Design and experimental characterization of a tunable vibration-based electromagnetic micro-generator

Vibration-based micro-generators, as an alternative source of energy, have become increasingly significant in the last decade. This paper presents a new tunable electromagnetic vibration-based micro-generator. Frequency tuning is realized by applying an axial tensile force to the micro-generator. The dimensions of the generator, especially the dimensions of the coil and the air gap between magnets, have been optimized to maximize the output voltage and power of the micro-generator. The resonant frequency has been successfully tuned from 67.6 to 98 Hz when various axial tensile forces were applied to the structure. The generator produced a power of 61.6–156.6 µW over the tuning range when excited at vibrations of 0.59 ms-2. The tuning mechanism has little effect on the total damping. When the tuning force applied on the generator becomes larger than the generator’s inertial force, the total damping increases resulting in reduced output power. The resonant frequency increases less than indicated from simulation and approaches that of a straight tensioned cable when the force associated with the tension in the beam becomes much greater than the beam stiffness. The test results agree with the theoretical analysis presented

Eukaryotic GCP1 is a conserved mitochondrial protein required for progression of embryo development beyond the globular stage in Arabidopsis thaliana.

GCPs (glycoproteases) are members of the HSP70 (beat-shock protein 70)/actin ATPase superfamily that are highly conserved in taxonomically diverse species from bacteria to man, suggesting an essential physiological role. Although originally identified and annotated as putative endopeptidases, a proteolytic activity could not be confirmed for these proteins. Our survey of genome databases revealed that all eukaryotic organisms contain two GCP genes [called GCP1 and GCP2/Kae1 (kinase-associated endopeptidase 1)], whereas prokaryotes have only one, either of the GCP1-(Bacteria) or the GMIKae1- (Archaea) type. GCP2/Kae1 is essential for telomere elongation and transcription of essential genes, although little is known about the localization, expression and physiological role of GCP1. In the present study on GCP1-type proteins from eukaryotic organisms we demonstrated that GCP1 is a mitochondrial protein in Homo sapiens [called here GCPI/OSGEPL1 (O-sialoglycoprotein endopeptidase)] and Arabidopsis thaliana, which is located/anchored to the mitochondrial inner membrane. Analysis of mRNA and protein levels revealed that the expression of GCP1/OSGEPL1 in A. thaliana and H. sapiens is tissue- and organ-specific and depends on the developmental stage, suggesting a more specialized function for this protein. We showed that homozygous A. thaliana GCP1 T-DNA (transferred DNA) insertion lines were embryonic lethal. Embryos in homozygous seeds were arrested at the globular stage and failed to undergo the transition into the heart stage. On the basis of these data we propose that the mitochondrial GCP1 is essential for embryonic development in plants

RC1339/APRc from <i>Rickettsia conorii</i> Is a Novel Aspartic Protease with Properties of Retropepsin-Like Enzymes

<div><p>Members of the species <i>Rickettsia</i> are obligate intracellular, gram-negative, arthropod-borne pathogens of humans and other mammals. The life-threatening character of diseases caused by many <i>Rickettsia</i> species and the lack of reliable protective vaccine against rickettsioses strengthens the importance of identifying new protein factors for the potential development of innovative therapeutic tools. Herein, we report the identification and characterization of a novel membrane-embedded retropepsin-like homologue, highly conserved in 55 <i>Rickettsia</i> genomes. Using <i>R. conorii</i> gene homologue RC1339 as our working model, we demonstrate that, despite the low overall sequence similarity to retropepsins, the gene product of <i>rc1339</i> APRc (for <u>A</u>spartic <u>P</u>rotease from <i><u>R</u>ickettsia <u>c</u>onorii</i>) is an active enzyme with features highly reminiscent of this family of aspartic proteases, such as autolytic activity impaired by mutation of the catalytic aspartate, accumulation in the dimeric form, optimal activity at pH 6, and inhibition by specific HIV-1 protease inhibitors. Moreover, specificity preferences determined by a high-throughput profiling approach confirmed common preferences between this novel rickettsial enzyme and other aspartic proteases, both retropepsins and pepsin-like. This is the first report on a retropepsin-like protease in gram-negative intracellular bacteria such as <i>Rickettsia</i>, contributing to the analysis of the evolutionary relationships between the two types of aspartic proteases. Additionally, we have also shown that APRc is transcribed and translated in <i>R. conorii</i> and <i>R. rickettsii</i> and is integrated into the outer membrane of both species. Finally, we demonstrated that APRc is sufficient to catalyze the <i>in vitro</i> processing of two conserved high molecular weight autotransporter adhesin/invasion proteins, Sca5/OmpB and Sca0/OmpA, thereby suggesting the participation of this enzyme in a relevant proteolytic pathway in rickettsial life-cycle. As a novel <i>bona fide</i> member of the retropepsin family of aspartic proteases, APRc emerges as an intriguing target for therapeutic intervention against fatal rickettsioses.</p></div

APRc can process rOmpA <i>in vitro</i>.

<p>Total membrane fractions of <i>E. coli</i> enriched in rOmpA were incubated with both activated APRc and the active site mutant form (D140A) for 16 h. The reaction products were then subjected to immunoblot analysis with anti-rOmpA Ab, confirming the disappearance of rOmpA in the presence of the active form of the enzyme. Molecular weight markers in kilodaltons (kDa) are shown on the left. Protein loading controls: Coomassie blue staining.</p

<i>T. pseudonana</i> protein termini identified by TAILS.

<p>(a) Schematic representation of the TAILS workflow. Proteins with free or naturally modified (black square) N termini are denatured, followed by chemical modification of all primary amines (grey triangle). Specific digestion with trypsin generates peptides amenable to mass spectrometric identification. N-terminal peptides are blocked, whereas internal or C-terminal peptides exhibit a trypsin-generated primary amine at their N terminus that is used to covalenty bind these peptides to an aldehyde-containing polymer which is subsequently removed by filtration. (b) Position of identified N-terminal peptides with respect to curated protein model. N termini matching the protein models at positions 1 and 2 are cytosolic proteins with intact (+M1) or removed (–M1) initiating Met. Black, acetylated N termini; dark grey, protein N termini present in both dimethylated and acetylated forms; light grey, free N termini identified as dimethylated peptides. (c) Sequence logoplot of the first 6 amino acids of 81 N termini of nuclear encoded proteins with intact initiating Met. (d) Sequence logoplot of 231 N termini of nuclear-encoded proteins starting at protein model position 2 because the initiating Met was removed. (e) Combined logoplot of N termini of 22 plastid-encoded proteins starting at position 2 after N-terminal Met excision plus 18 plastid-imported proteins with Met directly preceding the identified peptide.</p