RNA Interference of Plant MAPK Cascades for Functional Studies

RNA interference dynamics in juvenile Fasciola hepatica are altered during in vitro growth and development

10.1101/2020.06.06.137646 ◽

2020 ◽

Author(s):

Paul McCusker ◽

Wasim Hussain ◽

Paul McVeigh ◽

Erin McCammick ◽

Nathan G. Clarke ◽

...

Keyword(s):

Rna Interference ◽

Functional Genomics ◽

Liver Fluke ◽

Fasciola Hepatica ◽

Life Stage ◽

Life Stages ◽

Functional Studies ◽

Rnai Efficiency ◽

The Impact

AbstractFor over a decade RNA interference (RNAi) has been an important molecular tool for functional genomics studies in parasitic flatworms. Despite this, our understanding of RNAi dynamics in many flatworm parasites, such as the temperate liver fluke (Fasciola hepatica), remains rudimentary. The ability to maintain developing juvenile fluke in vitro provides the opportunity to perform functional studies during development of the key pathogenic life stage. Here, we investigate the RNAi competence of developing juvenile liver fluke. Firstly, all life stages examined possess, and express, core candidate RNAi effectors encouraging the hypothesis that all life stages of F. hepatica are RNAi competent. RNAi effector analyses supported growing evidence that parasitic flatworms have evolved a separate clade of RNAi effectors with unknown function. Secondly, we assessed the impact of growth / development during in vitro culture on RNAi in F. hepatica juveniles and found that during the first week post-excystment liver fluke juveniles exhibit quantitatively lower RNAi mediated transcript knockdown when maintained in growth inducing media. This did not appear to occur in older in vitro juveniles, suggesting that rapidly shifting transcript dynamics over the first week following excystment alters RNAi efficacy after a single 24 hour exposure to double stranded (ds)RNA. Finally, RNAi efficiency was found to be improved through use of a repeated dsRNA exposure methodology that has facilitated silencing of genes in a range of tissues, thereby increasing the utility of RNAi as a functional genomics tool in F. hepatica.

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Investigation of the role of TASK-2 channels in rat pulmonary arteries; pharmacological and functional studies following RNA interference procedures

British Journal of Pharmacology ◽

10.1038/sj.bjp.0706649 ◽

2006 ◽

Vol 147 (5) ◽

pp. 496-505 ◽

Cited By ~ 29

Author(s):

Mónika Gönczi ◽

Norbert Szentandrássy ◽

Ian T Johnson ◽

Anthony M Heagerty ◽

Arthur H Weston

Keyword(s):

Rna Interference ◽

Pulmonary Arteries ◽

Functional Studies

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Functional studies of an eyestalk molt inhibiting hormone (MIH-1) in black tiger shrimp Penaeus monodon by RNA interference

Frontiers in Neuroendocrinology ◽

10.1016/j.yfrne.2006.03.049 ◽

2006 ◽

Vol 27 (1) ◽

pp. 23

Author(s):

C.Y. Mak ◽

S.-M. Chan

Keyword(s):

Rna Interference ◽

Penaeus Monodon ◽

Black Tiger Shrimp ◽

Functional Studies ◽

Tiger Shrimp

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Motoring around the plant cell: insights from plant myosins

Biochemical Society Transactions ◽

10.1042/bst0380833 ◽

2010 ◽

Vol 38 (3) ◽

pp. 833-838 ◽

Cited By ~ 26

Author(s):

Imogen A. Sparkes

Keyword(s):

Plasma Membrane ◽

Rna Interference ◽

Nuclear Envelope ◽

Growth And Development ◽

Dominant Negative ◽

Organelle Movement ◽

Higher Plant ◽

Functional Studies ◽

Dynamic Movement

Organelle movement in plants cells is extremely dynamic. Movement is driven by the acto-myosin system. Higher plant myosins fall into two classes: classes XI and VIII. Localization studies have highlighted that myosins are present throughout the cytosol, label motile puncta and decorate the nuclear envelope and plasma membrane. Functional studies through expression of dominant-negative myosin variants, RNAi (RNA interference) and T-DNA insertional analysis have shown that class XI myosins are required for organelle movement. Intriguingly, organelle movement is also linked to Arabidopsis growth and development. The present review tackles current findings relating to plant organelle movement and the role of myosins.

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The low-resolution 3D-structure of porin, a channel-forming protein in E. coliouter membranes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100075932 ◽

1983 ◽

Vol 41 ◽

pp. 440-441

Author(s):

A. Engel ◽

D.L. Dorset ◽

A. Massalski ◽

J.P. Rosenbusch

Keyword(s):

Crystal Packing ◽

3D Structure ◽

Gram Negative Bacteria ◽

Protein Ratio ◽

Crystal Forms ◽

Large Molecules ◽

Functional Studies ◽

Voltage Dependent ◽

Planar Membranes ◽

Hexagonal Arrays

Porins represent a group of channel forming proteins that facilitate diffusion of small solutes across the outer membrane of Gram-negative bacteria, while excluding large molecules (>650 Da). Planar membranes reconstituted from purified matrix porin (OmpF protein) trimers and phospholipids have allowed quantitative functional studies of the voltage-dependent channels and revealed concerted activation of triplets. Under the same reconstitution conditions but using high protein concentrations porin aggregated to 2D lattices suitable for electron microscopy and image processing. Depending on the lipid-to- protein ratio three different crystal packing arrangements were observed: a large (a = 93 Å) and a small (a = 79 Å) hexagonal and a rectangular (a = 79 Å b = 139 Å) form with p3 symmetry for the hexagonal arrays. In all crystal forms distinct stain filled triplet indentations could be seen and were found to be morphologically identical within a resolution of (22 Å). It is tempting to correlate stain triplets with triple channels, but the proof of this hypothesis requires an analysis of the structure in 3 dimensions.

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Ionic homeostasis and subcellular element compartmentation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010013434x ◽

1990 ◽

Vol 48 (2) ◽

pp. 150-151

Author(s):

Ann LeFurgey ◽

Peter Ingram ◽

J.J. Blum ◽

M.C. Carney ◽

L.A. Hawkey ◽

...

Keyword(s):

Leishmania Major ◽

Ionic Homeostasis ◽

X Ray ◽

Functional Studies ◽

Cell Compartments ◽

X Ray Imaging ◽

Resolution Electron ◽

Multiple Cell ◽

Role Of Zn

Subcellular compartments commonly identified and analyzed by high resolution electron probe x-ray microanalysis (EPXMA) include mitochondria, cytoplasm and endoplasmic or sarcoplasmic reticulum. These organelles and cell regions are of primary importance in regulation of cell ionic homeostasis. Correlative structural-functional studies, based on the static probe method of EPXMA combined with biochemical and electrophysiological techniques, have focused on the role of these organelles, for example, in maintaining cell calcium homeostasis or in control of excitation-contraction coupling. New methods of real time quantitative x-ray imaging permit simultaneous examination of multiple cell compartments, especially those areas for which both membrane transport properties and element content are less well defined, e.g. nuclei including euchromatin and heterochromatin, lysosomes, mucous granules, storage vacuoles, microvilli. Investigations currently in progress have examined the role of Zn-containing polyphosphate vacuoles in the metabolism of Leishmania major, the distribution of Na, K, S and other elements during anoxia in kidney cell nuclel and lysosomes; the content and distribution of S and Ca in mucous granules of cystic fibrosis (CF) nasal epithelia; the uptake of cationic probes by mltochondria in cultured heart ceils; and the junctional sarcoplasmic retlculum (JSR) in frog skeletal muscle.

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