scholarly journals Unbiased high-content screening reveals Aβ- and tau-independent synaptotoxic activities in human brain homogenates from Alzheimer’s patients and high-pathology controls

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259335
Author(s):  
Hao Jiang ◽  
Thomas J. Esparza ◽  
Terrance T. Kummer ◽  
David L. Brody
Keyword(s):  
Human Brain ◽  
Brain Regions ◽  
Size Exclusion ◽  
Screening Assay ◽  
Synaptic Loss ◽  
Synapse Loss ◽  
Brain Extracts ◽  
Exclusion Chromatography ◽  
Cultured Neuron

Alzheimer’s disease (AD) is tightly correlated with synapse loss in vulnerable brain regions. It is assumed that specific molecular entities such as Aβ and tau cause synapse loss in AD, yet unbiased screens for synaptotoxic activities have not been performed. Here, we performed size exclusion chromatography on soluble human brain homogenates from AD cases, high pathology non-demented controls, and low pathology age-matched controls using our novel high content primary cultured neuron-based screening assay. Both presynaptic and postsynaptic toxicities were elevated in homogenates from AD cases and high pathology non-demented controls to a similar extent, with more modest synaptotoxic activities in homogenates from low pathology normal controls. Surprisingly, synaptotoxic activities were found in size fractions peaking between the 17–44 kDa size standards that did not match well with Aβ and tau immunoreactive species in these homogenates. The fractions containing previously identified high molecular weight soluble amyloid beta aggregates/”oligomers” were non-toxic in this assay. Furthermore, immunodepletion of Aβ and tau did not reduce synaptotoxic activity. This result contrasts with previous findings involving the same methods applied to 3xTg-AD mouse brain extracts. The nature of the synaptotoxic species has not been identified. Overall, our data indicates one or more potential Aβ and tau independent synaptotoxic activities in human AD brain homogenates. This result aligns well with the key role of synaptic loss in the early cognitive decline and may provide new insight into AD pathophysiology.

scholarly journals Metagenomic Profiling of Fecal-Derived Bacterial Membrane Vesicles in Crohn’s Disease Patients

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2795
Author(s):  
Nader Kameli ◽  
Heike E. F. Becker ◽  
Tessa Welbers ◽  
Daisy M. A. E. Jonkers ◽  
John Penders ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Membrane Vesicles ◽  
Amplicon Sequencing ◽  
Size Exclusion ◽  
Bacterial Membrane ◽  
Microbial Composition ◽  
Fecal Samples ◽  
Exclusion Chromatography

Background: In the past, many studies suggested a crucial role for dysbiosis of the gut microbiota in the etiology of Crohn’s disease (CD). However, despite being important players in host–bacteria interaction, the role of bacterial membrane vesicles (MV) has been largely overlooked in the pathogenesis of CD. In this study, we addressed the composition of the bacterial and MV composition in fecal samples of CD patients and compared this to the composition in healthy individuals. Methods: Fecal samples from six healthy subjects (HC) in addition to twelve CD patients (six active, six remission) were analyzed in this study. Fecal bacterial membrane vesicles (fMVs) were isolated by a combination of ultrafiltration and size exclusion chromatography. DNA was obtained from the fMV fraction, the pellet of dissolved feces as bacterial DNA (bDNA), or directly from feces as fecal DNA (fDNA). The fMVs were characterized by nanoparticle tracking analysis and cryo-electron microscopy. Amplicon sequencing of 16s rRNA V4 hypervariable gene regions was conducted to assess microbial composition of all fractions. Results: Beta-diversity analysis showed that the microbial community structure of the fMVs was significantly different from the microbial profiles of the fDNA and bDNA. However, no differences were observed in microbial composition between fDNA and bDNA. The microbial richness of fMVs was significantly decreased in CD patients compared to HC, and even lower in active patients. Profiling of fDNA and bDNA demonstrated that Firmicutes was the most dominant phylum in these fractions, while in fMVs Bacteroidetes was dominant. In fMV, several families and genera belonging to Firmicutes and Proteobacteria were significantly altered in CD patients when compared to HC. Conclusion: The microbial alterations of MVs in CD patients particularly in Firmicutes and Proteobacteria suggest a possible role of MVs in host-microbe symbiosis and induction or progression of inflammation in CD pathogenesis. Yet, the exact role for these fMV in the pathogenesis of the disease needs to be elucidated in future studies.

scholarly journals Pharmacoepigenetics of the role of DNA methylation in μ-opioid receptor expression in different human brain regions

Epigenomics ◽  
2016 ◽  
Vol 8 (12) ◽  
pp. 1583-1599 ◽  
Author(s):  
Claudia Knothe ◽  
Bruno G Oertel ◽  
Alfred Ultsch ◽  
Mattias Kettner ◽  
Peter Harald Schmidt ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Human Brain ◽  
Opioid Receptor ◽  
Brain Regions ◽  
Receptor Expression ◽  
Μ Opioid Receptor

scholarly journals The Two ADF-H Domains of Twinfilin Play Functionally Distinct Roles in Interactions with Actin Monomers

2002 ◽  
Vol 13 (11) ◽  
pp. 3811-3821 ◽  
Author(s):  
Pauli J. Ojala ◽  
Ville O. Paavilainen ◽  
Maria K. Vartiainen ◽  
Roman Tuma ◽  
Alan G. Weeds ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Activity ◽  
Size Exclusion ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Monomer ◽  
Wild Type ◽  
Native Page ◽  
Exclusion Chromatography

Twinfilin is a ubiquitous and abundant actin monomer–binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K D = 0.05 μM) than ATP-G-actin (K D = 0.47 μM) under physiological ionic conditions and forms a relatively stable (k off = 1.8 s−1) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.

Molecular Weight and Tacticity of Oligoacrylates by Capillary Electrophoresis - Mass Spectrometry

2010 ◽  
Vol 63 (8) ◽  
pp. 1219 ◽  
Author(s):  
Marianne Gaborieau ◽  
Tim J. Causon ◽  
Yohann Guillaneuf ◽  
Emily F. Hilder ◽  
Patrice Castignolles
Keyword(s):  
Capillary Electrophoresis ◽  
Raft Polymerization ◽  
Degree Of Polymerization ◽  
Size Exclusion ◽  
Separation Mechanism ◽  
Ionization Mass ◽  
Polymeric Particles ◽  
Reversible Addition ◽  
Exclusion Chromatography

Oligo(acrylic acid) efficiently stabilizes polymeric particles, especially particles produced by reversible addition–fragmentation chain transfer (RAFT) (as hydrophilic block of an amphiphilic copolymer). Capillary electrophoresis (CE) has a far higher resolution power to separate these oligomers than the commonly used size exclusion chromatography. Coupling CE to electrospray ionization mass spectrometric detection unravels the separation mechanism. CE separates these oligomers, not only according to their degree of polymerization, but also according to their tacticity, in agreement with NMR analysis. Such analysis will provide insight into the role of these oligomers as stabilizers in emulsion polymerization, and into the mechanism of the RAFT polymerization with respect to degree of polymerization and tacticity.

Oligomeric assembly and interactions within the human RuvB-like RuvBL1 and RuvBL2 complexes

2010 ◽  
Vol 429 (1) ◽  
pp. 113-125 ◽  
Author(s):  
Andrew Niewiarowski ◽  
Alison S. Bradley ◽  
Jayesh Gor ◽  
Adam R. McKay ◽  
Stephen J. Perkins ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Structural Model ◽  
Analytical Ultracentrifugation ◽  
Protein Complexes ◽  
Adenine Nucleotides ◽  
Size Exclusion ◽  
Molecular Architecture ◽  
Essential Components ◽  
Exclusion Chromatography

The two closely related eukaryotic AAA+ proteins (ATPases associated with various cellular activities), RuvBL1 (RuvB-like 1) and RuvBL2, are essential components of large multi-protein complexes involved in diverse cellular processes. Although the molecular mechanisms of RuvBL1 and RuvBL2 function remain unknown, oligomerization is likely to be important for their function together or individually, and different oligomeric forms might underpin different functions. Several experimental approaches were used to investigate the molecular architecture of the RuvBL1–RuvBL2 complex and the role of the ATPase-insert domain (domain II) for its assembly and stability. Analytical ultracentrifugation showed that RuvBL1 and RuvBL2 were mainly monomeric and each monomer co-existed with small proportions of dimers, trimers and hexamers. Adenine nucleotides induced hexamerization of RuvBL2, but not RuvBL1. In contrast, the RuvBL1–RuvBL2 complexes contained single- and double-hexamers together with smaller forms. The role of domain II in complex assembly was examined by size-exclusion chromatography using deletion mutants of RuvBL1 and RuvBL2. Significantly, catalytically competent dodecameric RuvBL1–RuvBL2, complexes lacking domain II in one or both proteins could be assembled but the loss of domain II in RuvBL1 destabilized the dodecamer. The composition of the RuvBL1–RuvBL2 complex was analysed by MS. Several species of mixed RuvBL1/2 hexamers with different stoichiometries were seen in the spectra of the RuvBL1–RuvBL2 complex. A number of our results indicate that the architecture of the human RuvBL1–RuvBL2 complex does not fit the recent structural model of the yeast Rvb1–Rvb2 complex.

Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2455-2462 ◽  
Author(s):  
Masaru Nagai ◽  
Maki Kawata ◽  
Hisayuki Watanabe ◽  
Machiko Ogawa ◽  
Kumiko Saito ◽  
...  
Keyword(s):  
Lentinula Edodes ◽  
Veratryl Alcohol ◽  
Size Exclusion ◽  
Melanin Synthesis ◽  
Sds Page ◽  
Diammonium Salt ◽  
Exclusion Chromatography ◽  
Homogeneous Preparation

A laccase (EC 1.10.3.2) was isolated from the fully browned gills of Lentinula edodes fruit bodies. The enzyme was purified to a homogeneous preparation using hydrophobic, cation-exchange and size-exclusion chromatography. SDS-PAGE analysis showed the purified laccase, Lcc 2, to be a monomeric protein of 58·0 kDa. The enzyme had an isoelectric point of around pH 6·9. The optimum pH for enzyme activity was around 3·0 against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), and it was most active at 40 °C and stable up to 50 °C. The enzyme contained 8·6 % carbohydrate and some copper atoms. The enzyme oxidized ABTS, p-phenylenediamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol and ferulic acid, but not veratryl alcohol and tyrosine. β-(3,4-Dihydroxyphenyl)alanine (l-DOPA), which was not oxidized by a laccase previously reported from the culture filtrate of L. edodes, was also oxidized by Lcc 2, and the oxidative product of l-dopa was identified as l-DOPA quinone by HPLC analysis. Lcc 2 was able to oxidize phenolic compounds extracted from fresh gills to brown-coloured products, suggesting a role for laccase in melanin synthesis in this strain.

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

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Alexander Moysa ◽  
Dietmar Hammerschmid ◽  
Roman H. Szczepanowski ◽  
Frank Sobott ◽  
Michal Dadlez
Keyword(s):  
Analytical Ultracentrifugation ◽  
Vascular Diseases ◽  
Native Mass Spectrometry ◽  
Full Length ◽  
Size Exclusion ◽  
Glycation End Products ◽  
Exclusion Chromatography ◽  
Hydrogen Deuterium

AbstractThe 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.

scholarly journals Photopatterning of PDMS Films: Challenging the Reaction between Benzophenone and Silicone Functional Groups

Materials ◽  
2021 ◽  
Vol 14 (8) ◽  
pp. 2027
Author(s):  
Arthur Stricher ◽  
Renaud G. Rinaldi ◽  
Laurent Chazeau ◽  
François Ganachaud
Keyword(s):  
Mechanical Properties ◽  
Nuclear Magnetic Resonance ◽  
Total Reflection ◽  
Attenuated Total Reflection ◽  
Size Exclusion ◽  
Reaction Pathways ◽  
Aromatic Solvent ◽  
Exclusion Chromatography ◽  
Simplified Procedure

Direct photopatterning of PDMS (Polydimethylsiloxane) through benzophenone photo-inhibition has received great interest in recent years. Indeed, the simplicity and versatility of this technique allows for easy processing of micro-canals, or local control of PDMS mechanical properties. Surprisingly, however, the chemical reactions between silicone hydride and/or silicone vinyl groups and benzophenone have only been assessed through qualitative methods (e.g., Attenuated total reflection fourier transform infrared). In this communication, the previously proposed reaction pathways are challenged, using nuclear magnetic resonance (NMR) spectroscopy and size exclusion chromatography (SEC) monitoring. A different mechanism depicting the role of benzophenone irradiation on the polyaddition reaction of silicone formulations is proposed, and a simplified procedure involving aromatic solvent is finally disclosed.

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