The quest to achieve the detailed structural and functional characterization of CymA

2012 ◽  
Vol 40 (6) ◽  
pp. 1291-1294 ◽  
Author(s):  
Ricardo O. Louro ◽  
Catarina M. Paquete
Keyword(s):  
Microbial Fuel Cells ◽  
Critical Role ◽  
Functional Characterization ◽  
Shewanella Oneidensis ◽  
Model Organism ◽  
Structural Behaviour ◽  
Metal Compounds ◽  
Potential Applications ◽  
And Function

Shewanella oneidensis MR-1 is a sediment organism capable of dissimilatory reduction of insoluble metal compounds such as those of Fe(II) and Mn(IV). This bacterium has been used as a model organism for potential applications in bioremediation of contaminated environments and in the production of energy in microbial fuel cells. The capacity of Shewanella to perform extracellular reduction of metals is linked to the action of several multihaem cytochromes that may be periplasmic or can be associated with the inner or outer membrane. One of these cytochromes is CymA, a membrane-bound tetrahaem cytochrome localized in the periplasm that mediates the electron transfer between the quinone pool in the cytoplasmic membrane and several periplasmic proteins. Although CymA has the capacity to regulate multiple anaerobic respiratory pathways, little is known about the structure and functional mechanisms of this focal protein. Understanding the structure and function of membrane proteins is hampered by inherent difficulties associated with their purification since the choice of the detergents play a critical role in the protein structure and stability. In the present mini-review, we detail the current state of the art in the characterization of CymA, and add recent information on haem structural behaviour for CymA solubilized in different detergents. These structural differences are deduced from NMR spectroscopy data that provide information on the geometry of the haem axial ligands. At least two different conformational forms of CymA are observed for different detergents, which seem to be related to the micelle size. These results provide guidance for the discovery of the most promising detergent that mimics the native lipid bilayer and is compatible with biochemical and structural studies.

scholarly journals Beyond classic editing: innovative CRISPR approaches for functional studies of long non-coding RNA

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Dahlia A Awwad
Keyword(s):  
Genetic Manipulation ◽  
Functional Characterization ◽  
The Novel ◽  
Functional Studies ◽  
Non Coding Rna ◽  
Rna Targeting ◽  
Potential Applications ◽  
And Function ◽  
Long Non Coding Rna

Abstract Long non-coding RNAs (lncRNAs) makeup a considerable part of the non-coding human genome and had been well-established as crucial players in an array of biological processes. In spite of their abundance and versatile roles, their functional characteristics remain largely undiscovered mainly due to the lack of suitable genetic manipulation tools. The emerging CRISPR/Cas9 technology has been widely adapted in several studies that aim to screen and identify novel lncRNAs as well as interrogate the functional properties of specific lncRNAs. However, the complexity of lncRNAs genes and the regulatory mechanisms that govern their transcription, as well as their unique functionality pose several limitations the utilization of classic CRISPR methods in lncRNAs functional studies. Here, we overview the unique characteristics of lncRNAs transcription and function and the suitability of the CRISPR toolbox for applications in functional characterization of lncRNAs. We discuss some of the novel variations to the classic CRISPR/Cas9 system that have been tailored and applied previously to study several aspects of lncRNAs functionality. Finally, we share perspectives on the potential applications of various CRISPR systems, including RNA-targeting, in the direct editing and manipulation of lncRNAs.

Functional characterization of human brown adipose tissue metabolism

2020 ◽  
Vol 477 (7) ◽  
pp. 1261-1286 ◽  
Author(s):  
Marie Anne Richard ◽  
Hannah Pallubinsky ◽  
Denis P. Blondin
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Functional Characterization ◽  
Human Infants ◽  
Positron Emission ◽  
Brown Adipose ◽  
Treatment Of Obesity ◽  
And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

Molecular and Functional Characterization of One Odorant-Binding Protein Gene OBP3 in Bemisia tabaci (Hemiptera: Aleyrodidae)

2019 ◽  
Author(s):  
Ran Wang ◽  
Yuan Hu ◽  
Peiling Wei ◽  
Cheng Qu ◽  
Chen Luo
Keyword(s):  
Host Plant ◽  
Bemisia Tabaci ◽  
Binding Protein ◽  
Insect Pest ◽  
Critical Role ◽  
Functional Characterization ◽  
Odorant Binding Protein ◽  
Binding Characteristics ◽  
And Function ◽  
Odorant Binding

Abstract Odorant binding proteins (OBPs) of insects play a critical role in chemical perceptions and choice of insect host plant. Bemisia tabaci is a notorious insect pest which can damage more than 600 plant species. In order to explore functions of OBPs in B. tabaci, here we investigated binding characteristics and function of odorant-binding protein 3 in B. tabaci (BtabOBP3). The results indicated that BtabOBP3 shows highly similar sequence with OBPs of other insects, including the typical signature motif of six cysteines. The recombinant BtabOBP3 protein was obtained, and the evaluation of binding affinities to tested volatiles of host plant was conducted, then the results indicated that β-ionone had significantly higher binding to BtabOBP3 among other tested plant volatiles. Furthermore, silencing of BtabOBP3 significantly altered choice behavior of B. tabaci to β-ionone. In conclusion, it has been demonstrated that BtabOBP3 exerts function as one carrier of β-ionone and the results could be contributed to reveal the mechanisms of choosing host plant in B. tabaci.

Functional characterization of the trypanosome translational repressor SCD6

2013 ◽  
Vol 457 (1) ◽  
pp. 57-67 ◽  
Author(s):  
Marina Cristodero ◽  
Bernd Schimanski ◽  
Manfred Heller ◽  
Isabel Roditi
Keyword(s):  
Functional Characterization ◽  
Stress Granule ◽  
Granule Formation ◽  
Translational Repressor ◽  
And Function

Trypanosomal SCD6 is a general repressor of translation. It is not required for stress granule formation and, unusually, does not interact with the helicase DHH1. We analysed domains involved in the localization and function of TbSCD6 and identified interacting partners.

scholarly journals A Mycobacterial Systems Resource for the Research Community

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
J. A. Judd ◽  
J. Canestrari ◽  
R. Clark ◽  
A. Joseph ◽  
P. Lapierre ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Protein Localization ◽  
Functional Characterization ◽  
Model Organism ◽  
Research Community ◽  
Mycobacterial Species ◽  
Biological Resource ◽  
Content Type ◽  
Core Genes

ABSTRACT Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more difficult to grow and genetically manipulate than model systems such as Escherichia coli and Bacillus subtilis. To facilitate functional characterization of mycobacterial proteins, we have established a Mycobacterial Systems Resource (MSR) using the model organism Mycobacterium smegmatis. This resource focuses specifically on 1,153 highly conserved core genes that are common to many mycobacterial species, including Mycobacterium tuberculosis, in order to provide the most relevant information and resources for the mycobacterial research community. The MSR includes both biological and bioinformatic resources. The biological resource includes (i) an expression plasmid library of 1,116 genes fused to a fluorescent protein for determining protein localization; (ii) a library of 569 precise deletions of nonessential genes; and (iii) a set of 843 CRISPR-interference (CRISPRi) plasmids specifically targeted to silence expression of essential core genes and genes for which a precise deletion was not obtained. The bioinformatic resource includes information about individual genes and a detailed assessment of protein localization. We anticipate that integration of these initial functional analyses and the availability of the biological resource will facilitate studies of these core proteins in many Mycobacterium species, including the less experimentally tractable pathogens M. abscessus, M. avium, M. kansasii, M. leprae, M. marinum, M. tuberculosis, and M. ulcerans. IMPORTANCE Diseases caused by mycobacterial species result in millions of deaths per year globally, and present a substantial health and economic burden, especially in immunocompromised patients. Difficulties inherent in working with mycobacterial pathogens have hampered the development and application of high-throughput genetics that can inform genome annotations and subsequent functional assays. To facilitate mycobacterial research, we have created a biological and bioinformatic resource (https://msrdb.org/) using Mycobacterium smegmatis as a model organism. The resource focuses specifically on 1,153 proteins that are highly conserved across the mycobacterial genus and, therefore, likely perform conserved mycobacterial core functions. Thus, functional insights from the MSR will apply to all mycobacterial species. We believe that the availability of this mycobacterial systems resource will accelerate research throughout the mycobacterial research community.

scholarly journals Identification and functional characterization of SlDronc in Spodoptera littoralis

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10329
Author(s):  
Hao Liu ◽  
Ke Zhou ◽  
Zhouning Yang
Keyword(s):  
Spodoptera Littoralis ◽  
Functional Characterization ◽  
Model Organism ◽  
Vital Role ◽  
Apoptotic Pathway ◽  
Effector Caspase ◽  
Baculovirus Infection ◽  
Wide Range ◽  
Induced Apoptosis ◽  
And Function

Background Apoptosis is responsible for eliminating damaged and virus-infected cells, regulating normal cell turnover, and maintaining the immune system’s development and function. Caspases play a vital role in both mammal and invertebrate apoptosis. Spodoptera littoralis is a generalist insect herbivore that is one of the most destructive pests in tropical and subtropical areas and attacks a wide range of commercially important crops. Although S. littoralis is a model organism in the study of baculovirus infection, its apoptotic pathway has not been explored. Methods We cloned a new caspase gene named sldronc in S. littoralis using Rapid Amplification of cDNA Ends (RACE). We then measured caspase activity on synthetic caspase substrates and S. littoralis’ effector caspase. SlDronc’s function in the apoptotic pathway and its interaction with caspase inhibitors were also tested in SL2 cells. Results We found that the initiator caspase SlDronc cleaved and activated effector caspase in S. littoralis. SlDronc overexpression induced apoptosis in SL2 cells, and Sldronc knockdown decreased apoptosis induced by UV irradiation in SL2 cells. Our results indicate that SlDronc acts as an apoptotic initiator caspase in S. littoralis. Additionally, we found that processed forms of SlDronc increased in the presence of N-terminally truncated S. littoralis inhibitors of apoptosis (SlIAP) and that SlDronc was inhibited by P49. This study contributes to the further understanding of S. littoralis’ apoptotic pathway and may facilitate future studies on baculovirus infection-induced apoptosis.

scholarly journals Ontogenic, phenotypic, and functional characterization of XCR1+ dendritic cells leads to a consistent classification of intestinal dendritic cells based on the expression of XCR1 and SIRPα

2014 ◽  
Author(s):  
Martina Becker ◽  
Steffen Güttler ◽  
Annabell Bachem ◽  
Evelyn Hartung ◽  
Ahmed Mora ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Functional Characterization ◽  
Cross Presentation ◽  
Intestinal Immune System ◽  
Lineage Marker ◽  
And Function ◽  
First Time

In the past, lack of lineage markers confounded the classification of dendritic cells (DC) in the intestine and impeded a full understanding of their location and function. We have recently shown that the chemokine receptor XCR1 is a lineage marker for cross-presenting DC in the spleen. Now we provide evidence that intestinal XCR1+ DC largely, but not fully, overlap with CD103+ CD11b- DC, the hypothesized correlate of “cross-presenting DC” in the intestine, and are selectively dependent in their development on the transcription factor Batf3. XCR1+ DC are located in the villi and epithelial crypts of the lamina propria of the small intestine, the T cell zones of Peyer’s Patches, and in the T cell zones and sinuses of the draining mesenteric lymph node. Functionally, we could demonstrate for the first time that XCR1+ / CD103+ CD11b- DC excel in the cross-presentation of orally applied antigen. Together, our data show that XCR1 is a lineage marker for cross-presenting DC also in the intestinal immune system. Further, extensive phenotypic analyses reveal that expression of the integrin SIRPα consistently demarcates the XCR1- DC population. We propose a simplified and consistent classification system for intestinal DC based on the expression of XCR1 and SIRPα.

Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish

Blood ◽  
2001 ◽  
Vol 98 (10) ◽  
pp. 3087-3096
Author(s):  
Graham J. Lieschke ◽  
Andrew C. Oates ◽  
Meredith O. Crowhurst ◽  
Alister C. Ward ◽  
Judith E. Layton
Keyword(s):  
Functional Characterization ◽  
Cell Mass ◽  
Myeloid Cells ◽  
Model Organism ◽  
Intermediate Cell ◽  
Adult Zebrafish ◽  
Hematopoietic Organ ◽  
Carbon Particles ◽  
And Function ◽  
A Site

The zebrafish is a useful model organism for developmental and genetic studies. The morphology and function of zebrafish myeloid cells were characterized. Adult zebrafish contain 2 distinct granulocytes, a heterophil and a rarer eosinophil, both of which circulate and are generated in the kidney, the adult hematopoietic organ. Heterophils show strong histochemical myeloperoxidasic activity, although weaker peroxidase activity was observed under some conditions in eosinophils and erythrocytes. Embryonic zebrafish have circulating immature heterophils by 48 hours after fertilization (hpf). A zebrafish myeloperoxidase homologue (myeloid-specificperoxidase; mpx) was isolated. Phylogenetic analysis suggested it represented a gene ancestral to the mammalian myeloperoxidase gene family. It was expressed in adult granulocytes and in embryos from 18 hpf, first diffusely in the axial intermediate cell mass and then discretely in a dispersed cell population. Comparison of hemoglobinized cell distribution,mpx gene expression, and myeloperoxidase histochemistry in wild-type and mutant embryos confirmed that the latter reliably identified a population of myeloid cells. Studies in embryos after tail transection demonstrated that mpx- and peroxidase-expressing cells were mobile and localized to a site of inflammation, indicating functional capability of these embryonic granulocytes. Embryonic macrophages removed carbon particles from the circulation by phagocytosis. Collectively, these observations have demonstrated the early onset of zebrafish granulopoiesis, have proved that granulocytes circulate by 48 hpf, and have demonstrated the functional activity of embryonic granulocytes and macrophages. These observations will facilitate the application of this genetically tractable organism to the study of myelopoiesis.

Functional characterization of long-chain prenyl diphosphate synthases from tomato

2013 ◽  
Vol 449 (3) ◽  
pp. 729-740 ◽  
Author(s):  
Matthew O. Jones ◽  
Laura Perez-Fons ◽  
Francesca P. Robertson ◽  
Peter M. Bramley ◽  
Paul D. Fraser
Keyword(s):  
Functional Characterization ◽  
Primary Metabolism ◽  
Virus Induced Gene Silencing ◽  
In Planta ◽  
Constitutive Overexpression ◽  
Prenyl Diphosphate ◽  
And Function ◽  
Leaf Appearance ◽  
Long Chain

The electron transfer molecules plastoquinone and ubiquinone are formed by the condensation of aromatic head groups with long-chain prenyl diphosphates. In the present paper we report the cloning and characterization of two genes from tomato (Solanum lycopersicum) responsible for the production of solanesyl and decaprenyl diphosphates. SlSPS (S. lycopersicum solanesyl diphosphate synthase) is targeted to the plastid and both solanesol and plastoquinone are associated with thylakoid membranes. A second gene [SlDPS (S. lycopersicum solanesyl decaprenyl diphosphate synthase)], encodes a long-chain prenyl diphosphate synthase with a different subcellular localization from SlSPS and can utilize geranyl, farnesyl or geranylgeranyl diphosphates in the synthesis of C45 and C50 prenyl diphosphates. When expressed in Escherichia coli, SlSPS and SlDPS extend the prenyl chain length of the endogenous ubiquinone to nine and ten isoprene units respectively. In planta, constitutive overexpression of SlSPS elevated the plastoquinone content of immature tobacco leaves. Virus-induced gene silencing showed that SlSPS is necessary for normal chloroplast structure and function. Plants silenced for SlSPS were photobleached and accumulated phytoene, whereas silencing SlDPS did not affect leaf appearance, but impacted on primary metabolism. The two genes were not able to complement silencing of each other. These findings indicate a requirement for two long-chain prenyl diphosphate synthases in the tomato.

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