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Article: A Single-Component Luminescent Biosensor for the SARS-CoV-2 Spike Protein

Ravalin, Matthew / Roh, Heegwang / Suryawanshi, Rahul / Kumar, G. Renuka / Pak, John E. / Ott, Melanie / Ting, Alice Y.

Journal of the American Chemical Society. 2022 July 25, v. 144, no. 30

2022

Abstract: Many existing protein detection strategies depend on highly functionalized antibody reagents. A simpler and easier to produce class of detection reagent is highly desirable. We designed a single-component, recombinant, luminescent biosensor that can be ... ...

Abstract	Many existing protein detection strategies depend on highly functionalized antibody reagents. A simpler and easier to produce class of detection reagent is highly desirable. We designed a single-component, recombinant, luminescent biosensor that can be expressed in laboratory strains of Escherichia coli and Saccharomyces cerevisiae. This biosensor is deployed in multiple homogeneous and immobilized assay formats to detect recombinant SARS-CoV-2 spike antigen and cultured virus. The chemiluminescent signal generated facilitates detection by an unaugmented cell phone camera. Binding-activated tandem split-enzyme (BAT) biosensors may serve as a useful template for diagnostics and reagents that detect SARS-CoV-2 antigens and other proteins of interest.
Keywords	Escherichia coli ; Saccharomyces cerevisiae ; Severe acute respiratory syndrome coronavirus 2 ; antibodies ; antigens ; biosensors ; cameras ; chemiluminescence ; diagnostic techniques ; mobile telephones ; viruses
Language	English
Dates of publication	2022-0725
Size	p. 13663-13672.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	3155-0
ISSN	1520-5126 ; 0002-7863
ISSN (online)	1520-5126
ISSN	0002-7863
DOI	10.1021/jacs.2c04192
Database	NAL-Catalogue (AGRICOLA)

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Article: Accelerating PERx Reaction Enables Covalent Nanobodies for Potent Neutralization of SARS-Cov-2 and Variants.

Yu, Bingchen / Li, Shanshan / Tabata, Takako / Wang, Nanxi / Kumar, G Renuka / Liu, Jun / Ott, Melanie M / Wang, Lei

bioRxiv : the preprint server for biology

2022

Abstract: The long-lasting COVID-19 pandemic and increasing SARS-CoV-2 variants demand effective drugs for prophylactics and treatment. Protein-based biologics offer high specificity yet their noncovalent interactions often lead to drug dissociation and incomplete ...

Abstract	The long-lasting COVID-19 pandemic and increasing SARS-CoV-2 variants demand effective drugs for prophylactics and treatment. Protein-based biologics offer high specificity yet their noncovalent interactions often lead to drug dissociation and incomplete inhibition. Here we developed covalent nanobodies capable of binding with SARS-CoV-2 spike protein irreversibly via proximity-enabled reactive therapeutic (PERx) mechanism. A novel latent bioreactive amino acid FFY was designed and genetically encoded into nanobodies to accelerate PERx reaction rate. After covalent engineering, nanobodies binding with the Spike in the down state, but not in the up state, were discovered to possess striking enhancement in inhibiting viral infection. In comparison with the noncovalent wildtype nanobody, the FFY-incorporated covalent nanobody neutralized both authentic SARS-CoV-2 and its Alpha and Delta variants with potency drastically increased over tens of folds. This PERx-enabled covalent nanobody strategy and uncovered insights on potency increase can be valuable to developing effective therapeutics for various viral infections.
Language	English
Publishing date	2022-03-14
Publishing country	United States
Document type	Preprint
DOI	10.1101/2022.03.11.483867
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: A single-component luminescent biosensor for the SARS-CoV-2 spike protein.

Ravalin, Matthew / Roh, Heegwang / Suryawanshi, Rahul / Kumar, G Renuka / Pak, John / Ott, Melanie / Ting, Alice Y

bioRxiv : the preprint server for biology

2022

Abstract	Many existing protein detection strategies depend on highly functionalized antibody reagents. A simpler and easier to produce class of detection reagent is highly desirable. We designed a single-component, recombinant, luminescent biosensor that can be expressed in laboratory strains of
Language	English
Publishing date	2022-06-15
Publishing country	United States
Document type	Preprint
DOI	10.1101/2022.06.15.496006
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: A Single-Component Luminescent Biosensor for the SARS-CoV-2 Spike Protein.

Ravalin, Matthew / Roh, Heegwang / Suryawanshi, Rahul / Kumar, G Renuka / Pak, John E / Ott, Melanie / Ting, Alice Y

Journal of the American Chemical Society

2022 Volume 144, Issue 30, Page(s) 13663–13672

Abstract	Many existing protein detection strategies depend on highly functionalized antibody reagents. A simpler and easier to produce class of detection reagent is highly desirable. We designed a single-component, recombinant, luminescent biosensor that can be expressed in laboratory strains of
MeSH term(s)	Biosensing Techniques ; COVID-19 ; Humans ; SARS-CoV-2 ; Spike Glycoprotein, Coronavirus/metabolism
Chemical Substances	Spike Glycoprotein, Coronavirus ; spike protein, SARS-CoV-2
Language	English
Publishing date	2022-07-25
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
ZDB-ID	3155-0
ISSN	1520-5126 ; 0002-7863
ISSN (online)	1520-5126
ISSN	0002-7863
DOI	10.1021/jacs.2c04192
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Mercapto-pyrimidines are reversible covalent inhibitors of the papain-like protease (PLpro) and inhibit SARS-CoV-2 (SCoV-2) replication.

Bajaj, Teena / Wehri, Eddie / Suryawanshi, Rahul K / King, Elizabeth / Pardeshi, Kundan Singh / Behrouzi, Kamyar / Khodabakhshi, Zahra / Schulze-Gahmen, Ursula / Kumar, G Renuka / Mofrad, Mohammad R K / Nomura, Daniel K / Ott, Melanie / Schaletzky, Julia / Murthy, Niren

RSC advances

2023 Volume 13, Issue 26, Page(s) 17667–17677

Abstract: The papain-like protease (PLpro) plays a critical role in SARS-CoV-2 (SCoV-2) pathogenesis and is essential for viral replication and for allowing the virus to evade the host immune response. Inhibitors of PLpro have great therapeutic potential, however, ...

Abstract	The papain-like protease (PLpro) plays a critical role in SARS-CoV-2 (SCoV-2) pathogenesis and is essential for viral replication and for allowing the virus to evade the host immune response. Inhibitors of PLpro have great therapeutic potential, however, developing them has been challenging due to PLpro's restricted substrate binding pocket. In this report, we screened a 115 000-compound library for PLpro inhibitors and identified a new pharmacophore, based on a mercapto-pyrimidine fragment that is a reversible covalent inhibitor (RCI) of PLpro and inhibits viral replication in cells. Compound 5 had an IC
Language	English
Publishing date	2023-06-12
Publishing country	England
Document type	Journal Article
ISSN	2046-2069
ISSN (online)	2046-2069
DOI	10.1039/d3ra01915b
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Novel RT-ddPCR Assays for determining the transcriptional profile of SARS-CoV-2.

Telwatte, Sushama / Kumar, Nitasha / Vallejo-Gracia, Albert / Kumar, G Renuka / Lu, Chuanyi M / Ott, Melanie / Wong, Joseph K / Yukl, Steven A

bioRxiv : the preprint server for biology

2021

Abstract: The exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive- ... ...

Abstract	The exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics.
Language	English
Publishing date	2021-01-12
Publishing country	United States
Document type	Preprint
DOI	10.1101/2021.01.12.425991
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Novel RT-ddPCR assays for simultaneous quantification of multiple noncoding and coding regions of SARS-CoV-2 RNA.

Telwatte, Sushama / Kumar, Nitasha / Vallejo-Gracia, Albert / Kumar, G Renuka / Lu, Chuanyi M / Ott, Melanie / Wong, Joseph K / Yukl, Steven A

Journal of virological methods

2021 Volume 292, Page(s) 114115

Abstract: A hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from ...

Abstract	A hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of sensitive, quantitative RT-ddPCR assays designed to target regions spanning the genome of SARS-CoV-2. Our assays target untranslated regions (5', 3') as well as different coding regions, including non-structural genes that are only found in full length (genomic) RNA and structural genes that are found in genomic as well as different subgenomic RNAs. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 gene expression and may also inform the development of improved diagnostic tools and therapeutics.
MeSH term(s)	COVID-19/diagnosis ; COVID-19 Nucleic Acid Testing/methods ; False Positive Reactions ; Humans ; Limit of Detection ; Open Reading Frames ; RNA, Viral/analysis ; Real-Time Polymerase Chain Reaction/methods ; SARS-CoV-2/genetics ; Viral Load
Chemical Substances	RNA, Viral
Language	English
Publishing date	2021-03-02
Publishing country	Netherlands
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2021.114115
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Novel RT-ddPCR assays for simultaneous quantification of multiple noncoding and coding regions of SARS-CoV-2 RNA

Telwatte, Sushama / Kumar, Nitasha / Vallejo-Gracia, Albert / Kumar, G. Renuka / Lu, Chuanyi M. / Ott, Melanie / Wong, Joseph K. / Yukl, Steven A.

Journal of virological methods. 2021 June, v. 292

2021

Abstract	A hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of sensitive, quantitative RT-ddPCR assays designed to target regions spanning the genome of SARS-CoV-2. Our assays target untranslated regions (5′, 3′) as well as different coding regions, including non-structural genes that are only found in full length (genomic) RNA and structural genes that are found in genomic as well as different subgenomic RNAs. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 gene expression and may also inform the development of improved diagnostic tools and therapeutics.
Keywords	Severe acute respiratory syndrome coronavirus 2 ; gene expression ; genomics ; polymerase chain reaction ; therapeutics
Language	English
Dates of publication	2021-06
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2021.114115
Database	NAL-Catalogue (AGRICOLA)

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Zs.A 1565: Show issues

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Jg. 1995 - 2021: Lesesall (1.OG)
ab Jg. 2022: Lesesaal (EG)

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Article ; Online: A covalent inhibitor targeting the papain-like protease from SARS-CoV-2 inhibits viral replication.

Han, Hesong / Gracia, Albert Vallejo / Røise, Joachim J / Boike, Lydia / Leon, Kristoffer / Schulze-Gahmen, Ursula / Stentzel, Michael R / Bajaj, Teena / Chen, Dake / Li, I-Che / He, Maomao / Behrouzi, Kamyar / Khodabakhshi, Zahra / Nomura, Daniel K / Mofrad, Mohammad R K / Kumar, G Renuka / Ott, Melanie / Murthy, Niren

RSC advances

2023 Volume 13, Issue 16, Page(s) 10636–10641

Abstract: Covalent inhibitors of the papain-like protease (PLpro) from SARS-CoV-2 have great potential as antivirals, but their non-specific reactivity with thiols has limited their development. In this report, we performed an 8000 molecule electrophile screen ... ...

Abstract	Covalent inhibitors of the papain-like protease (PLpro) from SARS-CoV-2 have great potential as antivirals, but their non-specific reactivity with thiols has limited their development. In this report, we performed an 8000 molecule electrophile screen against PLpro and identified an α-chloro amide fragment, termed compound 1, which inhibited SARS-CoV-2 replication in cells, and also had low non-specific reactivity with thiols. Compound 1 covalently reacts with the active site cysteine of PLpro, and had an IC50 of 18 μM for PLpro inhibition. Compound 1 also had low non-specific reactivity with thiols and reacted with glutathione 1-2 orders of magnitude slower than other commonly used electrophilic warheads. Finally, compound 1 had low toxicity in cells and mice and has a molecular weight of only 247 daltons and consequently has great potential for further optimization. Collectively, these results demonstrate that compound 1 is a promising lead fragment for future PLpro drug discovery campaigns.
Language	English
Publishing date	2023-04-04
Publishing country	England
Document type	Journal Article
ISSN	2046-2069
ISSN (online)	2046-2069
DOI	10.1039/d3ra00426k
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Nuclear accumulation of host transcripts during Zika Virus Infection.

Leon, Kristoffer E / Khalid, Mir M / Flynn, Ryan A / Fontaine, Krystal A / Nguyen, Thong T / Kumar, G Renuka / Simoneau, Camille R / Tomar, Sakshi / Jimenez-Morales, David / Dunlap, Mariah / Kaye, Julia / Shah, Priya S / Finkbeiner, Steven / Krogan, Nevan J / Bertozzi, Carolyn / Carette, Jan E / Ott, Melanie

PLoS pathogens

2023 Volume 19, Issue 1, Page(s) e1011070

Abstract: Zika virus (ZIKV) infects fetal neural progenitor cells (NPCs) causing severe neurodevelopmental disorders in utero. Multiple pathways involved in normal brain development are dysfunctional in infected NPCs but how ZIKV centrally reprograms these ... ...

Abstract	Zika virus (ZIKV) infects fetal neural progenitor cells (NPCs) causing severe neurodevelopmental disorders in utero. Multiple pathways involved in normal brain development are dysfunctional in infected NPCs but how ZIKV centrally reprograms these pathways remains unknown. Here we show that ZIKV infection disrupts subcellular partitioning of host transcripts critical for neurodevelopment in NPCs and functionally link this process to the up-frameshift protein 1 (UPF1). UPF1 is an RNA-binding protein known to regulate decay of cellular and viral RNAs and is less expressed in ZIKV-infected cells. Using infrared crosslinking immunoprecipitation and RNA sequencing (irCLIP-Seq), we show that a subset of mRNAs loses UPF1 binding in ZIKV-infected NPCs, consistent with UPF1's diminished expression. UPF1 target transcripts, however, are not altered in abundance but in subcellular localization, with mRNAs accumulating in the nucleus of infected or UPF1 knockdown cells. This leads to diminished protein expression of FREM2, a protein required for maintenance of NPC identity. Our results newly link UPF1 to the regulation of mRNA transport in NPCs, a process perturbed during ZIKV infection.
MeSH term(s)	Humans ; Brain/metabolism ; Brain/virology ; Neural Stem Cells/virology ; RNA Helicases/genetics ; RNA Helicases/metabolism ; Trans-Activators/metabolism ; Virus Replication ; Zika Virus/physiology ; Zika Virus Infection/genetics
Chemical Substances	RNA Helicases (EC 3.6.4.13) ; Trans-Activators ; UPF1 protein, human (EC 3.6.4.13)
Language	English
Publishing date	2023-01-05
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2205412-1
ISSN	1553-7374 ; 1553-7374
ISSN (online)	1553-7374
ISSN	1553-7374
DOI	10.1371/journal.ppat.1011070
Database	MEDical Literature Analysis and Retrieval System OnLINE

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