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Article ; Online: Developing a High-Throughput Assay for the Integral Membrane Glycerol 3-Phosphate Acyltransferase.

Tang, Yannan / Li, Dianfan

2019 Volume 17, Issue 6, Page(s) 267–274

Abstract: Phospholipid biosynthesis begins with the acylation of glycerol 3-phosphate (G3P). In most Gram-positive bacteria including many pathogens, a membrane protein called PlsY is the only acyltransferase that catalyzes this essential step, making it a ... ...

Abstract	Phospholipid biosynthesis begins with the acylation of glycerol 3-phosphate (G3P). In most Gram-positive bacteria including many pathogens, a membrane protein called PlsY is the only acyltransferase that catalyzes this essential step, making it a potential target for the development of antibiotics. A convenient enzymatic assay should facilitate such drug discovery activities. Previously, we developed a continuous assay by monitoring phosphate, one of the enzymatic product, using a fluorescently labeled phosphate binding protein in a bilayer environment called lipid cubic phase (LCP). However, some intrinsic characteristics of LCP, such as high viscosity, make the assay incompatible with common high-throughput liquid-handling platforms. Here, we adapted the assay by hosting PlsY in detergent micelles, enabling us to conduct the assay using standard multi-channel pipets in a high-throughput manner. With optimal enzyme loading, the reaction velocity was linear up to 30 min. PlsY showed Michaelis
MeSH term(s)	Bacteria/enzymology ; Cell Membrane/enzymology ; Dose-Response Relationship, Drug ; Drug Development ; Enzyme Inhibitors/chemical synthesis ; Enzyme Inhibitors/chemistry ; Enzyme Inhibitors/pharmacology ; Glycerol-3-Phosphate O-Acyltransferase/antagonists & inhibitors ; Glycerol-3-Phosphate O-Acyltransferase/isolation & purification ; Glycerol-3-Phosphate O-Acyltransferase/metabolism ; High-Throughput Screening Assays ; Lysophospholipids/chemical synthesis ; Lysophospholipids/chemistry ; Lysophospholipids/pharmacology ; Molecular Structure ; Structure-Activity Relationship
Chemical Substances	Enzyme Inhibitors ; Lysophospholipids ; Glycerol-3-Phosphate O-Acyltransferase (EC 2.3.1.15) ; lysophosphatidic acid (PG6M3969SG)
Language	English
Publishing date	2019-08-12
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1557-8127
ISSN (online)	1557-8127
DOI	10.1089/adt.2019.935
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Article ; Online: A stratified generalized additive model and permutation test for temporal heterogeneity of smoothed bivariate spatial effects.

Tang, Yannan / Vieira, Veronica M / Bartell, Scott M / Gillen, Daniel L

Statistics in medicine

2020 Volume 39, Issue 28, Page(s) 4187–4200

Abstract: Generalized additive models (GAMs) with bivariate smoothers are frequently used to map geographic disease risks in epidemiology studies. A challenge in identifying health disparities has been the lack of intuitive and computationally feasible methods to ... ...

Abstract	Generalized additive models (GAMs) with bivariate smoothers are frequently used to map geographic disease risks in epidemiology studies. A challenge in identifying health disparities has been the lack of intuitive and computationally feasible methods to assess whether the pattern of spatial effects varies over time. In this research, we accommodate time-stratified smoothers into the GAM framework to estimate time-specific spatial risk patterns while borrowing information from confounding effects across time. A backfitting algorithm for model estimation is proposed along with a permutation testing framework for assessing temporal heterogeneity of geospatial risk patterns across two or more time points. Simulation studies show that our proposed permuted mean squared difference (PMSD) test performs well with respect to type I error and power in various settings when compared with existing methods. The proposed model and PMSD test are used geospatial risk patterns of patent ductus arteriosus (PDA) in the state of Massachusetts over 2003-2009. We show that there is variation over time in spatial patterns of PDA risk, adjusting for other known risk factors, suggesting the presence of potential time-varying and space-related risk factors other than the adjusted ones.
MeSH term(s)	Algorithms ; Computer Simulation ; Humans
Language	English
Publishing date	2020-08-13
Publishing country	England
Document type	Journal Article
ZDB-ID	843037-8
ISSN	1097-0258 ; 0277-6715
ISSN (online)	1097-0258
ISSN	0277-6715
DOI	10.1002/sim.8718
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Article ; Online: High-level heterologous expression of the human transmembrane sterol Δ8,Δ7-isomerase in Pichia pastoris.

Cai, Hongmin / Yao, Hebang / Li, Tingting / Tang, Yannan / Li, Dianfan

Protein expression and purification

2019 Volume 164, Page(s) 105463

Abstract: Recombinant expression of human membrane proteins in large quantities remains a major challenge. Expression host is an important variable to screen for high-level production of membrane proteins. Using the green fluorescent protein (GFP) as a reporter, ... ...

Abstract	Recombinant expression of human membrane proteins in large quantities remains a major challenge. Expression host is an important variable to screen for high-level production of membrane proteins. Using the green fluorescent protein (GFP) as a reporter, we screened the expression of a human multi-pass membrane protein called sterol Δ8-Δ7 isomerase in three different hosts: Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. The expression of the His-tagged isomerase was exceptionally high in P. pastoris, reaching ~200 mg L
MeSH term(s)	Chromatography, Gel ; Gene Expression ; Humans ; Pichia/genetics ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Solubility ; Steroid Isomerases/chemistry ; Steroid Isomerases/genetics
Chemical Substances	Recombinant Proteins ; Steroid Isomerases (EC 5.3.3.-) ; EBP protein, human (EC 5.3.3.5)
Language	English
Publishing date	2019-08-02
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1055455-5
ISSN	1096-0279 ; 1046-5928
ISSN (online)	1096-0279
ISSN	1046-5928
DOI	10.1016/j.pep.2019.105463
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Conference proceedings: Verbesserte Kontrolle retinaler Flüssigkeit durch Faricimab in Phase-3-Studien bei DMÖ und nAMD

Koch, Frank / Dhoot, Dilsher / Haskova, Zdenka / Ives, Jane / Kotecha, Aachal / Margaron, Philippe / Souverain, Audrey / Tang, Yannan / Willis, Jeffery R.

2023 , Page(s) FP 3.15

Event/congress	35. Internationaler Kongress der Deutschen Ophthalmochirurgie (DOC); Nürnberg; 2023
Keywords	Medizin, Gesundheit
Publishing date	2023-06-13
Publisher	German Medical Science GMS Publishing House; Düsseldorf
Document type	Conference proceedings
DOI	10.3205/23doc032
Database	German Medical Science

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Article ; Online: An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification.

Cai, Hongmin / Yao, Hebang / Li, Tingting / Hutter, Cedric A J / Li, Yanfang / Tang, Yannan / Seeger, Markus A / Li, Dianfan

Communications biology

2020 Volume 3, Issue 1, Page(s) 753

Abstract: Green fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability. An ideal reporter should be stable itself and provide high sensitivity and yield. Here, we demonstrate that a coral (Galaxea ... ...

Abstract	Green fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability. An ideal reporter should be stable itself and provide high sensitivity and yield. Here, we demonstrate that a coral (Galaxea fascicularis) thermostable GFP (TGP) is by such reasons an improved tag compared to the conventional jellyfish GFPs. TGP faithfully reports membrane protein stability at temperatures near 90 °C (20-min heating). By contrast, the limit for the two popular GFPs is 64 °C and 74 °C. Replacing GFPs with TGP increases yield for all four test membrane proteins in four expression systems. To establish TGP as an affinity tag for membrane protein purification, several high-affinity synthetic nanobodies (sybodies), including a non-competing pair, are generated, and the crystal structure of one complex is solved. Given these advantages, we anticipate that TGP becomes a widely used tool for membrane protein structural studies.
MeSH term(s)	Chromatography, Affinity ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Gene Expression ; Genes, Reporter ; Green Fluorescent Proteins/chemistry ; Luminescent Proteins ; Membrane Proteins/chemistry ; Membrane Proteins/genetics ; Membrane Proteins/isolation & purification ; Models, Molecular ; Protein Conformation ; Protein Stability ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Recombinant Proteins/isolation & purification ; Recombinant Proteins/metabolism ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Single-Domain Antibodies/chemistry
Chemical Substances	Luminescent Proteins ; Membrane Proteins ; Recombinant Proteins ; Single-Domain Antibodies ; Green Fluorescent Proteins (147336-22-9)
Language	English
Publishing date	2020-12-10
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2399-3642
ISSN (online)	2399-3642
DOI	10.1038/s42003-020-01478-z
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Article: High performance low-k cyanate ester resins with a thermally stable cyclodextrin microsphere

Tang, Yannan / Gu, Aijuan / Liang, Guozheng / Yuan, Li

RSC advances. 2014 Apr. 01, v. 4, no. 31

2014

Abstract: A new biomass, cyclodextrin microsphere (M-CDP) with high thermal stability, small size and suitable content of –OH groups was synthesized and was then used to develop high performance low-k cyanate ester (CE) resins. The structure and properties of M- ... ...

Abstract	A new biomass, cyclodextrin microsphere (M-CDP) with high thermal stability, small size and suitable content of –OH groups was synthesized and was then used to develop high performance low-k cyanate ester (CE) resins. The structure and properties of M-CDP/CE resins were systematically studied. Compared with the CE resin, the M-CDP/CE system has better curing characteristics reflected by significantly decreased curing temperature and reaction enthalpy; moreover, M-CDP/CE resins show stable and lower dielectric constants as well as similar dielectric loss over the whole frequency range tested. The dielectric constant of M-CDP/CE resin with 5 wt% M-CDP is as low as 2.92 at 104 Hz. In addition, M-CDP/CE resins with suitable contents of M-CDP have better thermal stability than CE resins. These attractive performances of M-CDP/CE resins are attributed to the different networks induced by the unique structure of M-CDP. This investigation provides a new and easy method to prepare a thermally stable cyclodextrin derivative and corresponding low-k resins based on biomass materials.
Keywords	biomass ; cyanates ; cyclodextrins ; dielectric properties ; enthalpy ; microparticles ; resins ; temperature ; thermal stability
Language	English
Dates of publication	2014-0401
Size	p. 16136-16145.
Publishing place	The Royal Society of Chemistry
Document type	Article
ISSN	2046-2069
DOI	10.1039/c4ra00750f
Database	NAL-Catalogue (AGRICOLA)

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Conference proceedings: 2-Jahres-Ergebnisse der Studien YOSEMITE und RHINE zur Wirksamkeit, Wirkdauer und Sicherheit von Faricimab bei diabetischem Makulaödem

Mirshahi, Alireza / Wells, John A. / Danzig, Carl J. / Eichenbaum, David A. / Lim, Jennifer I. / Schlottmann, Patricio G. / Singh, Rishi P. / Mohan, Shaun / Tang, Yannan / Haskova, Zdenka

2023 , Page(s) FP 3.14

Event/congress	35. Internationaler Kongress der Deutschen Ophthalmochirurgie (DOC); Nürnberg; 2023
Keywords	Medizin, Gesundheit
Publishing date	2023-06-13
Publisher	German Medical Science GMS Publishing House; Düsseldorf
Document type	Conference proceedings
DOI	10.3205/23doc031
Database	German Medical Science

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Article ; Online: Structural insights into the committed step of bacterial phospholipid biosynthesis.

Li, Zhenjian / Tang, Yannan / Wu, Yiran / Zhao, Suwen / Bao, Juan / Luo, Yitian / Li, Dianfan

Nature communications

2017 Volume 8, Issue 1, Page(s) 1691

Abstract: The membrane-integral glycerol 3-phosphate (G3P) acyltransferase PlsY catalyses the committed and essential step in bacterial phospholipid biosynthesis by acylation of G3P, forming lysophosphatidic acid. It contains no known acyltransferase motifs, lacks ...

Abstract	The membrane-integral glycerol 3-phosphate (G3P) acyltransferase PlsY catalyses the committed and essential step in bacterial phospholipid biosynthesis by acylation of G3P, forming lysophosphatidic acid. It contains no known acyltransferase motifs, lacks eukaryotic homologs, and uses the unusual acyl-phosphate as acyl donor, as opposed to acyl-CoA or acyl-carrier protein for other acyltransferases. Previous studies have identified several PlsY inhibitors as potential antimicrobials. Here we determine the crystal structure of PlsY at 1.48 Å resolution, revealing a seven-transmembrane helix fold. Four additional substrate- and product-bound structures uncover the atomic details of its relatively inflexible active site. Structure and mutagenesis suggest a different acylation mechanism of 'substrate-assisted catalysis' that, unlike other acyltransferases, does not require a proteinaceous catalytic base to complete. The structure data and a high-throughput enzymatic assay developed in this work should prove useful for virtual and experimental screening of inhibitors against this vital bacterial enzyme.
MeSH term(s)	Amino Acid Substitution ; Bacteria/enzymology ; Bacteria/genetics ; Bacterial Proteins/chemistry ; Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; Biosynthetic Pathways/genetics ; Catalytic Domain ; Conserved Sequence ; Crystallography, X-Ray ; Genes, Bacterial ; Glycerol-3-Phosphate O-Acyltransferase/chemistry ; Glycerol-3-Phosphate O-Acyltransferase/genetics ; Glycerol-3-Phosphate O-Acyltransferase/metabolism ; Kinetics ; Models, Molecular ; Mutagenesis, Site-Directed ; Phospholipids/biosynthesis ; Protein Conformation ; Streptococcus pneumoniae/enzymology ; Streptococcus pneumoniae/genetics ; Substrate Specificity ; Thermotoga maritima/enzymology ; Thermotoga maritima/genetics ; Thermus thermophilus/enzymology ; Thermus thermophilus/genetics
Chemical Substances	Bacterial Proteins ; Phospholipids ; Glycerol-3-Phosphate O-Acyltransferase (EC 2.3.1.15)
Language	English
Publishing date	2017-11-22
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2041-1723
ISSN (online)	2041-1723
DOI	10.1038/s41467-017-01821-9
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Article ; Online: Faricimab Treat-and-Extend for Diabetic Macular Edema: Two-Year Results from the Randomized Phase 3 YOSEMITE and RHINE Trials.

Ophthalmology

2023

Abstract: Purpose: To evaluate the 2-year efficacy, durability, and safety of dual angiopoietin-2 and vascular endothelial growth factor (VEGF) A pathway inhibition with intravitreal faricimab according to a personalized treat-and-extend (T&E)-based regimen with ... ...

Abstract	Purpose: To evaluate the 2-year efficacy, durability, and safety of dual angiopoietin-2 and vascular endothelial growth factor (VEGF) A pathway inhibition with intravitreal faricimab according to a personalized treat-and-extend (T&E)-based regimen with up to every-16-week dosing in the YOSEMITE and RHINE (ClinicalTrials.gov identifiers, NCT03622580 and NCT03622593, respectively) phase 3 trials of diabetic macular edema (DME). Design: Randomized, double-masked, noninferiority phase 3 trials. Participants: Adults with visual acuity loss (best-corrected visual acuity [BCVA] of 25-73 letters) due to center-involving DME. Methods: Patients were randomized 1:1:1 to faricimab 6.0 mg every 8 weeks, faricimab 6.0 mg T&E (previously referred to as personalized treatment interval), or aflibercept 2.0 mg every 8 weeks. The T&E up to every-16-week dosing regimen was based on central subfield thickness (CST) and BCVA change. Main outcome measures: Included changes from baseline in BCVA and CST, number of injections, durability, absence of fluid, and safety through week 100. Results: In YOSEMITE and RHINE (n = 940 and 951, respectively), noninferior year 1 visual acuity gains were maintained through year 2; mean BCVA change from baseline at 2 years (weeks 92, 96, and 100 average) with faricimab every 8 weeks (YOSEMITE and RHINE, +10.7 letters and +10.9 letters, respectively) or T&E (+10.7 letters and +10.1 letters, respectively) were comparable with aflibercept every 8 weeks (+11.4 letters and +9.4 letters, respectively). The median number of study drug injections was lower with faricimab T&E (YOSEMITE and RHINE, 10 and 11 injections, respectively) versus faricimab every 8 weeks (15 injections) and aflibercept every 8 weeks (14 injections) across both trials during the entire study. In the faricimab T&E arms, durability was improved further during year 2, with > 60% of patients receiving every-16-week dosing and approximately 80% receiving every-12-week or longer dosing at week 96. Almost 80% of patients who achieved every-16-week dosing at week 52 maintained every-16-week dosing without an interval reduction through week 96. Mean CST reductions were greater (YOSEMITE/RHINE weeks 92/96/100 average: faricimab every 8 weeks -216.0/-202.6 µm, faricimab T&E -204.5/-197.1 µm, aflibercept every 8 weeks -196.3/-185.6 µm), and more patients achieved absence of DME (CST < 325 μm; YOSEMITE/RHINE weeks 92-100: faricimab every 8 weeks 87%-92%/88%-93%, faricimab T&E 78%-86%/85%-88%, aflibercept every 8 weeks 77%-81%/80%-84%) and absence of intraretinal fluid (YOSEMITE/RHINE weeks 92-100: faricimab every 8 weeks 59%-63%/56%-62%, faricimab T&E 43%-48%/45%-52%, aflibercept every 8 weeks 33%-38%/39%-45%) with faricimab every 8 weeks or T&E versus aflibercept every 8 weeks through year 2. Overall, faricimab was well tolerated, with a safety profile comparable with that of aflibercept. Conclusions: Clinically meaningful visual acuity gains from baseline, anatomic improvements, and extended durability with intravitreal faricimab up to every 16 weeks were maintained through year 2. Faricimab given as a personalized T&E-based dosing regimen supports the role of dual angiopoietin-2 and VEGF-A inhibition to promote vascular stability and to provide durable efficacy for patients with DME. Financial disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
Language	English
Publishing date	2023-12-28
Publishing country	United States
Document type	Journal Article
ZDB-ID	392083-5
ISSN	1549-4713 ; 0161-6420
ISSN (online)	1549-4713
ISSN	0161-6420
DOI	10.1016/j.ophtha.2023.12.026
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