Salts responsive nanovesicles through π-stacking induced self-assembly of backbone modified tripeptides

A set of backbone modified peptides of general formula Boc-Xx-m-ABA-Yy-OMe where m-ABA is meta-aminobenzoic acid and Xx and Yy are natural amino acids such as Phe, Gly, Pro, Leu, Ile, Tyr and Trp etc., are found to self-assemble into soft nanovesicular structures in methanol-water solution (9:1 by v/v). At higher concentration the peptides generate larger vesicles which are formed through fusion of smaller vesicles. The formation of vesicles has been facilitated through the participation of various noncovalent interactions such as aromatic pi-stacking, hydrogen bonding and hydrophobic interactions. Model study indicates that the pi-stacking induced self-assembly, mediated by m-ABA is essential for well structured vesicles formation. The presence of conformationally rigid m-ABA in the backbone of the peptides also helps to form vesicular structures by restricting the conformational entropy. The vesicular structures get disrupted in presence of various salts such as KCl, CaCl(2), N(n-Bu)(4)Br and (NH(4))(2)SO(4) in methanol-water solution. Fluorescence microscopy and UV studies reveal that the soft nanovesicles encapsulate organic dye molecules such as Rhodamine B and Acridine Orange which could be released through salts induced disruption of vesicles.

Realistic mean field forward predictions for the integration of co-registered EEG/fMRI

Brain activity can be measured non-invasively with functional imaging techniques. Each pixel in such an image represents a neural mass of about 105 to 107 neurons. Mean field models (MFMs) approximate their activity by averaging out neural variability while retaining salient underlying features, like neurotransmitter kinetics. However, MFMs incorporating the regional variability, realistic geometry and connectivity of cortex have so far appeared intractable. This lack of biological realism has led to a focus on gross temporal features of the EEG. We address these impediments and showcase a "proof of principle" forward prediction of co-registered EEG/fMRI for a full-size human cortex in a realistic head model with anatomical connectivity, see figure 1. MFMs usually assume homogeneous neural masses, isotropic long-range connectivity and simplistic signal expression to allow rapid computation with partial differential equations. But these approximations are insufficient in particular for the high spatial resolution obtained with fMRI, since different cortical areas vary in their architectonic and dynamical properties, have complex connectivity, and can contribute non-trivially to the measured signal. Our code instead supports the local variation of model parameters and freely chosen connectivity for many thousand triangulation nodes spanning a cortical surface extracted from structural MRI. This allows the introduction of realistic anatomical and physiological parameters for cortical areas and their connectivity, including both intra- and inter-area connections. Proper cortical folding and conduction through a realistic head model is then added to obtain accurate signal expression for a comparison to experimental data. To showcase the synergy of these computational developments, we predict simultaneously EEG and fMRI BOLD responses by adding an established model for neurovascular coupling and convolving "Balloon-Windkessel" hemodynamics. We also incorporate regional connectivity extracted from the CoCoMac database [1]. Importantly, these extensions can be easily adapted according to future insights and data. Furthermore, while our own simulation is based on one specific MFM [2], the computational framework is general and can be applied to models favored by the user. Finally, we provide a brief outlook on improving the integration of multi-modal imaging data through iterative fits of a single underlying MFM in this realistic simulation framework.

Alanine-rich amphiphilic peptide containing the RGD cell adhesion motif: a coating material for human fibroblast attachment and culture

We studied the self-assembly of peptide A6RGD (A: alanine, R: arginine, G: glycine, D: aspartic acid) in water, and the use of A6RGD substrates as coatings to promote the attachment of human cornea stromal fibroblasts (hCSFs). The self-assembled motif of A6RGD was shown to depend on the peptide concentration in water, where both vesicle and fibril formation were observed. Oligomers were detected for 0.7 wt% A6RGD, which evolved into short peptide fibres at 1.0 wt% A6RGD, while a co-existence of vesicles and long peptide fibres was revealed for 2–15 wt% A6RGD. A6RGD vesicle walls were shown to have a multilayer structure built out of highly interdigitated A6 units, while A6RGD fibres were based on β-sheet assemblies. Changes in the self-assembly motif with concentration were reflected in the cell culture assay results. Films dried from 0.1–1.0 wt% A6RGD solutions allowed hCSFs to attach and significantly enhanced cell proliferation relative to the control. In contrast, films dried from 2.5 wt% A6RGD solutions were toxic to hCSFs.

Effects of chlordiazepoxide on extinction and re-acquisition of operant behaviour in mice

Relatively little is known about the role of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in extinction of appetitively motivated tasks. The benzodiazepine (BZ) chlordiazepoxide (CDP) was administered during extinction and re-acquisition of lever pressing by mice following food reinforced discrete-trial fixed-ratio 5 (FR-5) training. Typical FR behaviour was established during baseline training and persisted for several extinction sessions. There were 15 extinction sessions in all, followed by six re-acquisition sessions where food reinforcement was re-introduced. In a 2x2x2 between-group design, CDP (15 mg/kg) or vehicle injections were given prior to either the last two food reinforcement sessions and the first 10 extinction sessions, or the final five extinction sessions, or the six re-acquisition sessions. Initially CDP had no effect on the rate of extinction, but after several extinction sessions it significantly facilitated it. Surprisingly, if CDP was administered only after several sessions of extinction, it immediately produced facilitation. Thus the delayed effects of CDP are not due to drug accumulation. These data suggest that some neural change must occur before CDP can affect extinction processes. In re-acquisition sessions, CDP facilitated the reinstatement of food-reinforced lever pressing. Implications for neural and behavioural accounts of operant extinction are discussed.

Untangling membrane rearrangement in the Nidovirales

All known positive sense single-stranded RNA viruses induce host cell membrane rearrangement for purposes of aiding viral genome replication and transcription. Members of the Nidovirales order are no exception, inducing intricate regions of double membrane vesicles and convoluted membranes crucial for the production of viral progeny. Although these structures have been well studied for some members of this order, much remains unclear regarding the biogenesis of these rearranged membranes. Here, we discuss what is known about these structures and their formation, compare some of the driving viral proteins behind this process across the nidovirus order, and examine possible routes of mechanism by which membrane rearrangement may occur.

Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca2+-dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca2+ responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca2+ channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A–siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca2+ current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

Computational analysis of the LRRK2 interactome

LRRK2 was identified in 2004 as the causative protein product of the Parkinson’s disease locus designated PARK8. In the decade since then, genetic studies have revealed at least 6 dominant mutations in LRRK2 linked to Parkinson’s disease, alongside one associated with cancer. It is now well established that coding changes in LRRK2 are one of the most common causes of Parkinson’s. Genome-wide association studies (GWAs) have, more recently, reported single nucleotide polymorphisms (SNPs) around the LRRK2 locus to be associated with risk of developing sporadic Parkinson’s disease and inflammatory bowel disorder. The functional research that has followed these genetic breakthroughs has generated an extensive literature regarding LRRK2 pathophysiology; however, there is still no consensus as to the biological function of LRRK2. To provide insight into the aspects of cell biology that are consistently related to LRRK2 activity, we analysed the plethora of candidate LRRK2 interactors available through the BioGRID and IntAct data repositories. We then performed GO terms enrichment for the LRRK2 interactome. We found that, in two different enrichment portals, the LRRK2 interactome was associated with terms referring to transport, cellular organization, vesicles and the cytoskeleton. We also verified that 21 of the LRRK2 interactors are genetically linked to risk for Parkin- son’s disease or inflammatory bowel disorder. The implications of these findings are discussed, with particular regard to potential novel areas of investigation.

Feline calicivirus p32, p39 and p30 proteins localize to the endoplasmic reticulum to initiate replication complex formation

In common with other positive-strand RNA viruses, replication of feline calicivirus (FCV) results in rearrangement of intracellular membranes and production of numerous membrane-bound vesicular structures on which viral genome replication is thought to occur. In this study, bioinformatics approaches have identified three of the FCV non-structural proteins, namely p32, p39 and p30, as potential transmembrane proteins. These proteins were able to target enhanced cyan fluorescent protein to membrane fractions where they behaved as integral membrane proteins. Immunofluorescence microscopy of these proteins expressed in cells showed co-localization with endoplasmic reticulum (ER) markers. Further electron microscopy analysis of cells co-expressing FCV p39 or p30 with a horseradish peroxidase protein containing the KDEL ER retention motif demonstrated gross morphological changes to the ER. Similar reorganization patterns, especially for those produced by p30, were observed in naturally infected Crandel-Rees feline kidney cells. Together, the data demonstrate that the p32, p39 and p30 proteins of FCV locate to the ER and lead to reorganization of ER membranes. This suggests that they may play a role in the generation of FCV replication complexes and that the endoplasmic reticulum may represent the potential source of the membrane vesicles induced during FCV infection.

Morphological characterization of periodontium-derived human stem cells

The aim of this study has been to characterize adult human somatic periodontium-derived stem cells (PDSCS) isolated from human periodontium and to follow their differentiation after cell culture. PDSCS were isolated from human periodontal tissue and cultured as spheres in serum-free medium. After 10 days the primary spheres were dissociated and the secondary spheres sub-cultured for another 1-2 weeks. Cells from different time points were analyzed, and immunohistochemical and electron microscopic investigations carried out. Histological analysis showed differentiation of spheres deriving from the PDSCS with central production of extracellular matrix beginning 3 days after sub-culturing. Isolated PDSCS developed pseudopodia which contained actin. Tubulin was found in the central portion of the cells. Pseudopodia between different cells anastomosed, indicating intercellular transport. Immunostaining for osteopontin demonstrated a positive reaction in primary spheres and within extracellular matrix vesicles after sub-culturing. In cell culture under serum-free conditions human PDSCS form spheres which are capable of producing extracellular matrix. Further investigations have do be carried out to investigate the capability of these cells to differentiate into osteogenic progenitor cells.

Simple method for sub-diffraction resolution imaging of cellular structures on standard confocal microscopes by three-photon absorption of quantum dots

This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts.

Regulation of cell migration in cancer: investigation into the regulation of cancer cell blebbing in the extracellular matrix

Cell migration is a highly coordinated process and any aberration in the regulatory mechanisms could result in pathological conditions such as cancer. The ability of cancer cells to disseminate to distant sites within the body has made it difficult to treat. Cancer cells also exhibit plasticity that makes them able to interconvert from an elongated, mesenchymal morphology to an amoeboid blebbing form under different physiological conditions. Blebs are spherical membrane protrusions formed by actomyosin-mediated contractility of cortical actin resulting in increased hydrostatic pressure and subsequent detachment of the membrane from the cortex. Tumour cells use blebbing as an alternative mode of migration by squeezing through preexisting gaps in the ECM, and bleb formation is believed to be mediated by the Rho-ROCK signaling pathway. However, the involvement of transmembrane water and ion channels in cell blebbing has not been examined. In the present study, the role of the transmembrane water channels, aquaporins, transmembrane ion transporters and lipid signaling enzymes in the regulation of blebbing was investigated. Using 3D matrigel matrix as an in vitro model to mimic normal extracellular matrix, and a combination of confocal and time-lapse microscopy, it was found that AQP1 knockdown by siRNA ablated blebbing of HT1080 and ACHN cells, and overexpression of AQP1-GFP not only significantly increased bleb size with a corresponding decrease in bleb numbers, but also induced bleb formation in non-blebbing cell lines. Importantly, AQP1 overexpression reduces bleb lifespan due to faster bleb retraction. This novel finding of AQP1-facilitated bleb retraction requires the activity of the Na+/H+ pump as inhibition of the ion transporter, which was found localized to intracellular vesicles, blocked bleb retraction in both cell lines. This study also demonstrated that a differential regulation of cell blebbing by AQP isoforms exists as knockdown of AQP5 had no effect on bleb formation. Data from this study also demonstrates that the lipid signaling PLD2 signals through PA in the LPA-LPAR-Rho-ROCK axis to positively regulate bleb formation in both cell lines. Taken together, this work provides a novel role of AQP1 and Na+/H+ pump in regulation of cell blebbing, and this could be exploited in the development of new therapy to treat cancer.

‘Symptomless’ infection by Botrytis cinerea

The study was carried out to clarify the nature of symptomless infection by Botrytis cinerea and to what extent it differs from aggressive necrotic infection in Lactuca sativa (lettuce) and Arabidopsis thaliana. Symptomless plants were produced by dry spore inoculation in plants growing in controlled environmental conditions or in glasshouses. Plating out of surface-disinfected and non-surface-disinfected samples of inoculated, apparently healthy, plants on selective medium revealed that the fungus was spreading from the initial inoculation site to newly developing plant organs both internally and externally. Similar findings were obtained in microscope experiments in which host plants were inoculated with GFP labelled B. cinerea and symptomless spreading was monitored under confocal laser scanning microscope. Spore germination on leaf surface was followed by development of sub-cuticular vesicles and plant cell damage in the infected epidermal cell and a few nearby cells. Sparsely branched long hyphae arose from the vesicles and spread on the leaf surface; spread was mostly on the outer surface of the epidermal layer but occasionally below the cuticle or epidermal cells. In the late symptomless phase, mycelium arising from single vesicles formed several mycelial networks on leaves. Experiments were carried out to compare the extent of gene expression in symptomless and necrotic infections, using RT-qPCR. Expression of selected genes was quantified in tissue samples based on the amount of mRNA of the respective genes found. In both host species, the mRNA concentration of signalling genes bcg1, bmp1 and calcineurin, and the pathogenicity genes bcsod1 and bcpg1 were similar to or slightly greater in symptomless samples than in necrotic samples. The mRNA of the signalling gene bac and pathogenicity genes bcbot1 and bcnep1, were not detected or detected in lower abundance than in necrosis. In lettuce, the leaves developing distant from the site of inoculation showed similar results to A. thaliana, but in healthy leaves close to the site of inoculation mRNA concentrations of bac and bcnep1 were similar to necrotic samples. Thus, in both host species, the fungus grew along with the plant and moved to newly growing plant parts without producing symptoms; during this growth some pathogenicity genes were less expressed than in necrotic infection.

Intracellular trafficking, localization, and mobilization of platelet-borne thiol isomerases

OBJECTIVE: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. APPROACH AND RESULTS: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13dJinx mice). CONCLUSIONS: Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.