Distribution Of Oligomers Research Articles

Cadherin Extracellular Domain Clustering in the Absence of Trans-Interactions

While both cis and trans (adhesive)-interactions cooperate in the assembly of intercellular adhesions, computational simulations have predicted that two-dimensional confinement may promote cis-oligomerization, in the absence of trans-interactions. Here, single molecule tracking of cadherin extracellular domains on supported lipid bilayers revealed the density-dependent formation of oligomers and cis-clusters in the absence of trans-interactions, as well as allowed direct observation of interactions via single molecule Förster Resonance Energy Transfer (FRET). Lateral oligomers were virtually eliminated by mutating a putative cis (lateral) binding interface. At low cadherin surface coverage, wild-type and mutant cadherin diffused rapidly, consistent with the motion of a lipid molecule within a cadherin-free supported bilayer and with cadherins diffusing as monomers. Although the diffusion of mutant cadherin did not change appreciably with increasing surface coverage, the average short-time diffusion coefficient of wild-type cadherin slowed significantly above a fractional surface coverage of ∼0.01 (∼1,100 molecules/µm2). A detailed analysis of molecular trajectories suggested the presence of a broad size distribution of cis-cadherin oligomers. These findings verify predictions that two-dimensional confinement promotes cis-oligomerization, in the absence of trans-interactions.

Correction of Systematic Bias in Single Molecule Photobleaching Measurements

Single molecule photobleaching is a powerful technique to measure the number of fluorescent units in subresolution molecular complexes, such as in toxic protein oligomers associated with amyloid diseases. However, photobleaching can occur before the sample is appropriately placed and focused. Such “prebleaching” can introduce a strong systematic bias toward smaller oligomers. Quantitative correction of prebleaching is known to be an ill-posed problem, limiting the utility of the technique. Here, we provide an experimental solution to improve its reliability. We chemically construct multimeric standards to estimate the prebleaching probability, B. We show that B can be used as a constraint to reliably correct the statistics obtained from a known distribution of standard oligomers. Finally, we apply this method to the data obtained from a heterogeneous oligomeric solution of human islet amyloid polypeptide. Our results show that photobleaching can critically skew the estimation of oligomeric distributions, so that low abundance monomers display a much higher apparent abundance. In summary, any inference from photobleaching experiments with B > 0.1 is likely to be unreliable, but our method can be used to quantitatively correct possible errors.

Oncogenic Gain of Function in Glioblastoma Is Linked to Mutant p53 Amyloid Oligomers

SummaryTumor-associated p53 mutations endow cells with malignant phenotypes, including chemoresistance. Amyloid-like oligomers of mutant p53 transform this tumor suppressor into an oncogene. However, the composition and distribution of mutant p53 oligomers are unknown and the mechanism involved in the conversion is sparse. Here, we report accumulation of a p53 mutant within amyloid-like p53 oligomers in glioblastoma-derived cells presenting a chemoresistant gain-of-function phenotype. Statistical analysis from fluorescence fluctuation spectroscopy, pressure-induced measurements, and thioflavin T kinetics demonstrates the distribution of oligomers larger than the active tetrameric form of p53 in the nuclei of living cells and the destabilization of native-drifted p53 species that become amyloid. Collectively, these results provide insights into the role of amyloid-like mutant p53 oligomers in the chemoresistance phenotype of malignant and invasive brain tumors and shed light on therapeutic options to avert cancer.

Effects of lipid membrane composition on the distribution of biocidal guanidine oligomer with solid supported lipid membranes.

Polyhexamethylene guanidine (PHMG) is a cationic antimicrobial oligomer that has been used prevalently over the past few decades. However, due to the lack of inhalation toxicity assessment of PHMG, it has caused severe health damage, including fatal lung fibrosis, after being used as one of the major active ingredients of humidifier disinfectants in Korea. Because the first step of the entry of PHMG into airway is its association with cell membranes, the distribution of PHMG between lipid membranes and water is very important to know the depositional flux in the respiratory systems and related toxic mechanisms. We developed a quantitative method to determine the distribution constant (Klipw) of PHMG between solid supported lipid membranes and water and evaluated the effects of lipid membrane compositions on the Klipw of PHMG. PHMG accumulated into anionic lipid membranes rapidly compared to into cationic or zwitterionic lipid membranes, suggesting fast adsorption of PHMG onto anionic lipid head groups. Klipw values with anionic/zwitterionic lipid mixtures were higher than Klipw values with anionic lipids only, potentially due to the later phase separation after preferential interaction between PHMG and anionic lipids in lipid mixtures. In addition, Klipw values increased with increasing single acyl chain lipid content in unsaturated lipids and decreasing cholesterol content. These results imply that changes in lipid spontaneous curvature and lipid bilayer packing density also affect the membrane distribution of PHMG.

Enhanced oligomerization of full-length RAGE by synergy of the interaction of its domains

The pattern recognition receptor RAGE (receptor for advanced glycation end-products) transmits proinflammatory signals in several inflammation-related pathological states, including vascular diseases, cancer, neurodegeneration and diabetes. Its oligomerization is believed to be important in signal transduction, but RAGE oligomeric structures and stoichiometries remain unclear. Different oligomerization modes have been proposed in studies involving different truncated versions of the extracellular parts of RAGE. Here, we provide basic characterization of the oligomerization patterns of full-length RAGE (including the transmembrane (TM) and cytosolic regions) and compare the results with oligomerization modes of its four truncated fragments. For this purpose, we used native mass spectrometry, analytical ultracentrifugation, and size-exclusion chromatography coupled with multi-angle light scattering. Our results confirm known oligomerization tendencies of separate domains and highlight the enhanced oligomerization properties of full-length RAGE. Mutational analyses within the GxxxG motif of the TM region show sensitivity of oligomeric distributions to the TM sequence. Using hydrogen–deuterium exchange, we mapped regions involved in TM-dependent RAGE oligomerization. Our data provide experimental evidence for the major role of the C2 and TM domains in oligomerization, underscoring synergy among different oligomerization contact regions along the RAGE sequence. These results also explain the variability of obtained oligomerization modes in RAGE fragments.

Novel in-situ preparation of nano sized Ni (0) catalyst for depolymerization of lignin-rich waste from industrial biorefinery

Lignin-rich waste are valuable sources of chemicals and other materials but the depolymerisation processes are still unworkable on industrial scale. One of the problems ongoing is the catalyst preparation which often requires laborious and time-consuming processes.In this paper, a first example of easy preparation of in-situ Ni(0) nano sized catalyst for lignin-rich industrial waste hydrogenolysis is reported. Ni(0) particles were produced by reduction of Ni(II) acetate dissolved in alcoholic suspension of lignin. Soda lignin was isolated from the solid stream of a second generation bioethanol industrial plant and was depolymerized to low molecular weight chemicals by Ni(0) and/or Pd/C in alcoholic media. In 1,2-propandiol at 210 °C, up to 70% of starting material was converted into soluble compounds with an overall yield of monolignol derivatives of about 18%. The remaining material is constituted by lignin fragments with molecular weight between 400 and 1400 Da. The oligomers formation and distribution was discussed.

Zinc Binding to Tau Influences Aggregation Kinetics and Oligomer Distribution.

Metal ions are well known modulators of protein aggregation and are key players in Alzheimer’s Disease, being found to be associated to pathologic protein deposits in diseased brains. Therefore, understanding how metals influence amyloid aggregation is critical in establishing molecular mechanisms that underlie disease onset and progression. Here, we report data on the interaction of full-length human Tau protein with calcium and zinc ions, evidencing that Tau self-assembly is differently regulated, depending on the type of bound metal ion. We established that Tau binds 4 Zn2+ and 1 Ca2+ per monomer while using native mass spectrometry analysis, without inducing order or substantial conformational changes in the intrinsically disordered Tau, as determined by structural analysis using circular dichroism and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopies. However, Tau aggregation is found to proceed differently in the calcium- and -zinc bound forms. While the rate of aggregation, as determined from thioflavin-T (ThT) fluorescence kinetics, is highly increased in both cases, the reaction proceeds via different mechanisms, as evidenced by the absence of the lag phase in the reaction of zinc-bound Tau. Monitoring Tau aggregation using native mass spectrometry indeed evidenced a distinct distribution of Tau conformers along the reaction, as confirmed by dynamic light scattering analysis. We propose that such differences arise from zinc binding at distinct locations within the Tau sequence that prompt both the rapid formation of seeding oligomers through interactions at high affinity sites within the repeat domains, as well as amorphous aggregation, through low affinity interactions with residues elsewhere in the sequence, including at the fuzzy coat domain.

Quantifying the inhibitory effect of Bcl-xl on the action of Mff using live-cell fluorescence imaging.

Mitochondrial fission regulates mitochondrial function and morphology, and has been linked to apoptosis. The mitochondrial fission factor (Mff), a tail‐anchored membrane protein, induces excessive mitochondrial fission, contributing to mitochondrial dysfunction and apoptosis. Here, we evaluated the inhibitory effect of Bcl‐xl, an antiapoptotic protein, on the action of Mff by using live‐cell fluorescence imaging. Microscopic imaging analysis showed that overexpression of Mff induced mitochondrial fragmentation and apoptosis, which were reversed by coexpression of Bcl‐xl. Microscopic imaging and live‐cell fluorescence resonance energy transfer analysis demonstrated that Bcl‐xl reconstructs the Mff network from punctate distribution of higher‐order oligomers to filamentous distribution of lower‐order oligomers. Live‐cell fluorescence resonance energy transfer two‐hybrid assay showed that Bcl‐xl interacted with Mff to form heterogenous oligomers with 1 : 2 stoichiometry in cytoplasm and 1 : 1 stoichiometry on mitochondria, indicating that two Bcl‐xl molecules primarily interact with four Mff molecules in cytoplasm, but with two Mff molecules on mitochondria.

Understanding the production of 5-hydroxymethylfurfural (HMF) from chitosan using solid acids

Herein, we demonstrate that 5-hydroxymethylfurfural (HMF) can be chemically derived from commercial chitosan (CScom) using solid acids in a reaction carried out using Nafion®50 resin (NR50) in a co-solvent system of methyl isobutyl ketone (MIBK) and DI water (V/V = 2) at 180 °C for 2 h. After reaction, the products can be well resolved by HPLC and GC-FID and identified by GC–MS. HMF yields could reach up to 32.6 ± 4.2 mol %, which is comparable to HMF yields obtained by using homogeneous organic/inorganic acids. We surmise that interactions between the chitosan chains and the perfluorinated backbone of NR50 could interrupt the hydrogen-bonding network within the chitosan to facilitate hydrolysis and further dehydration. In addition, to understand the interactions between the chitosan monomers/oligomers with the surface of NR50 resin, gel permeation chromatography (GPC) is employed to monitor the evolution of oligomer distribution and HMF production after the reaction. The results suggest that chitosan-derived monomers/oligomers might adsorb on NR50, and D-glucosamine might favorably dehydrated for HMF production once hydrolyzed from chitosan.

Amyloid Oligomers of Mutant p53 in Living Cells Result in Oncogenic Gain of Function in Glioblastoma

Tumor-associated p53 mutations endow cells with malignant phenotypes, including chemoresistance to standard treatments. Amyloid-like oligomeric species of mutant p53 have been attributed to transformation of the p53 tumor suppressor into an oncogene. However, the composition and distribution of mutant p53 oligomers linked to a chemoresistance phenotype are unknown. Knowledge of the mechanism involved in the conversion of p53 is also sparse. Here, we report the massive accumulation of a p53 mutant within amyloid-like p53 oligomers in brain cells from glioblastoma tumors leading into chemoresistance gain of function. This finding, along with statistical analysis of data from fluorescence fluctuation spectroscopy, pressure-induced measurements, and thioflavin T kinetics, demonstrates the distribution of oligomers larger than the active tetrameric form of p53 in the nuclei of living cells and the destabilization of native-drifted p53 species that become amyloid. Collectively, these results suggest the role of amyloid-like mutant p53 oligomers as participants in the chemoresistance phenotype of malignant and invasive brain tumors and shed light on new therapeutic options to avert cancer.

Deconstructing Oligomer Distributions: Discrete Species and Artificial Distributions

AbstractThe separation of an oligo(methyl acrylate) distribution, obtained from reversible addition–fragmentation chain transfer (RAFT) polymerization, in a discrete (dispersity=1) oligomeric library (degree of polymerization between 1 and 22) is presented. The properties of this library in terms of diffusivity, glass transition temperature, and viscosity are determined, filling a significant knowledge gap associated with these materials. The obtained oligomer library is used to construct artificial oligomer distributions on demand. These artificial oligomer distributions are used to highlight the potential to tailor physical properties of a material, while concomitantly demonstrating the limitations associated with size‐exclusion chromatography analysis of molecular weight and dispersity in particular.

Deconstructing Oligomer Distributions: Discrete Species and Artificial Distributions.

The separation of an oligo(methyl acrylate) distribution, obtained from reversible addition-fragmentation chain transfer (RAFT) polymerization, in a discrete (dispersity=1) oligomeric library (degree of polymerization between 1 and 22) is presented. The properties of this library in terms of diffusivity, glass transition temperature, and viscosity are determined, filling a significant knowledge gap associated with these materials. The obtained oligomer library is used to construct artificial oligomer distributions on demand. These artificial oligomer distributions are used to highlight the potential to tailor physical properties of a material, while concomitantly demonstrating the limitations associated with size-exclusion chromatography analysis of molecular weight and dispersity in particular.

Insight into the distribution of amino groups along the chain of chemically deacetylated hyaluronan

Deacetylated hyaluronan (daHA) containing reactive free amino groups is an important intermediate for further modification. Comparing direct and indirect NMR and HPLC to characterize the degree of HA deacetylation (DD), direct NMR approach using area ratio of anomeric CH and CH–NH2 groups was the most precise one. To describe the substitution pattern, daHA was selectively cleaved by nitrous acid generated in situ or hyaluronan lyase from Streptococcus pneumoniae. The resulting oligomers were identified by LC-ESI-MS. The experimental distribution of these oligomers was compared with theoretically expected random oligomer distribution. Independently on the starting HA molecular weight and deacetylation conditions, the experimental data differed from the random distribution model and suggested that deacetylation of certain N-acetyl-d-glucosamine had reduced the probability of deacetylation at the neighbouring disaccharide. This phenomenon was explained by conformational changes of HA caused by intra- and intermolecular interactions between positively charged amino and negatively charged carboxylic groups.

FISIK: Framework for the Inference of In Situ Interaction Kinetics from Single-Molecule Imaging Data

Recent experimental and computational developments have been pushing the limits of live-cell single-molecule imaging, enabling the monitoring of intermolecular interactions in their native environment with high spatiotemporal resolution. However, interactions are captured only for the labeled subset of molecules, which tends to be a small fraction. As a result, it has remained a challenge to calculate molecular interaction kinetics, in particular association rates, from live-cell single-molecule tracking data. To overcome this challenge, we developed a mathematical modeling-based Framework for the Inference of in Situ Interaction Kinetics (FISIK) from single-molecule imaging data with substoichiometric labeling. FISIK consists of (I) devising a mathematical model of molecular movement and interactions, mimicking the biological system and data-acquisition setup, and (II) estimating the unknown model parameters, including molecular association and dissociation rates, by fitting the model to experimental single-molecule data. Due to the stochastic nature of the model and data, we adapted the method of indirect inference for model calibration. We validated FISIK using a series of tests in which we simulated trajectories of diffusing molecules that interact with each other, considering a wide range of model parameters, and including resolution limitations, tracking errors, and mismatches between the model and the biological system it mimics. We found that FISIK has the sensitivity to determine association and dissociation rates, with accuracy and precision depending on the labeled fraction of molecules and the extent of molecule tracking errors. For cases where the labeled fraction is too low (e.g., to afford accurate tracking), combining dynamic but sparse single-molecule imaging data with almost-whole population oligomer distribution data improves FISIK’s performance. All in all, FISIK is a promising approach for the derivation of molecular interaction kinetics in their native environment from single-molecule imaging data with substoichiometric labeling.

Self‐Standing Membranes Consisting of Inherently Chiral Electroactive Oligomers: Electrosynthesis, Characterization and Preliminary Tests in Potentiometric Setups

AbstractSelf‐standing chiral electroactive synthetic membranes are presented, prepared by oxidative electro‐oligomerization of a thiophene‐based “inherently chiral” electroactive monomer on indium tin oxide (ITO) or fluorine‐doped tin oxide (FTO) electrodes, followed by detachment of the electrodeposited thin films in aqueous solution. The membranes, possibly mesoporous, consist of a mixture of open and cyclic “inherently chiral” oligomers, that is, in which both chirality and electroactivity originate from the same source, and this is the main conjugated backbone featuring a tailored torsion. Such a combination can afford outstanding chirality manifestations. The electrosynthesis conditions significantly modulate the oligomer distribution. Racemate films are compared to enantiopure ones. Circular dichroism confirms that (R)‐ or (S)‐ enantiopure films are obtained, starting from the corresponding (R)‐ or (S)‐ enantiopure monomers. Reliable transmembrane potential readings are obtained in preliminary tests in ion‐selective electrode (ISE)‐like setups, consistent with those predicted considering the membrane features, offering a first step towards extension of the protocol to chiral experiments.

Cadherin Extracellular Domain Clustering in the Absence of Trans-Interactions.

While both cis and trans (adhesive)-interactions cooperate in the assembly of intercellular adhesions, computational simulations have predicted that two-dimensional confinement may promote cis-oligomerization, in the absence of trans-interactions. Here, single-molecule tracking of cadherin extracellular domains on supported lipid bilayers revealed the density-dependent formation of oligomers and cis-clusters in the absence of trans-interactions. Lateral oligomers were virtually eliminated by mutating a putative cis (lateral) binding interface. At low cadherin surface coverage, wild-type and mutant cadherin diffused rapidly, consistent with the motion of a lipid molecule within a cadherin-free supported bilayer and with cadherins diffusing as monomers. Although the diffusion of mutant cadherin did not change appreciably with increasing surface coverage, the average short-time diffusion coefficient of wild-type cadherin slowed significantly above a fractional surface coverage of ∼0.01 (∼1100 molecules/μm2). A detailed analysis of molecular trajectories suggested the presence of a broad size distribution of cis-cadherin oligomers. These findings verify predictions that two-dimensional confinement promotes cis-oligomerization, in the absence of trans-interactions.

Kinetics of Radical Chain Polymerization: 1. Time-Dependent Distributions of Macroradicals and Oligomers

Numerical and analytical solutions of kinetic equations and the relevant equations for the moments of distribution of macroradicals and oligomers during radical chain polymerization are presented. It has been shown that the initial chain growth occurs in the ballistic mode involving first-generation primary radicals. The chain transfer in which a macroradical, reacting with a solvent, forms an oligomer and the primary radical of the next generation, causes a transition from the ballistic to the diffusion mode in which distributions of chains of different generations are mixed. Time-dependent distributions have been found for chain propagation and transfer rate constants independent of chain length.

(Trans)acetalization Reactions for the Synthesis of Oligomeric Oxymethylene Dialkyl Ethers Catalyzed by Zeolite BEA25

Oligomeric oxymethylene dimethyl ethers (DMEs; OMDMEn) are acetals of the type CH3O(CH2O)nCH3 with a high oxygen content and without carbon–carbon bonds in the molecular structure. Therefore, formation of soot and NOx emissions is largely suppressed during combustion. Oligomers with n = 3–5 exhibit properties similar to conventional diesel fuel and this stimulated extensive research in this field. Reactions of OMDME1, the corresponding ethyl derivative OMDEE1 and dimethyl ether (DME) with trioxane have been carried out employing zeolite BEA25 as acidic catalyst. Thus, oligomer mixtures with n = 1–5 are obtained and very small amounts of higher oligomers are also formed. OMDMEn- or OMDEEn-forming reactions from the starting materials take place as well as subsequent transacetalization reactions which lead usually to a product distribution according to a Schulz–Flory distribution. Several transacetalization reactions have been studied, e.g. reactions of OMDME1 with OMDME3 and OMDEE1 with OMDEE3, which lead to the typical mixtures with n = 1–5. Reactions of OMDME1 with OMDEE1 yielded ethoxymethoxymethane, i.e. the acetal with mixed end groups. This allows for the tuning of oligomer mixtures and adjustment of properties according to the required demands of the respective application. Regarding DME as starting compound, the transacetalization reaction with OMDME3 yielded only small amounts of OMDME1–5 while the reaction of DME with trioxane yielded considerable amounts of the oligomers. The reaction is comparatively slow enabling a kinetic control of the oligomer distribution which exhibits an elevated selectivity for the desired OMDME3–5 fraction in the beginning of the reaction.

Brisk Method for Fractionating and Separating Bio-Crude Oil

A new approach for the analysis of bio-crude oils (BCOs) has been qualitatively explored. The analytical scheme is based on the fractionation of BCO through precipitation in water, freeze-drying, solid phase extraction (SPE) and combinations of analytical techniques for the analysis of fractions. Monomeric components in BCO were characterised using gas chromatography coupled with mass spectrometer and flame ionisation detector (GC-MS/FID). The molecular mass distribution of oligomers was determined using gel permeation chromatography (GPC). The fractionation procedure appeared to have succeeded to a large extent as evident in the detection of various components along their expected fraction in the GC-MS/FID analysis. However, a quantitative analysis of the multiple components in different fractions appeared difficult owing to the loss of many volatile fractions through the fractionation procedure. And less than 7 wt. % of the sugar-related components in the acetylated polar SPE fraction were identified.

Wide distribution of alpha-synuclein oligomers in multiple system atrophy brain detected by proximity ligation (P3.8-010)

Wide distribution of alpha-synuclein oligomers in multiple system atrophy brain detected by proximity ligation (P3.8-010)