36 research outputs found
Fluctuation Methods To Study Protein Aggregation in Live Cells: Concanavalin A Oligomers Formation
No full textPrefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralleled cell morphology changes, indicating progressive cell compaction and death. Upon protein aggregation, we observed increased membrane water penetration as reported by Laurdan generalized polarization imaging
Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers
No full textAssembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Reduction of mobility is associated with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was observed on the seconds timescale that oligomerized in a concentration-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly.sponsorship: J. Hendrix is grateful for a postdoctoral fellowship and travel grant from the Research Foundation Flanders. D.C. Lamb wishes to acknowledge financial support of the Deutsche Forschungsgemeinschaft (DFG) through the Excellence Initiatives Nanosystems Initiative Munich, Center for Integrated Protein Science Munich, and SFB 1032 (Project B3) and the Ludwig-Maximilians-Universitat Munchen through the Center for Nano Science and the LMUinnovative Biolmaging Network. H.-G. Krausslich and B. Muller acknowledge DFG support through SFB638 (H.-G. Krausslich) and grant MU885/5-1 (B. Willer). H.-G. Krausslich and B. Willer are investigators of the CellNetworks Cluster of Excellence. This work was supported by the National Institutes of Health National Institutes of Health grants P41 GM103540 and P50-GM076516 (E. Gratton and M.A. Digman). (Research Foundation Flanders, Deutsche Forschungsgemeinschaft (DFG) through Excellence Initiatives Nanosystems Initiative Munich, Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universitat Munchen through Center for Nano Science, LMUinnovative BioImaging Network, DFG|SFB638, DFG|MU885/5-1, National Institutes of Health|P41 GM103540, National Institutes of Health|P50-GM076516, SFB 1032)status: Publishe
Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: Interplay between nanostructure and composition
No full textHere we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid-protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP-DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP-DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol-DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability. © 2013 Elsevier B.V
On the Effect of Downscaling in Inkjet Printed Life-Inspired Compartments
No full textThe fabrication of size-scalable liquid compartments is a topic of fundamental importance in synthetic
biology, aiming to mimic the structures and the functions of cellular compartments. Here, inkjet
printing is demonstrated as a customizable approach to fabricate aqueous compartments at different
size regimes (from nanoliter to femtoliter scale) revealing the crucial role of size in governing the
emerging of new properties. At first, inkjet printing is shown to produce homogenous aqueous
compartments stabilized by oil-confinement with mild surfactants down to the hundreds of picoliter
scale [1]. Raster Image Correlation Spectroscopy allows to monitor few intermolecular events by the
involvement of protein-binding assays within these compartments [2]. Subsequently, in order to
reduce droplet size at values below the nozzle size, a theoretical model from Eggers et al. [3] is
experimentally reproduced permitting to obtain femtoliter-scale aqueous droplets from picoliter-scale
microchannels [4]. As a remarkable difference to picoliter scale droplets, downscaling at the
femtoliter-size triggers the spontaneous formation of molecularly crowded shell structures at the
water/oil interface stabilized by a mixture of biocompatible surfactants. The shells have typical
thickness in order of hundreds of nanometers, in accordance with theoretical models [5]. Molecular
crowding effects in these systems are tested by using fluorescence lifetime imaging under the phasor
plot approach [6], revealing different characteristic lifetimes of specific probe molecules in the
confined volumes with respect to bulk solutions. The femtoliter-scale compartments autonomously
trigger the formation of unique features (e.g., up-concentration, spatial heterogeneity, molecular proximity) that are mediated by the intermolecular interactions in these novel environments, ultimately permitting to mimic the native conditions of sub-cellular scale compartments. The crowding conditions in femtoliter-scale droplets do not to affect the conformation variation of a model DNA hairpin in presence of molecular triggers and of a CYP2E1-catalyzed enzymatic reaction. Our results can be a first step towards the fabrication of size-scalable lab-on-a-chip compartments mimicking sub-cellular environments.
References
1. G. Arrabito, F. Cavaleri, V. Montalbano, V. Vetri, M. Leone, B. Pignataro, Lab on Chip, 2016, 16,
4666.
2. M.A. Digman, C. M. Brown, A. R. Horwitz,W.W. Mantulin, and E. Gratton, Biophysical Journal,
2016, 94, 2819.
3. J. Eggers, Phys. Rev. Lett. 1993, 71, 3458.
4. G. Arrabito, F. Cavaleri, A. Porchetta, F. Ricci, V. Vetri, M. Leone, B. Pignataro, Adv. Biosys.
2019, 1900023.
5. M. Staszak, J. Surfactants Deterg., 2016, 19, 297.
6. C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P.J. Donovan, and E. Gratton, Proc. Natl.
Acad. Sci. USA 2011, 108, 13582
Recommended from our members
Author Correction: Automated segmentation and tracking of mitochondria in live-cell time-lapse images
No full textIn the version of this article originally published, there was an omission in the Acknowledgements section. The text “This work was also supported in part by American Cancer Society Institutional Research Grant 134045-IRG-19-145-16-IRG” has now been added to the HTML and PDF versions of the article
The relationship between forgiveness and personality factors of military service soldiers with and without experience in internacional military operations
No full textŠī bakalaura darba mērķis bija noskaidrot: 1. Vai pastāv sakarības starp piedošanas aspektiem un personības faktoriem militārā dienesta karavīriem? 2. Vai pastāv atšķirības piedošanas aspektu un personības faktoru sakarību mērījos karavīriem bez un ar pieredzi starptautiskajās militārajās operācijās? Pētījumos ir pierādīts, ka veiksmīgas attiecības un sadarbību ir iespējams panākt ar piedošanu, kur lielāku vai mazāku iespēju piedošanai nosaka personības faktori. Mūsdienās piedošanas un personības faktoru (iezīmju) saistība zinātniskajos pētījumos pētīta izmantojot vienu no atzītākajiem mūsdienu personības iezīmju struktūras modeļiem „Lielā Piecinieka” (Big Five) modeli jeb piecu faktoru modeli (Five Factor Model), kura autors ir Digmans (Digman, 1990). Pētījumā piedalījās 50 militārā dienesta karavīri, vecuma diapazonā no 23 līdz 47 gadiem. Lai atbildētu uz pētījuma jautājumiem, respondenti aizpildīja divas aptaujas - Latvijas personības aptauju (LPA-v3) Pašraksturojuma forma, kura ietver 6 skalas – neirotisms, ekstraversija, atvērtība pieredzei, apzinīgums, labvēlīgums, godīgums-pieticība (V. Perepjolkina, V.Reņģe, 2013) un Piedošanas aptauju (Heartland Forgiveness Scale, HFS, Thomson, 2002, 2005), kura ietver 3 skalas – piedošana sev, piedošana citiem un piedošana situācijai. Pētījums apstiprināja, ka piedošanas aspektiem ir saistība ar šādiem personības faktoriem – neirotismu, ekstraversiju, apzinīgumu un labvēlīgumu, un minētās sakarības ir apstiprinājušās arī citos pētījumos. Savukārt, par atvērtību pieredzei un godīgumu - pieticību citos pētījumos sakarības netika atrastas. Neatkarīgi no tā vai karavīram ir bijusi vai nav bijusi pieredze starptautiskajās operācijās, šī pētījuma rezultāti liecina, ka atšķirības piedošanas aspektos un personības faktoros “neirotisms”, ekstraversija”, “labvēlīgums” un “atvērtība pieredzei” nepastāv.The aim of the given This Bachelor thesis was to find out: 1. Is there a relationship between aspects of forgiveness and personality traits military service soldiers? 2. Are there is forgiveness aspects and personality traits realtionship military soldiers without and with experience in international military operations? Studies have shown that good relations and cooperation is possible with forgiveness, where more or less personality factors determined the possibility of forgiveness. Today, forgivenss and personality factors scientific studies investigated the relationship with one of the most respected contemporary personality traits structure models Big Five or the model of the five factors (Five Factor Model), whose author is Digman (Digman, 1990). This research included 50 respondents, age range from 23 to 47 years old. The research used The personality survey (LPA-3), which revealed 6 scale – neuroticism, extraversion, agreeableness, conscientiousness, openness to experience, honesty-humility (V. Perepjolkina, V.Reņģe, 2013) and Heartland Forgiveness Scale, HFS, Thomson (2002, 2005), which includes 3 scale – forgiveness yourself, forgiveness ohers and forgiveness situations. The study confirmed that forgiveness is the relationship with the forgiveness aspects of the personality traits – neuroticism, extraversion, conscientiousness and agreeableness, and those connections are confirmedin other studies. The openness to experience and honesty-humility contentment in other studies of the connections werw not found. Regardless of whether a soldier has been or had no experience ininternational operationsthe results of this study show that theaspects of forgiveness and personality factors does not exist. Keywords: forgiveness, personality traits, military soldiers
The potential for natural attenuation of petroleum hydrocarbons in groundwater: Shell Newport Terminal, Victoria
No full textDeposited with permission of the author. © 2000 Amelia Jayne LavisGroundwater in the fractured and jointed Quaternary Newer Volcanics basaltic aquifer system beneath the Shell Newport Terminal has been contaminated by petroleum hydrocarbons. This petroleum hydrocarbon contamination has resulted from a number of different spill incidents over the terminal's long operation.
Petroleum hydrocarbon contamination has led to the formation of a light nonaqueous phase liquid (LNAPL) floating on the water table. Associated dissolved and vapour phases have also developed within the basaltic aquifer. The effectiveness of natural attenuation processes to remediate the petroleum hydrocarbon contamination has been evaluated based on the gas chromatography / mass spectroscopy analysis of LNAPL samples and changes in groundwater geochemistry.
Three distinct LNAPL plumes were identified within the Shell Newport Terminal jet facility plume (kerosene source); off-site plume along High 8t (mixed source of leaded petrol, kerosene, and diesel); and black oil fuel gantry plume, which is migrating off-site to Digman Reserve (mixed source of leaded petrol, shell sol A, and diesel). LNAPL ratio analysis revealed only that the samples were degraded compared to their estimated source composition. Further interpretation of ratios was difficult due to constraints involved in source compositions, sampling limitations, heterogeneity of the groundwater system, and high transport velocities of the groundwater compared to LNAPL migration. (For complete abstract open document
Quantitative Live-Cell Imaging of Human Immunodeficiency Virus (HIV-1) Assembly
No full textAdvances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site
Creating green consumer loyalty; How to strategically market CSR and obtain consumer preference
No full textSustainability is an important social theme that is taking on increasing significance in many areas, including the supermarket sector. This report presents the findings of a literature study looking at the opportunities available to supermarkets to gain a preferential position in the eyes of consumers for their efforts regarding sustainability. Duurzaamheid is een belangrijk maatschappelijk thema, dat ook in de supermarktbranche van steeds grotere betekenis wordt. Dit rapport presenteert de bevindingen van een literatuurstudie naar de aangrijpingspunten die supermarkten hebben om voor hun duurzaamheidsinspanningen een voorkeurspositie bij de consument te verkrijgen
Huntington's disease cerebrospinal fluid seeds aggregation of mutant huntingtin
No full textHuntington's disease (HD), a progressive neurodegenerative disease, is caused by an expanded CAG triplet repeat producing a mutant huntingtin protein (mHTT) with a polyglutamine-repeat expansion. Onset of symptoms in mutant huntingtin gene-carrying individuals remains unpredictable. We report that synthetic polyglutamine oligomers and cerebrospinal fluid (CSF) from BACHD transgenic rats and from human HD subjects can seed mutant huntingtin aggregation in a cell model and its cell lysate. Our studies demonstrate that seeding requires the mutant huntingtin template and may reflect an underlying prion-like protein propagation mechanism. Light and cryo-electron microscopy show that synthetic seeds nucleate and enhance mutant huntingtin aggregation. This seeding assay distinguishes HD subjects from healthy and non-HD dementia controls without overlap (blinded samples). Ultimately, this seeding property in HD patient CSF may form the basis of a molecular biomarker assay to monitor HD and evaluate therapies that target mHTT
