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Article ; Online: Protocol for assessing translational regulation in mammalian cell lines by OP-Puro labeling

Jack Chun-Chieh Hsu / Joanna B. Pawlak / Maudry Laurent-Rolle / Peter Cresswell

STAR Protocols, Vol 3, Iss 3, Pp 101654- (2022)

2022

Abstract: ... to Hsu et al. (2021) and Hsu et al. (2022). : Publisher’s note: Undertaking any experimental protocol ...

Abstract	Summary: Translational regulation is a fundamental step in gene expression with critical roles in biological processes within a cell. Here, we describe a protocol to assess translation activity in mammalian cells by incorporation of O-propargyl-puromycin (OP-Puro). OP-Puro is a puromycin analog that is incorporated into newly synthesized proteins and is detected by click chemistry reaction. We use OP-Puro labeling to assess translation activity between different cell types or cells under different growth conditions by confocal microscopy and flow cytometry.For complete details on the use and execution of this protocol, please refer to Hsu et al. (2021) and Hsu et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Keywords	Cell biology ; Molecular biology ; Gene expression ; Molecular/Chemical probes ; Science (General) ; Q1-390
Language	English
Publishing date	2022-09-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Protocol for assessing translational regulation in mammalian cell lines by OP-Puro labeling.

Hsu, Jack Chun-Chieh / Pawlak, Joanna B / Laurent-Rolle, Maudry / Cresswell, Peter

STAR protocols

2022 Volume 3, Issue 3, Page(s) 101654

Abstract: ... to Hsu et al. (2021) and Hsu et al. (2022). ...

Abstract	Translational regulation is a fundamental step in gene expression with critical roles in biological processes within a cell. Here, we describe a protocol to assess translation activity in mammalian cells by incorporation of O-propargyl-puromycin (OP-Puro). OP-Puro is a puromycin analog that is incorporated into newly synthesized proteins and is detected by click chemistry reaction. We use OP-Puro labeling to assess translation activity between different cell types or cells under different growth conditions by confocal microscopy and flow cytometry. For complete details on the use and execution of this protocol, please refer to Hsu et al. (2021) and Hsu et al. (2022).
MeSH term(s)	Animals ; Cell Line ; Click Chemistry/methods ; Mammals/metabolism ; Proteomics ; Puromycin/analogs & derivatives ; Puromycin/pharmacology
Chemical Substances	O-propargyl-puromycin ; Puromycin (4A6ZS6Q2CL)
Language	English
Publishing date	2022-08-29
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2022.101654
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Translational shutdown and evasion of the innate immune response by SARS-CoV-2 NSP14 protein.

Hsu, Jack Chun-Chieh / Laurent-Rolle, Maudry / Pawlak, Joanna B / Wilen, Craig B / Cresswell, Peter

Proceedings of the National Academy of Sciences of the United States of America

2021 Volume 118, Issue 24

Abstract: The ongoing COVID-19 pandemic has caused an unprecedented global health crisis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Subversion of host protein synthesis is a common strategy that pathogenic ... ...

Abstract	The ongoing COVID-19 pandemic has caused an unprecedented global health crisis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Subversion of host protein synthesis is a common strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to shut down host protein synthesis and that SARS-CoV-2 nonstructural protein NSP14 exerts this activity. We show that the translation inhibition activity of NSP14 is conserved in human coronaviruses. NSP14 is required for virus replication through contribution of its exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) activities. Mutations in the ExoN or N7-MTase active sites of SARS-CoV-2 NSP14 abolish its translation inhibition activity. In addition, we show that the formation of NSP14-NSP10 complex enhances translation inhibition executed by NSP14. Consequently, the translational shutdown by NSP14 abolishes the type I interferon (IFN-I)-dependent induction of interferon-stimulated genes (ISGs). Together, we find that SARS-CoV-2 shuts down host innate immune responses via a translation inhibitor, providing insights into the pathogenesis of SARS-CoV-2.
MeSH term(s)	Animals ; COVID-19/immunology ; Chlorocebus aethiops ; Exoribonucleases/immunology ; Humans ; Immune Evasion ; Immunity, Innate ; Protein Biosynthesis/immunology ; SARS-CoV-2/immunology ; Vero Cells ; Viral Nonstructural Proteins/immunology
Chemical Substances	Viral Nonstructural Proteins ; Exoribonucleases (EC 3.1.-) ; NSP14 protein, SARS-CoV-2 (EC 3.1.-)
Language	English
Publishing date	2021-05-27
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.2101161118
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Article ; Online: CMPK2 restricts Zika virus replication by inhibiting viral translation.

Joanna B Pawlak / Jack Chun-Chieh Hsu / Hongjie Xia / Patrick Han / Hee-Won Suh / Tyler L Grove / Juliet Morrison / Pei-Yong Shi / Peter Cresswell / Maudry Laurent-Rolle

PLoS Pathogens, Vol 19, Iss 4, p e

2023 Volume 1011286

Abstract: Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can ... ...

Abstract	Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention. Type I interferon (IFN-I) production in response to microbial products is one of the host's first line of defense against invading pathogens. Cytidine/uridine monophosphate kinase 2 (CMPK2) is a type I interferon-stimulated gene (ISG) that exerts antiviral effects. However, the molecular mechanism by which CMPK2 inhibits viral replication is unclear. Here, we report that CMPK2 expression restricts Zika virus (ZIKV) replication by specifically inhibiting viral translation and that IFN-I- induced CMPK2 contributes significantly to the overall antiviral response against ZIKV. We demonstrate that expression of CMPK2 results in a significant decrease in the replication of other pathogenic flaviviruses including dengue virus (DENV-2), Kunjin virus (KUNV) and yellow fever virus (YFV). Importantly, we determine that the N-terminal domain (NTD) of CMPK2, which lacks kinase activity, is sufficient to restrict viral translation. Thus, its kinase function is not required for CMPK2's antiviral activity. Furthermore, we identify seven conserved cysteine residues within the NTD as critical for CMPK2 antiviral activity. Thus, these residues may form an unknown functional site in the NTD of CMPK2 contributing to its antiviral function. Finally, we show that mitochondrial localization of CMPK2 is required for its antiviral effects. Given its broad antiviral activity against flaviviruses, CMPK2 is a promising potential pan-flavivirus inhibitor.
Keywords	Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
Subject code	570
Language	English
Publishing date	2023-04-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Prolonged incubation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a patient on rituximab therapy.

Koff, Alan G / Laurent-Rolle, Maudry / Hsu, Jack Chun-Chieh / Malinis, Maricar

Infection control and hospital epidemiology

2020 Volume 42, Issue 10, Page(s) 1286–1288

Abstract: The incubation period of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is rarely >14 days. We report a patient with hypogammaglobulinemia who developed coronavirus disease 2019 (COVID-19) with a confirmed incubation period of at least 21 ... ...

Abstract	The incubation period of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is rarely >14 days. We report a patient with hypogammaglobulinemia who developed coronavirus disease 2019 (COVID-19) with a confirmed incubation period of at least 21 days. These findings raise concern for a prolonged presymptomatic transmission phase, necessitating a longer quarantine duration in this patient population.
MeSH term(s)	COVID-19 ; Humans ; Quarantine ; Rituximab/therapeutic use ; SARS-CoV-2 ; Time Factors
Chemical Substances	Rituximab (4F4X42SYQ6)
Keywords	covid19
Language	English
Publishing date	2020-10-07
Publishing country	United States
Document type	Case Reports ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	639378-0
ISSN	1559-6834 ; 0195-9417 ; 0899-823X
ISSN (online)	1559-6834
ISSN	0195-9417 ; 0899-823X
DOI	10.1017/ice.2020.1239
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Article ; Online: CMPK2 restricts Zika virus replication by inhibiting viral translation.

Pawlak, Joanna B / Hsu, Jack Chun-Chieh / Xia, Hongjie / Han, Patrick / Suh, Hee-Won / Grove, Tyler L / Morrison, Juliet / Shi, Pei-Yong / Cresswell, Peter / Laurent-Rolle, Maudry

PLoS pathogens

2023 Volume 19, Issue 4, Page(s) e1011286

Abstract	Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention. Type I interferon (IFN-I) production in response to microbial products is one of the host's first line of defense against invading pathogens. Cytidine/uridine monophosphate kinase 2 (CMPK2) is a type I interferon-stimulated gene (ISG) that exerts antiviral effects. However, the molecular mechanism by which CMPK2 inhibits viral replication is unclear. Here, we report that CMPK2 expression restricts Zika virus (ZIKV) replication by specifically inhibiting viral translation and that IFN-I- induced CMPK2 contributes significantly to the overall antiviral response against ZIKV. We demonstrate that expression of CMPK2 results in a significant decrease in the replication of other pathogenic flaviviruses including dengue virus (DENV-2), Kunjin virus (KUNV) and yellow fever virus (YFV). Importantly, we determine that the N-terminal domain (NTD) of CMPK2, which lacks kinase activity, is sufficient to restrict viral translation. Thus, its kinase function is not required for CMPK2's antiviral activity. Furthermore, we identify seven conserved cysteine residues within the NTD as critical for CMPK2 antiviral activity. Thus, these residues may form an unknown functional site in the NTD of CMPK2 contributing to its antiviral function. Finally, we show that mitochondrial localization of CMPK2 is required for its antiviral effects. Given its broad antiviral activity against flaviviruses, CMPK2 is a promising potential pan-flavivirus inhibitor.
MeSH term(s)	Virus Replication ; Zika Virus/physiology ; Vero Cells ; Chlorocebus aethiops ; Animals ; Humans ; Nucleoside-Phosphate Kinase/metabolism ; Interferon Type I/metabolism ; Flavivirus/physiology ; Mitochondria ; Protein Biosynthesis
Chemical Substances	CMPK2 protein, human (EC 2.7.4.14) ; Nucleoside-Phosphate Kinase (EC 2.7.4.4) ; Interferon Type I
Language	English
Publishing date	2023-04-19
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2205412-1
ISSN	1553-7374 ; 1553-7374
ISSN (online)	1553-7374
ISSN	1553-7374
DOI	10.1371/journal.ppat.1011286
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to downregulate MHC-I surface expression.

Arshad, Najla / Laurent-Rolle, Maudry / Ahmed, Wesam S / Hsu, Jack Chun-Chieh / Mitchell, Susan M / Pawlak, Joanna / Sengupta, Debrup / Biswas, Kabir H / Cresswell, Peter

bioRxiv : the preprint server for biology

2022

Abstract: Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small protein β : Significance statement: Viruses may down-regulate MHC class I expression on infected cells ... ...

Abstract	Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small protein β Significance statement: Viruses may down-regulate MHC class I expression on infected cells to avoid elimination by cytotoxic T cells. We report that the accessory proteins ORF7a and ORF3a of SARS-CoV-2 mediate this function and delineate the two distinct mechanisms involved. While ORF3a inhibits global protein trafficking to the cell surface, ORF7a acts specifically on MHC-I by competing with β
Language	English
Publishing date	2022-05-17
Publishing country	United States
Document type	Preprint
DOI	10.1101/2022.05.17.492198
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Article ; Online: SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to down-regulate MHC-I surface expression.

Arshad, Najla / Laurent-Rolle, Maudry / Ahmed, Wesam S / Hsu, Jack Chun-Chieh / Mitchell, Susan M / Pawlak, Joanna / Sengupta, Debrup / Biswas, Kabir H / Cresswell, Peter

Proceedings of the National Academy of Sciences of the United States of America

2022 Volume 120, Issue 1, Page(s) e2208525120

Abstract: Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small-protein ... ...

Abstract	Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small-protein β
MeSH term(s)	Humans ; Antigen Presentation ; COVID-19 ; Histocompatibility Antigens Class I/genetics ; Histocompatibility Antigens Class I/metabolism ; HLA Antigens ; Peptides ; SARS-CoV-2/metabolism ; Viral Regulatory and Accessory Proteins/metabolism
Chemical Substances	Histocompatibility Antigens Class I ; HLA Antigens ; Peptides ; Viral Regulatory and Accessory Proteins ; ORF3a protein, SARS-CoV-2 ; ORF7a protein, SARS-CoV-2
Language	English
Publishing date	2022-12-27
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.2208525120
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: A Progressively Expanded Database for Automated Lung Sound Analysis

Fu-Shun Hsu / Shang-Ran Huang / Chien-Wen Huang / Yuan-Ren Cheng / Chun-Chieh Chen / Jack Hsiao / Chung-Wei Chen / Feipei Lai

Applied Sciences, Vol 12, Iss 7623, p

An Update

2022 Volume 7623

Abstract: We previously established an open-access lung sound database, HF_Lung_V1, and developed deep learning models for inhalation, exhalation, continuous adventitious sound (CAS), and discontinuous adventitious sound (DAS) detection. The amount of data used ... ...

Abstract	We previously established an open-access lung sound database, HF_Lung_V1, and developed deep learning models for inhalation, exhalation, continuous adventitious sound (CAS), and discontinuous adventitious sound (DAS) detection. The amount of data used for training contributes to model accuracy. In this study, we collected larger quantities of data to further improve model performance and explored issues of noisy labels and overlapping sounds. HF_Lung_V1 was expanded to HF_Lung_V2 with a 1.43× increase in the number of audio files. Convolutional neural network–bidirectional gated recurrent unit network models were trained separately using the HF_Lung_V1 (V1_Train) and HF_Lung_V2 (V2_Train) training sets. These were tested using the HF_Lung_V1 (V1_Test) and HF_Lung_V2 (V2_Test) test sets, respectively. Segment and event detection performance was evaluated. Label quality was assessed. Overlap ratios were computed between inhalation, exhalation, CAS, and DAS labels. The model trained using V2_Train exhibited improved performance in inhalation, exhalation, CAS, and DAS detection on both V1_Test and V2_Test. Poor CAS detection was attributed to the quality of CAS labels. DAS detection was strongly influenced by the overlapping of DAS with inhalation and exhalation. In conclusion, collecting greater quantities of lung sound data is vital for developing more accurate lung sound analysis models.
Keywords	auscultation ; convolutional neural network ; deep learning ; gated recurrent unit ; lung sound ; Technology ; T ; Engineering (General). Civil engineering (General) ; TA1-2040 ; Biology (General) ; QH301-705.5 ; Physics ; QC1-999 ; Chemistry ; QD1-999
Subject code	780
Language	English
Publishing date	2022-07-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Prolonged incubation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a patient on rituximab therapy

Koff, Alan G. / Laurent-Rolle, Maudry / Hsu, Jack Chun-Chieh / Malinis, Maricar

Infection Control & Hospital Epidemiology

2020 , Page(s) 1–2

Abstract: Abstract The incubation period of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is rarely >14 days. We report a patient with hypogammaglobulinemia who developed coronavirus disease 2019 (COVID-19) with a confirmed incubation period of at ... ...

Abstract	Abstract The incubation period of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is rarely >14 days. We report a patient with hypogammaglobulinemia who developed coronavirus disease 2019 (COVID-19) with a confirmed incubation period of at least 21 days. These findings raise concern for a prolonged presymptomatic transmission phase, necessitating a longer quarantine duration in this patient population.
Keywords	Microbiology (medical) ; Epidemiology ; Infectious Diseases ; covid19
Language	English
Publisher	Cambridge University Press (CUP)
Publishing country	uk
Document type	Article ; Online
ZDB-ID	639378-0
ISSN	1559-6834 ; 0195-9417 ; 0899-823X
ISSN (online)	1559-6834
ISSN	0195-9417 ; 0899-823X
DOI	10.1017/ice.2020.1239
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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