Development and Validation of Arc Nanobodies: New Tools for Probing Arc Dynamics and Function

Neurochemical Research - Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral...     

Scotia Arc Acari: antiquity and origin

The Scotia Arc comprises South Georgia, South Sandwich, South Orkney and South Shetland Islands together with the Antarctic Peninsula. Free-living Acari of the Scotia Arc contain immigrant and endemic elements. Transport of immigrant species to and within the Maritime Antarctic has been via Holocene storms and ocean currents. Most immigrants are Gamasida, Oribatida and Acaridida while Actinedida dominate the endemic element. Immigrant species on South Georgia share common 'sub-Antarctic' affinities with South Indian and, to a lesser extent, South Pacific Ocean island faunas. In contrast, immigrants to the rest of the Scotia Arc and Bouvetøya on the mid-Atlantic Ridge, form a robust 'Maritime Antarctic' Province group. The endemic component is of largely Tertiary origin and, like that of Continental Antarctica, dominated by a few cosmopolitan families and genera of Actinedida. There are no bona fide pan-Antarctic species and little evidence that Continental and Maritime Antarctic faunas have a common ancestry, indeed the Continental endemic fauna is entirely montane while that of Maritime Antarctica is coastal. The presence of common Maritime Antarctic/South Pacific island genera corroborate the Antarctic Peninsula as being derived from a South Pacific island archipelago which collided with Continental Antarctica during the Tertiary period.     

Arc1p: Anchoring, routing, coordinating

Accurate synthesis of aminoacyl-tRNAs (aa-tRNA) by aminoacyl-tRNA synthetases (aaRS) is an absolute requirement for errorless decoding of the genetic code and is studied since more than four decades. In all three kingdoms of life aaRSs are capable of assembling into multi-enzymatic complexes that are held together by auxiliary non-enzymatic factors, but the role of such macromolecular assemblies is still poorly understood. In the yeast Saccharomyces cerevisiae, Arc1p holds cytosolic methionyl-tRNA synthetase (_cMRS) and glutamyl-tRNA synthetase (_cERS) together and plays an important role in fine tuning several cellular processes like aminoacylation, translation and carbon source adaptation.     

Arc/Arg3.1: linking gene expression to synaptic plasticity and memory

Arc/Arg3.1 is an effector immediate-early gene implicated in the consolidation of memories. Although cloned a decade ago, the physiological role of Arc/Arg3.1 in the brain has remained elusive. Four papers in this issue of Neuron address this function. These studies show that Arc/Arg3.1 regulates endophilin 3 and dynamin 2, two components of the endocytosis machinery. Genetic ablation of Arc/Arg3.1 in mice or overexpression in culture suggest that Arc/Arg3.1 regulates AMPA receptor trafficking and synaptic plasticity. Finally, Arc/Arg3.1 knockout mice show memory retention deficits. These recent developments provide new insights into the function of this popular activity-dependent neuronal marker.     

NMR studies of Arc repressor mutants: proton assignments, secondary structure, and long-range contacts for the thermostable proline-8----leucine variant of Arc

Arc repressor is a 53-residue sequence-specific DNA binding protein. We report the assignment of the proton NMR spectrum and the secondary structure for the thermostable PL8 variant of Arc. This mutant, which differs from wild type by a Pro-8----Leu substitution, was chosen for study because its enhanced stability allows spectra to be acquired at elevated temperatures where spectral resolution is higher. The first five residues of the protein play important roles in DNA binding but appear to be disordered in solution. Residues 6-14 form the remaining part of the N-terminal DNA binding region of the protein and assume an antiparallel beta-conformation. This indicates that Arc is a member of a new class of DNA binding proteins. The observed interresidue nuclear Overhauser effects are consistent with a beta-strand, gamma-turn, beta-strand structure for the residue 6-14 region, although other structures are also consistent with the data. The remaining portion of the protein is predominantly alpha-helical. Residues 16-26 and 35-50 form amphipathic alpha-helices which may pack together in a four-helix bundle in the protein dimer.     

Experimental Observation and Numerical Analysis of the Arc Plasma Axial Motion in a Magnetically Rotating Arc Plasma Generator

Plasma Physics Reports - To better understand the arc plasma axial motion under an axial magnetic field, a high-speed charge-coupled device camera was used to observe the arc plasma images in a...     

Crystallization of the Arc repressor

Arc, a represser from Salmonella phage P22 has been crystallized from ammonium phosphate at pH 8.0. The crystals form in space group P2₁2₁2₁ with $\text{a = 90.26}\text{A}\text{?}\text{, b = 52.88}\text{A}\text{?}\text{and}\text{c = 47.58}\text{A}\text{?}$. The crystals diffract to 2.2 Å resolution.     

Cardiac Exposure in the Dynamic Conformal Arc Therapy,Intensity-Modulated Radiotherapy and Volumetric Modulated Arc Therapy of Lung Cancer

Purpose

To retrospectively evaluate the cardiac exposure in three cohorts of lung cancer patients treated with dynamic conformal arc therapy (DCAT), intensity-modulated radiotherapy (IMRT), or volumetric modulated arc therapy (VMAT) at our institution in the past seven years.

Methods and Materials

A total of 140 lung cancer patients were included in this institutional review board approved study: 25 treated with DCAT, 70 with IMRT and 45 with VMAT. All plans were generated in a same commercial treatment planning system and have been clinically accepted and delivered. The dose distribution to the heart and the effects of tumor laterality, the irradiated heart volume and the beam-to-heart distance on the cardiac exposure were investigated.

Results

The mean dose to the heart among all 140 plans was 4.5 Gy. Specifically, the heart received on average 2.3, 5.2 and 4.6 Gy in the DCAT, IMRT and VMAT plans, respectively. The mean heart doses for the left and right lung tumors were 4.1 and 4.8 Gy, respectively. No patients died with evidence of cardiac disease. Three patients (2%) with preexisting cardiac condition developed cardiac disease after treatment. Furthermore, the cardiac exposure was found to increase linearly with the irradiated heart volume while decreasing exponentially with the beam-to-heart distance.

Conclusions

Compared to old technologies for lung cancer treatment, modern radiotherapy treatment modalities demonstrated better heart sparing. But the heart dose in lung cancer radiotherapy is still higher than that in the radiotherapy of breast cancer and Hodgkin’s disease where cardiac complications have been extensively studied. With strong correlations of mean heart dose with beam-to-heart distance and irradiated heart volume, cautions should be exercised to avoid long-term cardiac toxicity in the lung cancer patients undergoing radiotherapy.     

Equilibrium dissociation and unfolding of the Arc repressor dimer

The equilibrium unfolding reaction of Arc repressor, a dimeric DNA binding protein encoded by bacteriophage P22, can be monitored by fluorescence or circular dichroism changes. The stability of Arc is concentration dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The stability of the protein is decreased at low pH and increased by high salt concentration. The salt dependence suggests that two ions bind preferentially to the folded protein. In 10 mM potassium phosphate (pH 7.3) and 100 mM KCl, the unfolding free energy reaches a maximum near room temperature. The results suggest that at the low protein concentrations where operator DNA binding is normally measured, Arc is predominantly monomeric and unfolded.     

Virtual Tangential-fields Arc Therapy (ViTAT) for whole breast irradiation: Technique optimization and validation

PurposeTo test the performances of a volumetric arc technique named ViTAT (Virtual Tangential-fields Arc Therapy) mimicking tangential field irradiation for whole breast radiotherapy.MethodsViTAT plans consisted in 4 arcs whose starting/ending position were established based on gantry angle distribution of clinical plans for right and left-breast. The arcs were completely blocked excluding the first and last 20°. Different virtual bolus densities and thicknesses were preliminarily evaluated to obtain the best plan performances. For 40 patients with tumor laterality equally divided between right and left sides, ViTAT plans were optimized considering the clinical DVHs for OARs (resulting from tangential field manual planning) to constrain them: ViTAT plans were compared with the clinical tangential-fields in terms of DVH parameters for both PTV and OARs.ResultsDistal angle values were suggested in the ranges [220°,240°] for the right-breast and [115°,135°] for the left-breast cases; medial angles were [60°,40°] for the right side and [295°,315°] for the left side, limiting the risk of collision. The optimal virtual bolus had −500 HU density and 1.5 cm thickness. ViTAT plans generated dose distributions very similar to the tangential-field plans, with significantly improved PTV homogeneity. The mean doses of ipsilateral OARs were comparable between the two techniques with minor increase of the low-dose spread in the range 2–15 Gy (few % volume); contralateral OARs were slightly better spared with ViTAT.ConclusionViTAT dose distributions were similar to tangential-fields. ViTAT should allow automatic plan optimization by developing knowledge-based DVH prediction models of patients treated with tangential-fields.     

Serotonin2 Receptors in the Nucleus Tractus Solitarius: Characterization and Role in the Baroreceptor Reflex Arc

1. There is a general agreement concerning the key role of the baroreceptor reflex in blood pressure homeostasis. It is also well accepted that baroreceptor afferent messages are first integrated within the nucleus tractus solitarius (NTS) and that an excitatory amino acid, probably glutamate, is the principal neurotransmitter of corresponding afferents fibers. However, important points concerning the processing of baroreceptor messages within the NTS remain to be clarified, in particular the possible modulatory role of other neuroactive substances at this particular level in the medulla oblongata.2. In this context, the present review focuses on serotonin, and the possible facilitatory influence of NTS serotonergic afferents and receptors on the baroreceptor reflex arc. Relevant pharmacological, electrophysiological, immunohistochemical, and biochemical data, are presented and discussed. They can be summarized as follows.3. The selective destruction of the nodose ganglion-NTS serotonergic pathway produces a long-term increase in blood pressure variability, similar to that caused by baroreceptor denervation.4. Microinjection of picomolar doses of 5-HT into the NTS elicits the typical responses of baroreceptor activation.5. The cardiovascular effects elicited by local microinjections of specific agonists and antagonists into the NTS of intact rats and of animals that underwent nodose ganglionectomy indicate that the baroreceptor-like effects of locally administered 5-HT are mediated by the activation of postsynaptic 5-HT₂ receptors.6. The medullary pathways which mediate NTS 5-HT₂ receptor-evoked responses are similar to those involved in the baroreceptor reflex arc.7. Pharmacological and electrophysiological studies suggest that the cardiovascular effects of intra-NTS 5-HT involve the 5-HT_2A receptor subtype expressed by NTS barosensitive neurons that receive polysynaptic vagal afferents.8. Intra-NTS microinjection of a subthreshold dose of DOI, a 5-HT₂ receptor agonist, which, on its own, does not produce any cardiovascular changes, significantly enhances the bradycardiac component of the baroreflex.9. Altogether, the data summarized above show that, in the NTS, 5-HT acting at 5-HT_2A receptors exerts a facilitatory influence on the baroreceptor reflex, especially on the cardiac component of this reflex.10. Convergent pharmacological and electrophysiological data indicate that, in the NTS, functional interactions between NMDA- and 5-HT_2A-receptors coexpressed by the same neurons probably underlie the facilitatory influence of 5-HT upon the baroreceptor reflex.11. Under physiological conditions, the 5-HT_2A receptor-mediated facilitatory modulation of the cardiovagal component of the baroreflex might be triggered by 5-HT released from nodose ganglion-NTS serotoninergic afferent neurons and/or for serotoninergic projections originating in raphe nuclei. The latter possibility might notably occur during recovery after physical exercise and/or during the freezing reaction in stressed animals.     

Hydrogen exchange studies of the Arc repressor: Evidence for a monomeric folding intermediate

The hydrogen exchange rates of the backbone amide and labile side-chain protons of the dimeric Arc repressor have been measured. For the slowly exchanging amides in the α-helical regions, these rates show a concentration dependence. To account for this dependence, the role of the monomer–dimer equilibrium was considered. Extrapolating the observed exchange rates to zero dimer concentration provides estimates of these rates in the monomer and shows that they are significantly retarded compared to those of an unfolded polypeptide. This suggests that the monomer is in a structured “molten globule” like state. In particular, the two helices of Arc retain a high degree of their secondary structure and it is proposed that the two amphiphilic helices are packed together with their hydrophobic faces. Evidence for a partially folded structure in the Arc monomer was reported earlier in two other studies [J. L. Silva, C. F. Silveira, A. Correia, Jr., and L. Pontes (1992) Journal of Molecular Biology, Vol. 223, pp. 545–555: X. Peng, J. Jonas, and J. L. Silva (1993) Proceedings of the National Academy of Science USA, Vol. 90, pp. 1776–1780]. By combining the results of these studies and ours, a folding pathway of the dimeric Arc repressor involving four different stages is proposed. Due to the low concentration of Arc repressor in the cell, the protein is present either as a free monomer or it is bound to DNA presumably as a tetramer. Therefore the folding pathway can be regarded as an integral part of the overall DNA binding process. © 1995 John Wiley & Sons, Inc.     

Wildfires in the Eastern Arc Mountains of Tanzania: Burned areas,underlying causes and management challenges

The Eastern Arc Mountains are one of the most important ecosystems that conserve biodiversity in the world. These ecosystems are threatened by the increasing occurrence of wildfires. Nevertheless, there is inadequate information useful for the development of effective strategies to prevent or respond to future fires. This paper analyses the current extent of dry season fires, underlying causes and the effectiveness of the fire management strategy being implemented in and around the Uluguru Nature Forest Reserve (UNFR) between 2016 and 2021. Differenced Normalised Burn Ratio derived from Landsat satellite images was applied to determine the extent of burned areas, and focus group discussions were held to determine the underlying causes of fires and the extent of implementation of fire management strategies. About 2% (472 ha) of reserved UNFR and 5% (2,854 ha) of unreserved forests were burned in 2017. Some of the fires impacted on 60% (370 ha) of the grassy Lukwangule plateau, which is home to a fire‐sensitive endemic species. The underlying causes of fires varied spatially across the mountains but generally, fire escaping from farm preparation and hunting activities were found to be the most prevalent. On average, survey participants perceived that fire management strategy objectives were achieved by only 29% mainly constrained by a shortage of financial and human resources. Our findings suggest that ignitions and fire spread in UNFR could be prevented or controlled through sustainable funding of fire management activities and the effective engagement of local communities in the management of the reserve.     

Spatial Structure and Dynamics of a Pulsed Arc Discharge in Vacuum

Plasma Physics Reports - The process of switching a short vacuum gap using an auxiliary discharge over the surface of a dielectric is studied by the high-speed recording of images of the plasma in...     

MicroRNA Regulation of the Synaptic Plasticity-Related Gene Arc

Expression of activity-regulated cytoskeleton associated protein (Arc) is crucial for diverse types of experience-dependent synaptic plasticity and long-term memory in mammals. However, the mechanisms governing Arc-specific translation are little understood. Here, we asked whether Arc translation is regulated by microRNAs. Bioinformatic analysis predicted numerous candidate miRNA binding sites within the Arc 3'-untranslated region (UTR). Transfection of the corresponding microRNAs in human embryonic kidney cells inhibited expression of an Arc 3'UTR luciferase reporter from between 10 to 70% across 16 microRNAs tested. Point mutation and deletion of the microRNA-binding seed-region for miR-34a, miR-326, and miR-19a partially or fully rescued reporter expression. In addition, expression of specific microRNA pairs synergistically modulated Arc reporter expression. In primary rat hippocampal neuronal cultures, ectopic expression of miR-34a, miR-193a, or miR-326, downregulated endogenous Arc protein expression in response to BDNF treatment. Conversely, treatment of neurons with cell-penetrating, peptide nucleic acid (PNA) inhibitors of miR-326 enhanced Arc mRNA expression. BDNF dramatically upregulated neuronal expression of Arc mRNA and miR-132, a known BDNF-induced miRNA, without affecting expression of Arc-targeting miRNAs. Developmentally, miR-132 was upregulated at day 10 in vitro whereas Arc-targeting miRNAs were downregulated. In the adult brain, LTP induction in the dentate gyrus triggered massive upregulation of Arc and upregulation of miR-132 without affecting levels of mature Arc-targeting miRNAs. Turning to examine miRNA localization, qPCR analysis of dentate gyrus synaptoneurosome and total lysates fractions demonstrated synaptic enrichment relative to small nucleolar RNA. In conclusion, we find that Arc is regulated by multiple miRNAs and modulated by specific miRNA pairs in vitro. Furthermore, we show that, in contrast to miR-132, steady state levels of Arc-targeting miRNAs do not change in response to activity-dependent expression of Arc in hippocampal neurons in vitro or during LTP in vivo.     

Statistical modeling of mRNP transport in dendrites: A comparative analysis of β-actin and Arc mRNP dynamics

Localization of messenger RNA (mRNA) in dendrites is crucial for regulating gene expression during long-term memory formation. mRNA binds to RNA-binding proteins (RBPs) to form messenger ribonucleoprotein (mRNP) complexes that are transported by motor proteins along microtubules to their target synapses. However, the dynamics by which mRNPs find their target locations in the dendrite have not been well understood. Here, we investigated the motion of endogenous β-actin and Arc mRNPs in dissociated mouse hippocampal neurons using the MS2 and PP7 stem-loop systems, respectively. By evaluating the statistical properties of mRNP movement, we found that the aging Lévy walk model effectively describes both β-actin and Arc mRNP transport in proximal dendrites. A critical difference between β-actin and Arc mRNPs was the aging time, the time lag between transport initiation and measurement initiation. The longer mean aging time of β-actin mRNP (~100 s) compared with that of Arc mRNP (~30 s) reflects the longer half-life of constitutively expressed β-actin mRNP. Furthermore, our model also permitted us to estimate the ratio of newly generated and pre-existing β-actin mRNPs in the dendrites. This study offers a robust theoretical framework for mRNP transport, which provides insight into how mRNPs locate their targets in neurons.     

Polyynes and Cyanopolyynes: Their Synthesis with the Carbon Arc Gives the Same Abundances Occurring in Carbon-Rich Stars

Carbon vapour generated from a carbon arc or by laser ablation of graphite is reactive with simple molecules and atoms producing end-capped polyyne chains. With these techniques both hydrogen-terminated polyynes as well as monocyano- and dicyanopolyynes have been produced. Experiments based on arcing graphite electrodes can reproduce the molecular distribution of polyynes existing around carbon-rich AGB stars. In fact, it has been found that the relative abundances of the polyynes produced in carbon arc in vacuum decreases by a factor between 3 and 5 as the chain length increases by a C₂ unit. An analogous trend has been observed both for polyynes and cyanopolyynes in the circumstellar environment around carbon-rich stars. This fact suggests that the mechanism of formation of the polyynes in the carbon arc may be similar to that occurring in the surroundings of the carbon-rich stars. Polyynes and cyanopolyynes represent authentic prebiotic molecules which appear quite ubiquitous in the cosmos and should have played a role in the early organic chemistry preceding the appearance of life. Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005.     

Building arks for tRNA: Structure and function of the Arc1p family of non-catalytic tRNA-binding proteins

Following the intricate architecture of the eukaryotic cell, protein synthesis involves formation of many macromolecular assemblies, some of which are composed by tRNA-aminoacylation enzymes. Protein-protein and protein-tRNA interactions in these complexes can be facilitated by non-catalytic tRNA-binding proteins. This review focuses on the dissection of the molecular, structural and functional properties of a particular family of such proteins: yeast Arc1p and its homologues in prokaryotes and higher eukaryotes. They represent paradigms of the strategies employed for the organization of sophisticated and dynamic nanostructures supporting spatio-temporal cellular organization.