Molecular and biochemical characterization of hydroxyjasmonate and flavonoid sulfotransferases from Arabidopsis thaliana

Recently, we initiated a functional genomics project with the objective of characterizing the biological function of all the sulfotransferase (ST) coding genes of A. thaliana . Based on amino acid sequence alignment with previously characterized soluble STs, we have identified 17 genes coding for putative STs in the genome of this plant. Prior to this work, only one ST-coding gene ( AtST1 ) has been characterized from A. thaliana . AtST1 was shown to sulfonate brassinosteroids and was proposed to be involved in the modulation of their biological activity. This thesis presents the characterization of two more ST-coding genes from A. thaliana , namely AtST2a and AtST3a . The recombinant AtST3a protein was found to exhibit strict specificity for position 7 of flavonoids. In contrast with the previously characterized flavonol 7-ST from Flaveria bidentis that sulfonates only flavonol disulfates, AtST3a was found to accept a number of flavonols and flavone aglycones, as well as their monosulfate derivatives. The AtST3a is expressed only at the earlier stage of seedling development. In contrast, the expression pattern of the flavonol 3-ST from Flaveria species is detectable at all stages of plant development, with highest activities found in the terminal buds and first pair of leaves. The natural occurrence of a ST exhibiting high specificity for flavonoids in A. thaliana suggests that sulfated flavonoids may be of more common occurrence in the plant kingdom than once thought. In this study, we demonstrate that the AtST2a from A. thaliana encodes a sulfotransferase specific for 11- and 12-hydroxyjasmonic acid. Jasmonic acid and its derivatives, commonly named jasmonates, are of ubiquitous occurrence in the plant kingdom and they play an important role in the plant response to biotic and abiotic stresses. More recently, it has been demonstrated that jasmonates are also involved in the control of key developmental processes such as anther development. AtST2a is not expressed in plants growing in the light but is induced 8 hours after their transfer to the dark. Overexpression of AtST2a in transgenic Arabidopsis leads to a delayed flowering phenotype observed only when the plants are growing under long-days. In contrast, decreasing AtST2a expression by expressing antisense AtST2a RNA leads to an early flowering phenotype observed only in short day-grown transgenic plants. Our results suggest that the function of AtST2a is to sulfonate hydroxylated jasmonic acids under growth conditions that do not favor flowering. This data also suggest that hydroxylated jasmonic acids act as signals that promote the transition from vegetative to reproductive growth when A. thaliana is exposed to an inductive photoperiod

Organisational Agility of Social Enterprises: A Phenomenological Study of Microfinance Institutions in Ethiopia

This doctoral study examines whether MFIs in Ethiopia have developed the managerial readiness to face emerging threats and seize the opportunities within the context of unpredictable changes and turbulences. The interpretative phenomenological approach (IPA) has been used to collect, shape, and interpret the lived experiences, intuitions, and insights of 10 CEOs of MFIs. The data were interpreted using the double hermeneutic and analysed through the lenses of the theory of organisational agility and the Cynefin framework to make decisions. The premise of this study is that MFIs without agile organisational capability may fail to prepare and respond to changes in the external environment. The study results show multiple impediments that restrict MFIs from being adaptive towards achieving double bottom lines, i.e., the creation of social and financial value. MFIs in Ethiopia suffer from “pain points” such as inflation, illiquidity, and turbulences. The challenges include: the weak governance practice of nominal shareholders, outdated decision-making processes causing delays, staff turnover reducing enterprising capacity, and MFIs lacking sufficient digital and technological infrastructure. The study found most MFIs are incapable of responding quickly and innovatively to seize opportunities or to overcome adversities. The conclusion is that MFIs in Ethiopia have inadequate agile organisational capability to make strategic choices and execute operational processes during multiple and complex changes. The findings of the research are important, and pertinent for a better understanding of the organisational agility of social enterprises. The study has provided five recommendations to enhance policies and practical actions to build the agile organisational capabilities of social enterprises

Localization of the Carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex

<p>Abstract</p> <p>Background</p> <p><it>Carnation Italian ringspot virus </it>(CIRV) is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36), are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined.</p> <p>Results</p> <p>Here we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs) and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC) that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM), but not with the sorting and assembly machinery (SAM).</p> <p>Conclusion</p> <p>Our results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer membrane also sheds light on the mechanisms involved in sorting of host-cell membrane proteins to mitochondria, a process that has been largely unexplored in plants.</p

Deglutitive Inhibition, Latency Between Swallow and Esophageal Contractions and Primary Esophageal Motor Disorders

Swallowing induces an inhibitory wave that is followed by a contractile wave along the esophageal body. Deglutitive inhibition in the skeletal muscle of the esophagus is controlled in the brain stem whilst in the smooth muscle, an intrinsic peripheral control mechanism is critical. The latency between swallow and contractions is determined by the pattern of activation of the inhibitory and excitatory vagal pathways, the regional gradients of inhibitory and excitatory myenteric nerves, and the intrinsic properties of the smooth muscle. A wave of inhibition precedes a swallow-induced peristaltic contraction in the smooth muscle part of the human oesophagus involving both circular and longitudinal muscles in a peristaltic fashion. Deglutitive inhibition is necessary for drinking liquids which requires multiple rapid swallows (MRS). During MRS the esophageal body remains inhibited until the last of the series of swallows and then a peristaltic contraction wave follows. A normal response to MRS requires indemnity of both inhibitory and excitatory mechanisms and esophageal muscle. MRS has recently been used to assess deglutitive inhibition in patients with esophageal motor disorders. Examples with impairment of deglutitive inhibition are achalasia of the LES and diffuse esophageal spasm

New Insights Into Sunflower (Helianthus annuus L.) FatA and FatB Thioesterases, Their Regulation, Structure and Distribution

Sunflower seeds (Helianthus annuus L.) accumulate large quantities of triacylglycerols (TAG) between 12 and 28 days after flowering (DAF). This is the period of maximal acyl-acyl carrier protein (acyl-ACP) thioesterase activity in vitro, the enzymes that terminate the process of de novo fatty acid synthesis by catalyzing the hydrolysis of the acyl-ACPs synthesized by fatty acid synthase. Fatty acid thioesterases can be classified into two families with distinct substrate specificities, namely FatA and FatB. Here, some new aspects of these enzymes have been studied, assessing how both enzymes contribute to the acyl composition of sunflower oil, not least through the changes in their expression during the process of seed filling. Moreover, the binding pockets of these enzymes were modeled based on new data from plant thioesterases, revealing important differences in their volume and geometry. Finally, the subcellular location of the two enzymes was evaluated and while both possess an N-terminal plastid transit peptide, only in FatB contains a hydrophobic sequence that could potentially serve as a transmembrane domain. Indeed, using in vivo imaging and organelle fractionation, H. annuus thioesterases, HaFatA and HaFatB, appear to be differentially localized in the plastid stroma and membrane envelope, respectively. The divergent roles fulfilled by HaFatA and HaFatB in oil biosynthesis are discussed in the light of our data.España MINECO y FEDER Projects AGL2014- 53537-R y AGL2017-83449-

Design Development and Analysis Of Two Wheeler Eco Friendly Plastic Carburetor With Rapid Prototyping

The design development and analysis of carburetor has been carry out by applying structural and thermal loads. The present work particularly deals with the drafting and designing of carburetor using plastic materials which can be manufactured with rapid prototyping to increase mass production. My main aim is to prevent the component from getting corroded or undergoing corrosion. Replacing metal components with plastic ones can offer some important bondage. Unlike metals, plastic materials can be modified to better suit. And also manufactured by using RP technique the life of the product increases of course when fuel injectors are replaced it as the main fuel input system, it had evolve into a complicated, sophisticated, expensive system. Carburetors are still found on automobiles, many small engines like those on lawn movers and model airplanes still use carburetors. It is to keep the cost of the engine down and it is very cheap to manufacture while fuel injectors requires more costly control systems

Protein–Protein Interaction Network and Subcellular Localization of the Arabidopsis Thaliana ESCRT Machinery

The endosomal sorting complex required for transport (ESCRT) consists of several multi-protein subcomplexes which assemble sequentially at the endosomal surface and function in multivesicular body (MVB) biogenesis. While ESCRT has been relatively well characterized in yeasts and mammals, comparably little is known about ESCRT in plants. Here we explored the yeast two-hybrid protein interaction network and subcellular localization of the Arabidopsis thaliana ESCRT machinery. We show that the Arabidopsis ESCRT interactome possesses a number of protein–protein interactions that are either conserved in yeasts and mammals or distinct to plants. We show also that most of the Arabidopsis ESCRT proteins examined at least partially localize to MVBs in plant cells when ectopically expressed on their own or co-expressed with other interacting ESCRT proteins, and some also induce abnormal MVB phenotypes, consistent with their proposed functional role(s) as part of the ESCRT machinery in Arabidopsis. Overall, our results help define the plant ESCRT machinery by highlighting both conserved and unique features when compared to ESCRT in other evolutionarily diverse organisms, providing a foundation for further exploration of ESCRT in plants

Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope

Background: Tail-anchored (TA) proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope. Methodology/Principal Findings: Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34) and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9). Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein. Conclusions/Significance: Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie the delivery of TA proteins to their proper intracellular locations in general

Using length data to derive biological reference points for kiddi shrimp Parapenaeopsis stylifera (Milne Edwards, 1837) from the south-eastern Arabian Sea, India

Parapenaeopsis stylifera, a major commercial penaeid shrimp fishery resource in the Indian Ocean, has lacked adequate information on life history parameters for nearly two decades. In this study, growth and mortality parameters of P. stylifera from the southwest coast of India were estimated using length data and used to derive biological reference points for the species. The asymptotic length for females was L∞ = 131 mm; k = 1.1 y−1 and for males L∞ = 117 mm; k = 1.25 y−1. Mortality parameter estimates were Z = 4.42, M = 1.24, F = 3.18 y−1 and exploitation rate E = 0.72 for females; Z = 5.76, M = 1.39, F = 4.37 y−1 and E = 0.76 for males. Thomson and Bell yield biomass, Beverton and Holt yield per recruit, and relative yield per recruit models were applied to predict the stock status and length cohort analysis for estimating the stock size. The Beverton and Holt analysis gave Emax = 0.69 in females and 0.75 for males, which is below the Ecurrent values obtained for the sexes. The Thomson and Bell analysis indicated that if Fcurrent at which the yield is 121 460 t in females and in males 128 064 t is further increased, rise in yield will be modest. B/B0 and SB/SB0 at Fcurrent were 24% and 18% for females and 21% and 16% for males, respectively. Target reference point F0.1 and F0.5 at different levels of age at capture tc (0.5, 0.6, 0.7 and 0.8 yrs) was estimated by Beverton and Holt yield per recruit model. The outcome from these models forms integral inputs for multispecies/multigear tropical fisheries management. Parapenaeopsis stylifera is one of the inshore penaeid shrimp identified by the Marine Stewardship Council for certification from the region and, moreover, biological reference points are a prerequisite to assessment and management of tropical multispecies fisheries for ecosystem-based fisheries management