 3I14 to bnAbs from several defined classes provide insights to the bias selection of VH3-30 antibodies and reveals that 3I14 represents a novel structural solution within the VH3-30 repertoire. The structures reported here improve our understanding of cross-group heterosubtypic binding activity, providing the basis for advancing immunogen designs aimed at eliciting a broadly protective response to IAV.

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Comparative Metabolite Profile, Biological Activity and Overall Quality of Three Lettuce (Lactuca sativa L., Asteraceae) Cultivars in Response to Sulfur Nutrition

Pharmaceutics ◽

10.3390/pharmaceutics13050713 ◽

2021 ◽

Vol 13 (5) ◽

pp. 713

Author(s):

Muna Ali Abdalla ◽

Fengjie Li ◽

Arlette Wenzel-Storjohann ◽

Saad Sulieman ◽

Deniz Tasdemir ◽

...

Keyword(s):

Secondary Metabolites ◽

Plant Growth ◽

Sesquiterpene Lactones ◽

Critical Role ◽

Soluble Sugars ◽

Radical Scavenging ◽

Metabolite Profile ◽

Water Soluble ◽

Radical Scavenger ◽

Higher Plant

The main objective of the present study was to assess the effects of sulfur (S) nutrition on plant growth, overall quality, secondary metabolites, and antibacterial and radical scavenging activities of hydroponically grown lettuce cultivars. Three lettuce cultivars, namely, Pazmanea RZ (green butterhead, V1), Hawking RZ (green multi-leaf lettuce, V2), and Barlach RZ (red multi-leaf, V3) were subjected to two S-treatments in the form of magnesium sulfate (+S) or magnesium chloride (−S). Significant differences were observed under −S treatments, especially among V1 and V2 lettuce cultivars. These responses were reflected in the yield, levels of macro- and micro-nutrients, water-soluble sugars, and free inorganic anions. In comparison with the green cultivars (V1 and V2), the red-V3 cultivar revealed a greater acclimation to S starvation, as evidenced by relative higher plant growth. In contrast, the green cultivars showed higher capabilities in production and superior quality attributes under +S condition. As for secondary metabolites, sixteen compounds (e.g., sesquiterpene lactones, caffeoyl derivatives, caffeic acid hexose, 5-caffeoylquinic acid (5-OCQA), quercetin and luteolin glucoside derivatives) were annotated in all three cultivars with the aid of HPLC-DAD-MS-based untargeted metabolomics. Sesquiterpene lactone lactucin and anthocyanin cyanidin 3-O-galactoside were only detected in V1 and V3 cultivars, respectively. Based on the analyses, the V3 cultivar was the most potent radical scavenger, while V1 and V2 cultivars exhibited antibacterial activity against Staphylococcus aureus in response to S provision. Our study emphasizes the critical role of S nutrition in plant growth, acclimation, and nutritional quality. The judicious-S application can be adopted as a promising antimicrobial prototype for medical applications.

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A non-toxic, reversibly released imaging probe for oral cancer that is derived from natural compounds

Scientific Reports ◽

10.1038/s41598-021-93408-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Magda Ghanim ◽

Nicola Relitti ◽

Gavin McManus ◽

Stefania Butini ◽

Andrea Cappelli ◽

...

Keyword(s):

Oral Cancer ◽

Cancer Progression ◽

Intraoperative Imaging ◽

Critical Role ◽

Water Soluble ◽

Stem Cell Marker ◽

Squamous Cells ◽

Tissue Identification ◽

Tumorigenic Potential ◽

The Impact

AbstractCD44 is emerging as an important receptor biomarker for various cancers. Amongst these is oral cancer, where surgical resection remains an essential mode of treatment. Unfortunately, surgery is frequently associated with permanent disfigurement, malnutrition, and functional comorbidities due to the difficultly of tumour removal. Optical imaging agents that can guide tumour tissue identification represent an attractive approach to minimising the impact of surgery. Here, we report the synthesis of a water-soluble fluorescent probe, namely HA-FA-HEG-OE (compound 1), that comprises components originating from natural sources: oleic acid, ferulic acid and hyaluronic acid. Compound 1 was found to be non-toxic, displayed aggregation induced emission and accumulated intracellularly in vesicles in SCC-9 oral squamous cells. The uptake of 1 was fully reversible over time. Internalization of compound 1 occurs through receptor mediated endocytosis; uniquely mediated through the CD44 receptor. Uptake is related to tumorigenic potential, with non-tumorigenic, dysplastic DOK cells and poorly tumorigenic MCF-7 cells showing only low intracellular levels and highlighting the critical role of endocytosis in cancer progression and metastasis. Together, the recognised importance of CD44 as a cancer stem cell marker in oral cancer, and the reversible, non-toxic nature of 1, makes it a promising agent for real time intraoperative imaging.

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Synthesis of Fluorescent Carbon Dots and Their Application in Ascorbic Acid Detection

Molecules ◽

10.3390/molecules26051246 ◽

2021 ◽

Vol 26 (5) ◽

pp. 1246

Author(s):

Tengfei Wang ◽

Hui Luo ◽

Xu Jing ◽

Jiali Yang ◽

Meijun Huo ◽

...

Keyword(s):

Ascorbic Acid ◽

Fluorescence Intensity ◽

Carbon Dots ◽

Ph Dependence ◽

Scale Up ◽

Water Soluble ◽

Jujube Fruit ◽

Fluorescent Carbon Dots ◽

Fluorescent Carbon

Water-soluble fluorescent carbon dots (CDs) were synthesized by a hydrothermal method using citric acid as the carbon source and ethylenediamine as the nitrogen source. The repeated and scale-up synthetic experiments were carried out to explore the feasibility of macroscopic preparation of CDs. The CDs/Fe3+ composite was prepared by the interaction of the CDs solution and Fe3+ solution. The optical properties, pH dependence and stability behavior of CDs or the CDs/Fe3+ composite were studied by ultraviolet spectroscopy and fluorescence spectroscopy. Following the principles of fluorescence quenching after the addition of Fe3+ and then the fluorescence recovery after the addition of asorbic acid, the fluorescence intensity of the carbon dots was measured at λex = 360 nm, λem = 460 nm. The content of ascorbic acid was calculated by quantitative analysis of the changing fluorescence intensity. The CDs/Fe3+ composite was applied to the determination of different active molecules, and it was found that the composite had specific recognition of ascorbic acid and showed an excellent linear relationship in 5.0–350.0 μmol·L−1. Moreover, the detection limit was 3.11 μmol·L−1. Satisfactory results were achieved when the method was applied to the ascorbic acid determination in jujube fruit. The fluorescent carbon dots composites prepared in this study may have broad application prospects in a rapid, sensitive and trace determination of ascorbic acid content during food processing.

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Considering treatment-as-prevention scale-up for Australian prisons: a qualitative sub-study of expert stakeholders from the Australian ‘surveillance and treatment of prisoners with hepatitis C’ project (SToP-C)

Harm Reduction Journal ◽

10.1186/s12954-021-00494-4 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Jake Rance ◽

◽

Lise Lafferty ◽

Carla Treloar

Keyword(s):

Hepatitis C ◽

Primary Prevention ◽

Critical Role ◽

Scale Up ◽

Treatment As Prevention ◽

Structured Interviews ◽

Hcv Treatment ◽

Public Health Response ◽

Direct Acting ◽

Levels Of Support

Abstract Background With direct-acting antivirals dramatically reshaping the public health response to the hepatitis C virus (HCV), prisons are set to play a critical role in elimination efforts. Despite the theoretical demonstration of HCV treatment-as-prevention in prison in mathematical modeling, limited empirical data exist. The Australian ‘Surveillance and Treatment of Prisoners with Hepatitis C’ project (SToP-C) is the world’s first trial of HCV treatment-as-prevention in prison. Drawing on interviews with HCV expert stakeholders, this paper explores the factors respondents identified as crucial to the success of future scale-up. Accounting for such perspectives matters because of the influence expert discourse has in shaping implementation. Methods Semi-structured interviews were conducted with nineteen HCV experts working across key policy, advocacy, research and clinical dimensions of the Australian HCV response. Data were coded using qualitative data management software (NVivo 11). Analysis proceeded via a hybrid deductive and inductive approach. Results Notwithstanding concerns regarding the lack of primary prevention in Australian prisons, stakeholders reported broad levels of support for the intervention and for the future scale-up of HCV treatment. A number of considerations, both external and internal to the prison system, were identified as key. The principal external factor was an enabling political-cum-policy environment; internal factors included: obtaining support from prisons’ executive and custodial staff; promoting health within a security-first institutional culture; allocating time for treatment within prisoners’ tightly regulated schedules; ensuring institutional stability during treatment given the routine movement of prisoners between prisons; prioritizing the availability of retreatment given the paucity of primary prevention; and securing sufficient clinical space for treatment. Conclusion The challenges to implementation are considerable, ranging from macrolevel concerns to in-prison logistical matters. Nonetheless, we argue that prisons remain an obvious setting for treatment scale-up, not only for prevention and potential elimination benefit, but for the treatment opportunities they afford a socially disadvantaged and underserved population. While noting widespread concerns among respondents regarding the paucity of primary prevention in Australian prisons, results indicate broad levels of support among expert stakeholders for HCV treatment scale-up in prison.

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Crystallization of and selenomethionine phasing strategy for a SETMAR–DNA complex

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16012723 ◽

2016 ◽

Vol 72 (9) ◽

pp. 713-719 ◽

Cited By ~ 2

Author(s):

Qiujia Chen ◽

Millie Georgiadis

Keyword(s):

Dna Binding ◽

Catalytic Domain ◽

Specific Binding ◽

Critical Role ◽

Binding Activity ◽

Structural Basis ◽

Unexpected Finding ◽

Terminal Inverted Repeat ◽

Dna Binding Activity ◽

New Genes

Transposable elements have played a critical role in the creation of new genes in all higher eukaryotes, including humans. Although the chimeric fusion protein SETMAR is no longer active as a transposase, it contains both the DNA-binding domain (DBD) and catalytic domain of theHsmar1transposase. The amino-acid sequence of the DBD has been virtually unchanged in 50 million years and, as a consequence, SETMAR retains its sequence-specific binding to the ancestralHsmar1terminal inverted repeat (TIR) sequence. Thus, the DNA-binding activity of SETMAR is likely to have an important biological function. To determine the structural basis for the recognition of TIR DNA by SETMAR, the design of TIR-containing oligonucleotides and SETMAR DBD variants, crystallization of DBD–DNA complexes, phasing strategies and initial phasing experiments are reported here. An unexpected finding was that oligonucleotides containing two BrdUs in place of thymidines produced better quality crystals in complex with SETMAR than their natural counterparts.

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