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  1. Article ; Online: In vivo Validation of Hsp90 Trans-splicing in Giardia lamblia: Highlighting the Role of Cis-elements.

    Tushir, Sheetal / Jhanwar, Pratima / Benda, Martin / Horáčková, Vendula / Doležal, Pavel / Tatu, Utpal

    Journal of molecular biology

    2024  Volume 436, Issue 4, Page(s) 168440

    Abstract: Giardia lambliacauses giardiasis, one of the most common human infectious diseases globally. Previous studies from our lab have shown that hsp90 gene ofGiardia is split into two halves, namely hspN and hspC. The independent pre-mRNAs of these split genes ...

    Abstract Giardia lambliacauses giardiasis, one of the most common human infectious diseases globally. Previous studies from our lab have shown that hsp90 gene ofGiardia is split into two halves, namely hspN and hspC. The independent pre-mRNAs of these split genes join by trans-splicing, producing a full-length Hsp90 (FlHsp90) mRNA. Genetic manipulation of the participating genes is necessary to understand the mechanism and significance of such trans-splicing based expression of Hsp90. In this study, we have performed transfection based exogenous expression of hspN and/or hspC in G. lamblia. We electroporated a plasmid containing the Avi-tagged hspN component of Hsp90 and examined its fate in G. lamblia. We show that the exogenously expressed hspN RNA gets trans-spliced to endogenously expressed hspC RNA, giving rise to a hybrid-FlHsp90. We highlight the importance of cis-elements in this trans-splicing reaction through mutational analysis. The episomal plasmid carrying deletions in the intronic region of hspN, showed inhibition of the trans-splicing reaction.Additionally, exogenous hspC RNA also followed the same fate as of exogenous hspN, while upon co-transfection with episomal hspN, they underwent trans-splicing with each other. Using eGFP as a test protein, we have shown that intronic sequences of hsp90 gene can guide trans-splicing mediated repair of any associated exonic sequences. Our study provides in vivo validation of Hsp90 trans-splicing, showing crucial role of cis-elements and importantly highlights the potential of hsp90 intronic sequences to function as a minimal splicing tool.
    MeSH term(s) Giardia lamblia/genetics ; Introns/genetics ; RNA Precursors/genetics ; Trans-Splicing/genetics ; HSP90 Heat-Shock Proteins/genetics ; Protozoan Proteins/genetics
    Chemical Substances RNA Precursors ; HSP90 Heat-Shock Proteins ; Protozoan Proteins
    Language English
    Publishing date 2024-01-11
    Publishing country Netherlands
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 80229-3
    ISSN 1089-8638 ; 0022-2836
    ISSN (online) 1089-8638
    ISSN 0022-2836
    DOI 10.1016/j.jmb.2024.168440
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: The role of nuclear organization in trans-splicing based expression of heat shock protein 90 in Giardia lamblia.

    Iyer, Vinithra / Tushir, Sheetal / Verma, Shreekant / Majumdar, Sudeshna / Gayen, Srimonta / Mishra, Rakesh / Tatu, Utpal

    PLoS neglected tropical diseases

    2021  Volume 15, Issue 9, Page(s) e0009810

    Abstract: Hsp90 gene of G. lamblia has a split nature comprising two ORFs separated by 777 kb on chromosome 5. The ORFs of the split gene on chromosome 5 undergo transcription to generate independent pre-mRNAs that join by a unique trans-splicing reaction that ... ...

    Abstract Hsp90 gene of G. lamblia has a split nature comprising two ORFs separated by 777 kb on chromosome 5. The ORFs of the split gene on chromosome 5 undergo transcription to generate independent pre-mRNAs that join by a unique trans-splicing reaction that remains partially understood. The canonical cis-acting nucleotide elements such as 5'SS-GU, 3'SS-AG, polypyrimidine tract and branch point adenine are present in the independent pre-mRNAs and therefore trans-splicing of Hsp90 must be assisted by spliceosomes in vivo. Using an approach of RNA-protein pull down, we show that an RNA helicase selectively interacts with HspN pre-mRNA. Our experiments involving high resolution chromosome conformation capture technology as well as DNA FISH show that the trans-spliced genes of Giardia are in three-dimensional spatial proximity in the nucleus. Altogether our study provides a glimpse into the in vivo mechanisms involving protein factors as well as chromatin structure to facilitate the unique inter-molecular post-transcriptional stitching of split genes in G. lamblia.
    MeSH term(s) Giardia lamblia/genetics ; Giardia lamblia/metabolism ; HSP90 Heat-Shock Proteins/genetics ; HSP90 Heat-Shock Proteins/metabolism ; Humans ; Open Reading Frames ; RNA Precursors/genetics ; RNA Precursors/metabolism ; RNA Splicing ; Spliceosomes/genetics ; Spliceosomes/metabolism ; Trans-Splicing
    Chemical Substances HSP90 Heat-Shock Proteins ; RNA Precursors
    Language English
    Publishing date 2021-09-24
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2429704-5
    ISSN 1935-2735 ; 1935-2727
    ISSN (online) 1935-2735
    ISSN 1935-2727
    DOI 10.1371/journal.pntd.0009810
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: The role of nuclear organization in trans-splicing based expression of heat shock protein 90 in Giardia lamblia.

    Vinithra Iyer / Sheetal Tushir / Shreekant Verma / Sudeshna Majumdar / Srimonta Gayen / Rakesh Mishra / Utpal Tatu

    PLoS Neglected Tropical Diseases, Vol 15, Iss 9, p e

    2021  Volume 0009810

    Abstract: Hsp90 gene of G. lamblia has a split nature comprising two ORFs separated by 777 kb on chromosome 5. The ORFs of the split gene on chromosome 5 undergo transcription to generate independent pre-mRNAs that join by a unique trans-splicing reaction that ... ...

    Abstract Hsp90 gene of G. lamblia has a split nature comprising two ORFs separated by 777 kb on chromosome 5. The ORFs of the split gene on chromosome 5 undergo transcription to generate independent pre-mRNAs that join by a unique trans-splicing reaction that remains partially understood. The canonical cis-acting nucleotide elements such as 5'SS-GU, 3'SS-AG, polypyrimidine tract and branch point adenine are present in the independent pre-mRNAs and therefore trans-splicing of Hsp90 must be assisted by spliceosomes in vivo. Using an approach of RNA-protein pull down, we show that an RNA helicase selectively interacts with HspN pre-mRNA. Our experiments involving high resolution chromosome conformation capture technology as well as DNA FISH show that the trans-spliced genes of Giardia are in three-dimensional spatial proximity in the nucleus. Altogether our study provides a glimpse into the in vivo mechanisms involving protein factors as well as chromatin structure to facilitate the unique inter-molecular post-transcriptional stitching of split genes in G. lamblia.
    Keywords Arctic medicine. Tropical medicine ; RC955-962 ; Public aspects of medicine ; RA1-1270
    Subject code 570
    Language English
    Publishing date 2021-09-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Proteo-Genomic Analysis of SARS-CoV-2: A Clinical Landscape of Single-Nucleotide Polymorphisms, COVID-19 Proteome, and Host Responses.

    Tushir, Sheetal / Kamanna, Sathisha / Nath, Sujith S / Bhat, Aishwarya / Rose, Steffimol / Aithal, Advait R / Tatu, Utpal

    Journal of proteome research

    2021  Volume 20, Issue 3, Page(s) 1591–1601

    Abstract: A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and continues to be a global health challenge. To understand viral disease biology, we have carried out proteo-genomic ... ...

    Abstract A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and continues to be a global health challenge. To understand viral disease biology, we have carried out proteo-genomic analysis using next-generation sequencing (NGS) and mass spectrometry on nasopharyngeal swabs of COVID-19 patients to examine the clinical genome and proteome. Our study confirms the mutability of SARS-CoV-2 showing multiple single-nucleotide polymorphisms. NGS analysis detected 27 mutations, of which 14 are synonymous, 11 are missense, and 2 are extragenic in nature. Phylogenetic analysis of SARS-CoV-2 isolates indicated their close relation to a Bangladesh isolate and multiple origins of isolates within the country. Our proteomic analysis, for the first time, identified 13 different SARS-CoV-2 proteins from the clinical swabs. Of the total 41 peptides captured by high-resolution mass spectrometry, 8 matched to nucleocapsid protein, 2 to ORF9b, and 1 to spike glycoprotein and ORF3a, with remaining peptides mapping to ORF1ab polyprotein. Additionally, host proteome analysis revealed several key host proteins to be uniquely expressed in COVID-19 patients. Pathway analysis of these proteins points toward modulation in immune response, especially involving neutrophil and IL-12-mediated signaling. Besides revealing the aspects of host-virus pathogenesis, our study opens new avenues to develop better diagnostic markers and therapeutic approaches.
    MeSH term(s) COVID-19/virology ; Coronavirus Nucleocapsid Proteins/genetics ; Genome, Viral ; Genomics ; High-Throughput Nucleotide Sequencing ; Host Microbial Interactions/genetics ; Host Microbial Interactions/physiology ; Humans ; Mutation ; Pandemics ; Phosphoproteins/genetics ; Phylogeny ; Polymorphism, Single Nucleotide ; Polyproteins/genetics ; Proteome ; Proteomics ; SARS-CoV-2/genetics ; SARS-CoV-2/pathogenicity ; SARS-CoV-2/physiology ; Spike Glycoprotein, Coronavirus/genetics ; Viral Proteins/genetics ; Viroporin Proteins/genetics
    Chemical Substances Coronavirus Nucleocapsid Proteins ; ORF1ab polyprotein, SARS-CoV-2 ; ORF3a protein, SARS-CoV-2 ; Phosphoproteins ; Polyproteins ; Proteome ; Spike Glycoprotein, Coronavirus ; Viral Proteins ; Viroporin Proteins ; nucleocapsid phosphoprotein, SARS-CoV-2 ; spike protein, SARS-CoV-2
    Language English
    Publishing date 2021-02-08
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2078618-9
    ISSN 1535-3907 ; 1535-3893
    ISSN (online) 1535-3907
    ISSN 1535-3893
    DOI 10.1021/acs.jproteome.0c00808
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 6189: Show issues Location:
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  5. Article: Co-extrusion of food grains-banana pulp for nutritious snacks: optimization of process variables.

    Mridula, D / Sethi, Swati / Tushir, Surya / Bhadwal, Sheetal / Gupta, R K / Nanda, S K

    Journal of food science and technology

    2017  Volume 54, Issue 9, Page(s) 2704–2716

    Abstract: Present study was undertaken to optimize the process conditions for development of food grains (maize, defatted soy flour, sesame seed)-banana based nutritious expanded snacks using extrusion processing. Experiments were designed using Box-Behnken design ...

    Abstract Present study was undertaken to optimize the process conditions for development of food grains (maize, defatted soy flour, sesame seed)-banana based nutritious expanded snacks using extrusion processing. Experiments were designed using Box-Behnken design with banana pulp (8-24 g), screw speed (300-350 rpm) and feed moisture (14-16% w.b.). Seven responses viz. expansion ratio (ER), bulk density (BD), water absorption index (WAI), protein, minerals, iron and sensory acceptability were considered for optimizing independent parameters. ER, BD, WAI, protein content, total minerals, iron content, and overall acceptability ranged 2.69-3.36, 153.43-238.83 kg/m
    Language English
    Publishing date 2017-07-05
    Publishing country India
    Document type Journal Article
    ZDB-ID 242498-8
    ISSN 0975-8402 ; 0022-1155
    ISSN (online) 0975-8402
    ISSN 0022-1155
    DOI 10.1007/s13197-017-2707-4
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  6. Article ; Online: Proteo-genomic analysis of SARS-CoV-2: A clinical landscape of SNPs, COVID-19 proteome and host responses

    Tushir, Sheetal / Kamanna, Sathisha / Nath, Sujith S / Bhat, Aishwarya / Rose, Steffimol / Aithal, Advait R / Tatu, Utpal

    medRxiv

    Abstract: A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 and continues to be a global health challenge. To understand viral disease biology, we have carried out proteo-genomic analysis using next generation ... ...

    Abstract A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 and continues to be a global health challenge. To understand viral disease biology, we have carried out proteo-genomic analysis using next generation sequencing (NGS) and mass-spectrometry on nasopharyngeal swabs of COVID-19 patients to examine clinical genome and proteome. Our study confirms the hyper mutability of SARS-CoV-2 showing multiple SNPs. NGS analysis detected 27 mutations of which 14 are synonymous, 11 are missense and 2 are extragenic in nature. Phylogenetic analysis of SARS-CoV-2 isolates indicated their close relation to Bangladesh isolate and multiple origins of isolates within a country. Our proteomic analysis, for the first time identified 13 different SARS-CoV-2 proteins from the clinical swabs. Of the total 41 peptides captured by HRMS, 8 matched to nucleocapsid protein, 2 to ORF9b, 1 to spike glycoprotein and ORF3a, with remaining mapping to ORF1ab polyprotein. Additionally, host proteome analysis revealed several key host proteins to be uniquely expressed in COVID-19 patients. Pathway analysis of these proteins points towards modulation in immune response, especially involving neutrophil and IL-12 mediated signaling. Besides revealing the aspects of host-virus pathogenesis, our study opens new avenues to develop better diagnostic markers and therapeutics.
    Keywords covid19
    Language English
    Publishing date 2020-11-30
    Publisher Cold Spring Harbor Laboratory Press
    Document type Article ; Online
    DOI 10.1101/2020.11.27.20237032
    Database COVID19

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  7. Article: Co-extrusion of food grains-banana pulp for nutritious snacks: optimization of process variables

    Mridula, D / Swati Sethi / Surya Tushir / Sheetal Bhadwal / R. K. Gupta / S. K. Nanda

    Journal of food science and technology. 2017 Aug., v. 54, no. 9

    2017  

    Abstract: Present study was undertaken to optimize the process conditions for development of food grains (maize, defatted soy flour, sesame seed)-banana based nutritious expanded snacks using extrusion processing. Experiments were designed using Box–Behnken ... ...

    Abstract Present study was undertaken to optimize the process conditions for development of food grains (maize, defatted soy flour, sesame seed)-banana based nutritious expanded snacks using extrusion processing. Experiments were designed using Box–Behnken design with banana pulp (8–24 g), screw speed (300–350 rpm) and feed moisture (14–16% w.b.). Seven responses viz. expansion ratio (ER), bulk density (BD), water absorption index (WAI), protein, minerals, iron and sensory acceptability were considered for optimizing independent parameters. ER, BD, WAI, protein content, total minerals, iron content, and overall acceptability ranged 2.69–3.36, 153.43–238.83 kg/m³, 4.56–4.88 g/g, 15.19–15.52%, 2.06–2.27%, 4.39–4.67 mg/100 g (w.b.) and 6.76–7.36, respectively. ER was significantly affected by all three process variables while BD was influenced by banana pulp and screw speed only. Studied process variables did not affected colour quality except ‘a’ value with banana pulp and screw speed. Banana pulp had positive correlation with water solubility index, total minerals and iron content and negative with WAI, protein and overall acceptability. Based upon multiple response analysis, optimized conditions were 8 g banana pulp, 350 rpm screw speed and 14% feed moisture indicating the protein, calorie, iron content and overall sensory acceptability in sample as 15.46%, 401 kcal/100 g, 4.48 mg/100 g and 7.6 respectively.
    Keywords absorption ; bananas ; bulk density ; color ; corn ; defatted products ; extrusion ; food grains ; iron ; minerals ; protein content ; pulp ; sesame seed ; snacks ; soy flour ; water solubility
    Language English
    Dates of publication 2017-08
    Size p. 2704-2716.
    Publishing place Springer India
    Document type Article
    ZDB-ID 242498-8
    ISSN 0975-8402 ; 0022-1155
    ISSN (online) 0975-8402
    ISSN 0022-1155
    DOI 10.1007/s13197-017-2707-4
    Database NAL-Catalogue (AGRICOLA)

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