Assessment of whole-body MRI including diffusion-weighted sequences in the initial staging of breast cancer patients at high risk of metastases in comparison with PET-CT: a prospective cohort study.

OBJECTIVE: The aim of this study was to assess the diffusion-weighted whole-body-MRI (WBMRI) in the initial staging of breast cancer at high risk of metastases in comparison with positron emission tomography (PET)-CT. METHODS: Forty-five women were prospectively enrolled. The inclusion criteria were female gender, age >18, invasive breast cancer, an initial PET-CT, and a performance status of 0-2. The exclusion criteria were contraindication to WB-MRI and breast cancer recurrence. The primary outcome was the concordance of WB-MRI and PET-CT in the diagnosis of distant metastases, whereas secondary outcomes included their concordance for the primary tumor and regional lymph nodes (LN), as well as the agreement of WB-MRI interpretation between two radiologists. RESULTS: The mean age was 51.2 years with a median size of the primary tumor of 30 mm. Concordance between the two modalities was almost perfect for metastases staging, all sites included (k = 0.862), with excellent interobserver agreement. The accuracy of WB-MRI for detecting regional LN, distant LN, lung, liver, or bone metastases ranged from 91 to 96%. In 2 patients, WB-MRI detected bone metastases that were overlooked by PET-CT. WB-MRI showed a substantial agreement with PET-CT for staging the primary tumor, regional LN status, and stage (k = 0.766, k = 0.756, and k = 0.785, respectively) with a high interobserver agreement. CONCLUSION: WB-MRI including DWI could be a reliable and reproducible examination in the initial staging of breast cancer patients at high risk of metastases, especially for bone metastases and therefore could be used as a surrogate to PET-CT. CLINICAL RELEVANCE STATEMENT: Whole-body-MRI including DWI is a promising technique for detecting metastases in the initial staging of breast cancer at high risk of metastases. KEY POINTS: Whole-body-MRI (WB-MRI) was effective for detecting metastases in the initial staging of 45 breast cancer patients at high risk of metastases in comparison with PET-CT. Concordance between WB-MRI and PET-CT was almost perfect for metastases staging, all sites included, with excellent interobserver agreement. The accuracy of WB-MRI for detecting bone metastases was 92%.

Learning curve for fetal postmortem ultrasound.

OBJECTIVE: To determine the learning curve of fetal postmortem ultrasound (PMUS) and evaluate the evolution of its diagnostic performance over the past 8 years. METHODS: PMUS was performed by two fetal medicine specialists and two experts on 100 unselected fetuses of 12-38 weeks of gestation in a prospective, double-blind manner. 21 pre-defined internal structures were analyzed consecutively by the trainee alone and the expert, with a comparison of diagnosis and immediate feedback. The learning curves for examination duration, non-recognition of structures and final diagnoses were computed using cumulative summation analysis. Secondly, the expert PMUS diagnostic accuracy using autopsy as the gold standard was compared to the previously published data. RESULTS: The trainees reached expert level of PMUS at 28-36 cases for examination duration (12.1 ± 5.2 min), non-diagnostic rate (6.5%, 137/2100), and abnormality diagnosis. In a group of 33 fetuses ≥20 weeks who had an autopsy, the experts PMUS performance was improved after 8 years with a reduction of all organs non-diagnostic rate (6.5 %VS 11.4%, p < 0.01) and higher sensitivity for the heart (100% VS 40.9%, p < 0.01) and the abdomen (100%VS 56.5%, p < 0.05). CONCLUSION: PMUS offers a short learning curve for fetal medicine specialists and on-going improvement of diagnostic accuracy over time.

Postmortem imaging of fetuses at early gestations: A comparison of microfocus computed tomography with postmortem magnetic resonance at 9.4 T and postmortem ultrasound.

OBJECTIVE: To compare the diagnostic performance of postmortem ultrasound (PMUS), 9.4 T magnetic resonance imaging (MRI) and microfocus computed tomography (micro-CT) for the examination of early gestation fetuses. METHOD: Eight unselected fetuses (10-15 weeks gestational age) underwent at least 2 of the 3 listed imaging examinations. Six fetuses underwent 9.4 T MRI, four underwent micro-CT and six underwent PMUS. All operators were blinded to clinical history. All imaging was reported according to a prespecified template assessing 36 anatomical structures, later grouped into five regions: brain, thorax, heart, abdomen and genito-urinary. RESULTS: More anatomical structures were seen on 9.4 T MRI and micro-CT than with PMUS, with a combined frequency of identified structures of 91.9% and 69.7% versus 54.5% and 59.6 (p < 0.001; p < 0.05) respectively according to comparison groups. In comparison with 9.4 T MRI, more structures were seen on micro-CT (90.2% vs. 83.3%, p < 0.05). Anatomical structures were described as abnormal on PMUS in 2.7%, 9.4 T MRI in 6.1% and micro-CT 7.7% of all structures observed. However, the accuracy test could not be calculated because conventional autopsy was performed on 6 fetuses of that only one structure was abnormal. CONCLUSION: Micro-CT appears to offer the greatest potential as an imaging adjunct or non-invasive alternative for conventional autopsies in early gestation fetuses.

TrkA mediates effect of novel KIDINS220 mutation in human brain ventriculomegaly.

Congenital hydrocephalus is a potentially devastating, highly heterogeneous condition whose genetic subset remains incompletely known. We here report a consanguineous family where three fetuses presented with brain ventriculomegaly and limb contractures and shared a very rare homozygous variant of KIDINS220, consisting of an in-frame deletion of three amino acids adjacent to the fourth transmembrane domain. Fetal brain imaging and autopsy showed major ventriculomegaly, reduced brain mass, and with no histomorphologic abnormalities. We demonstrate that the binding of KIDINS220 to TrkA is diminished by the deletion mutation. This family is the second that associates a KIDINS220 genetic variant with human ventriculomegaly and limb contractures, validating causality of the gene and indicating TrkA as a likely mediator of the phenotype.

Interactions of REF1 and SRPP1 rubber particle proteins from Hevea brasiliensis with synthetic phospholipids: Effect of charge and size of lipid headgroup.

According to the fatty acid and headgroup compositions of the phospholipids (PL) from Hevea brasiliensis latex, three synthetic PL were selected (i.e. POPA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and POPG: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) to investigate the effect of PL headgroup on the interactions with two major proteins of Hevea latex, i.e. Rubber Elongation Factor (REF1) and Small Rubber Particle Protein (SRPP1). Protein/lipid interactions were screened using two models (lipid vesicles in solution or lipid monolayers at air/liquid interface). Calcein leakage, surface pressure, ellipsometry, microscopy and spectroscopy revealed that both REF1 and SRPP1 displayed stronger interactions with anionic POPA and POPG, as compared to zwitterionic POPC. A particular behavior of REF1 was observed when interacting with POPA monolayers (i.e. aggregation + modification of secondary structure from α-helices to ß-sheets, characteristic of its amyloid aggregated form), which might be involved in the irreversible coagulation mechanism of Hevea rubber particles.

Transport of hydrocortisone in targeted layers of the skin by multi-lamellar liposomes.

Hydrocortisone (HyC), a hydrophobic pharmaceutical active, was encapsulated in multi-lamellar liposomes (MLLs) composed of P100, a mixture of phospholipids, and Tween®80. Three different HyC-loaded formulations were designed to target the stratum corneum, the living epidermis and the hypodermis. The impact of encapsulation on their size, elasticity and zeta potential, the three key factors controlling MLLs skin penetration, was studied. Raman mapping of phospholipids and HyC allowed the localisation of both components inside an artificial skin, Strat-M®, demonstrating the efficiency of the targeting. Percutaneous permeation profiles through excised human skin were performed over 48 h, supporting results on artificial skin. Their modelling revealed that HyC encapsulated in MLLs, designed to target the stratum corneum and living epidermis, exhibited a non-Fickian diffusion process. In contrast, a Fickian diffusion was found for HyC administered in solution, in a pharmaceutical cream and in transdermal MLLs. These results allowed us to propose a mechanism of interaction between HyC-containing MLLs and the skin.

Chemical Imaging of RNA-Tau Amyloid Fibrils at the Nanoscale Using Tip-Enhanced Raman Spectroscopy.

In the presence of cofactors, tau protein can form amyloid deposits in the brain which are implicated in many neurodegenerative disorders. Heparin, lipids, and RNA are used to recreate tau aggregates in vitro from recombinant protein. However, the mechanism of interaction of these cofactors and the interactions between cofactors and tau are poorly understood. Herein, we use tip-enhanced Raman spectroscopy (TERS) to visualize the spatial distribution of adenine, protein secondary structure, and amino acids (arginine, lysine and histidine) in single polyadenosine (polyA)-induced tau fibrils with nanoscale spatial resolution (<10-20 nm). Based on reference unenhanced and surface-enhanced Raman spectra, we show that the polyA anionic cofactor is incorporated in the fibril structure and seems to be superficial to the ß-sheet core, but nonetheless enveloped within the random-coiled fuzzy coat. TERS images also prove the colocalization of positively charged arginine, lysine, and histidine amino acids and negatively charged polyA, which constitutes an important step forward to better comprehend the action of RNA cofactors in the mechanism of formation of toxic tau fibrils. TERS appears as a powerful technique for the identification of cofactors in individual tau fibrils and their mode of interaction.

Value of diffusion-weighted MRI in predicting early response to neoadjuvant chemotherapy of breast cancer: comparison between ROI-ADC and whole-lesion-ADC measurements.

OBJECTIVE: The aim of the study was to assess DWI with ROI-ADC and WL-ADC measurements in early response after NAC in breast cancer. METHODS: Between January 2016 and December 2019, 55 women were enrolled in this prospective single-center study. MRI was performed at three time points for each patient: before treatment (MRI 1: DW and DCE MRI), after one cycle of NAC (MRI 2: noncontrast DW MRI), and after completion of NAC before surgery (MRI 3: DW and DCE MRI). ROI-ADC and WL-ADC measurements were obtained on MRI and were compared to histology findings and to the RCB class. Patients were categorized as having pCR or non-pCR. RESULTS: Among 48 patients, 9 experienced pCR. An increase of ROI-ADC between MRI 1 and 2 of more than 47.5% had a sensitivity of 88.9% and a specificity of 63.4% in predicting pCR, whereas WL-ADC did not predict pCR. An increase of ROI-ADC between MRI 1 and 2 of more than 47.5% had a sensitivity of 83.3% and a specificity of 64.9% in predicting radiologic complete response. An increase of WL-ADC between MRI 1 and 2 of more than 25.5% had a sensitivity of 83.3% and a specificity of 75.5% in predicting radiologic complete response. CONCLUSION: After one cycle of NAC, a significant increase in breast tumor ROI-ADC at DWI predicted complete pathologic and radiologic responses. KEY POINTS: • An increase of WL-ADC between MRI 1 and 2 of more than 25.5% had a sensitivity of 83.3% and a specificity of 75.5% in predicting radiologic complete response. • An increase of ROI-ADC between MRI 1 and 2 of more than 47.5% had a sensitivity of 88.9% and a specificity of 63.4% in predicting pCR, and a sensitivity of 83.3% and a specificity of 64.9% in predicting radiologic complete response. • A significant increase in breast tumor ROI-ADC at DWI predicted complete pathologic and radiologic responses.

PiP2 favors an α-helical structure of non-recombinant Hsp12 of Saccharomyces cerevisiae.

Hsp12 is a small heat shock protein of Saccharomyces cerevisiae upregulated in response to various stresses. Non recombinant Hsp12 has been purified and characterized. Using circular dichroism (CD), Isothermal Titration Calorimetry (ITC) and Differential Scanning Calorimetry (DSC), it has been demonstrated that the native Hsp12 is monomeric and intrinsically disordered (IDP). Hsp12 gains in structure in the presence of specific lipids (PiP2). The helical form binds to liposomes models membrane with high affinity, leading to their rigidification. These results suggest that hydrophobic and ionic interactions are involved. Hsp12 is most likely a membrane chaperone expressed during stresses in Saccharomyces cerevisiae.

Lipid selectivity in detergent extraction from bilayers.

Despite numerous studies on detergent-induced solubilization of membranes and on the underlying mechanisms associated with this process, very little is known regarding the selectivity of detergents for lipids during their extraction from membranes. To get insights about this phenomenon, solubilization of model bilayers prepared from binary lipid mixtures by different detergents was examined. Three commonly used detergents were used: the non-ionic Triton X-100 (TX), the negatively-charged sodium dodecylsulfate (SDS), and the positively-charged n-dodecyltrimethylammonium chloride (DTAC). Two model membranes were used in order to identify if specific intermolecular interactions can lead to lipid selectivity: bilayers made of a binary mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and of a binary mixture of POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG). Therefore, it was possible to describe systems presenting a combination of detergents bearing different charges with bilayers with different polymorphic propensities and charge. In conditions for which partial solubilization was observed, the composition of the extracted lipid phase was quantified with Liquid Chromatography coupled to Mass Spectrometry to elucidate whether a lipid selectivity occurred in the solubilization process. On one hand, it is found that repulsive or attractive electrostatic interactions did not lead to any lipid selectivity. On the other hand, POPE was systematically less extracted than POPC, regardless of the detergent nature. We propose that this lipid selectivity is inherent to the molecular shape of POPE unsuited for micelles curvature properties.

Study of G-Protein Coupled Receptor Signaling in Membrane Environment by Plasmon Waveguide Resonance.

Here we describe an experimental technique, termed plasmon waveguide resonance (PWR) spectroscopy that enables the characterization of molecular interactions occurring at the level of anisotropic thin films as lipid membranes and therein inserted or interacting molecules. PWR allows one to characterize such molecular interactions at different levels: (1) acquire binding curves and calculate dissociation constants; (2) obtain kinetic information; (3) obtain information about associated anisotropy changes and changes in membrane thickness; (4) obtain insight about lateral homogeneity (formation of domains). Points 1, 2, and 4 can be directly obtained from the data. Point 3 requires spectral fitting procedures so that the different optical parameters characterizing thin films as proteolipid membranes, namely refractive index and extinction coefficient for both p- (TM component of light that is parallel to the incident light) and s- (TE component of light that is perpendicular to the incident light) polarizations and thickness, can be determined. When applied to membrane proteins as the G-protein coupled receptor (GPCR) family, both ligand-induced conformational changes of the receptor can be followed as well as interactions with effectors (e.g., G-proteins). Additionally, by either altering the lipid composition in cellular membranes or specifically controlling its composition in the case of lipid model membranes with reconstituted proteins, the role of the lipid environment in receptor activation and signaling can be determined. Additionally, the eventual partition of receptors in different lipid microdomains (e.g., lipid rafts) can be followed. Such information can be obtained  ex cellulo with mammalian cell membrane fragments expressing the protein of interest and/or in vitro with lipid model systems where the protein under investigation has been reconstituted. Moreover, PWR can also be applied to directly follow the reconstitution of membrane proteins in lipid model membranes. The measurements are performed directly (no labeling of molecular partners), in real time and with very high sensitivity. Here we will discuss different aspects of GPCR activation and signaling where PWR brought important information in parallel with other approaches. The utility of PWR is not limited to GPCRs but can be applied to any membrane protein. PWR is also an excellent tool to characterize the interaction of membrane active molecules (as cell penetrating, antimicrobial, viral and amyloid peptides) with lipids. A brief section is dedicated to such applications, with particular emphasis on amyloid peptides. To finalize, as PWR is a homemade technology, ongoing instrument developments aiming at breaking current experimental limitations are briefly discussed, namely, the coupling of PWR with electrochemical measurements and the expansion of measurements from the visible to the infrared region.

Molecular architecture of bacterial amyloids in Bacillus biofilms.

The formation of biofilms provides structural and adaptive bacterial response to the environment. In Bacillus species, the biofilm extracellular matrix is composed of exopolysaccharides, hydrophobins, and several functional amyloid proteins. We report, using multiscale approaches such as solid-state NMR (SSNMR), electron microscopy, X-ray diffraction, dynamic light scattering, attenuated total reflection Fourier transform infrared (FTIR), and immune-gold labeling, the molecular architecture of B. subtilis and pathogenic B. cereus functional amyloids. SSNMR data reveal that the major amyloid component TasA in its fibrillar amyloid form contain ß-sheet and α-helical secondary structure, suggesting a nontypical amyloid architecture in B. subtilis. Proteinase K digestion experiments indicate the amyloid moiety is ∼100 aa long, and subsequent SSNMR and FTIR signatures for B. subtilis and B. cereus TasA filaments highlight a conserved amyloid fold, albeit with substantial differences in structural polymorphism and secondary structure composition. Structural analysis and coassembly data on the accessory protein TapA in B. subtilis and its counterpart camelysin in B. cereus reveal a catalyzing effect between the functional amyloid proteins and a common structural architecture, suggesting a coassembly in the context of biofilm formation. Our findings highlight nontypical amyloid behavior of these bacterial functional amyloids, underlining structural variations between biofilms even in closely related bacterial species.-El Mammeri, N., Hierrezuelo, J., Tolchard, J., Cámara-Almirón, J., Caro-Astorga, J., Álvarez-Mena, A., Dutour, A., Berbon, M., Shenoy, J., Morvan, E., Grélard, A., Kauffmann, B., Lecomte, S., de Vicente, A., Habenstein, B., Romero, D., Loquet, A. Molecular architecture of bacterial amyloids in Bacillus biofilms.

Tip-Enhanced Raman Spectroscopy: A Tool for Nanoscale Chemical and Structural Characterization of Biomolecules.

Due to its high molecular sensitivity and spatial optical resolution down to sub-nanometer values, tip-enhanced Raman spectroscopy (TERS) has emerged as a powerful microscopy technique for nanoscale characterization. Progress in TERS instrumentation and in the manufacturing of efficient TERS tips allow for chemical and structural analysis under various experimental conditions (different wavelengths, substrates, and surrounding media). Many biological species have been examined by using this technique. Nucleic acids (individual nucleobases, DNA, and RNA) can show specific TERS features that reveal their composition, conformation, and defects. TERS studies on peptides and proteins (such as amyloid fibrils) provide relevant information on their morphology and structure, leading to valuable insight to their functions and behavior. Finally, lipid layers and membranes, viruses, bacteria, and cells can also be finely characterized. Generalizing TERS measurements in liq- uid medium to study biological systems is the main future challenge.

Necrotizing Microascus tracheobronchitis in a bilateral lung transplant recipient.

Invasive fungal infections are a major cause of mortality among solid organ transplant recipients. Scopulariopsis species and their teleomorph Microascus are molds found in soil and decaying organic matter. We report here the case of a woman who underwent bilateral lung transplantation for severe emphysema. On day 25 after transplantation, endobronchial green-black lesions were detected during routine endoscopy. Endobronchial swabs, biopsies, and bronchoalveolar lavage samples were positive for Microascus cirrosus. This fungal infection developed despite voriconazole given for previous persistent invasive aspergillosis. Treatment consisted of a combination of antifungal medication (voriconazole, terbinafine, amphotericin B, and caspofungin) and endoscopic resection of necrosed bronchial mucosa. A favorable clinical outcome was achieved after 7 weeks of treatment. Seven cases of Scopulariopsis/Microascus infection have been previously described in solid organ transplant recipients. Only two survived after treatment with an antifungal combination therapy including echinocandins, posaconazole, and terbinafine. In immunocompromised patients, infection by Microascus species is a rare but life-threatening event because of innate resistance to most common antifungal drugs. Our patient was successfully cured by combined therapy including intravenous voriconazole and caspofungin, oral terbinafine, and inhaled voriconazole and amphotericin B administered for 7 weeks in association with iterative endoscopic debridement to reduce fungal inoculum.

Single lipoaminoglycoside promotes efficient intracellular antibody delivery: A comprehensive insight into the mechanism of action.

Delivery of biologically active proteins into cells is emerging as important strategy for many applications. Previous experiments have shown that lipoaminoglycosides were capable of delivery of the anti-cytokeratin8 antibody (anti-K8) but only when formulated with lipid helpers potentially leading to toxicity from excess lipids. Here, we optimized anti-K8 delivery with various lipoaminoglycosides in the absence of a lipid helper. Results led to the identification of the aminoglycoside lipid dioleyl phosphoramido ribostamycin (DOPRI) as a potent intracellular delivery system for anti-K8. Electron microscopy revealed that delivered anti-K8 molecules were bound to intermediate filaments in cells. Anti-K8 was bound to the surface of DOPRI vesicles without perturbing lipid organization. Macropinocytosis and caveolin mediated endocytosis contributed to anti-K8 internalization and to filament labeling with a major contribution being made by the caveolin pathway. The results showed that the unique properties of DOPRI were sufficient for efficient intracellular protein delivery without requiring lipid helpers.

PIP2 Phospholipid-Induced Aggregation of Tau Filaments Probed by Tip-Enhanced Raman Spectroscopy.

The morphology and secondary structure of peptide fibers formed by aggregation of tubulin-associated unit (Tau) fragments (K18), in the presence of the inner cytoplasmic membrane phosphatidylinositol component (PIP2 ) or heparin sodium (HS) as cofactors, are determined with nanoscale (<10 nm) spatial resolution. By means of tip-enhanced Raman spectroscopy (TERS), the inclusion of PIP2 lipids in fibers is determined based on the observation of specific C=O ester vibration modes. Moreover, analysis of amide I and amide III bands suggests that the parallel ß-sheet secondary structure content is lower and the random coil content is higher for fibers grown from the PIP2 cofactor instead of HS. These observations highlight the occurrence of some local structural differences between these fibers. This study constitutes the first nanoscale structural characterization of Tau/phospholipid aggregates, which are implicated in deleterious mechanisms on neural membranes in Alzheimer's disease.

Hevea brasiliensis prohevein possesses a conserved C-terminal domain with amyloid-like properties in vitro.

Prohevein is a wound-induced protein and a main allergen from latex of Hevea brasiliensis (rubber tree). This 187 amino-acid protein is cleaved in two fragments: a N-terminal 43 amino-acids called hevein, a lectin bearing a chitin-binding motif with antifungal properties and a C-terminal domain (C-ter) far less characterized. We provide here new insights on the characteristics of prohevein, hevein and C-terminal domain. Using complementary biochemical (ThT/CR/chitin binding, agglutination) and structural (modeling, ATR-FTIR, TEM, WAXS) approaches, we show that this domain clearly displays all the characteristics of an amyloid-like proteins in vitro, that could confer agglutination activity in synergy with its chitin-binding activity. Additionally, this C-ter domain is highly conserved and present in numerous plant prohevein-like proteins or pathogenesis-related (PR and WIN) proteins. This could be the hallmark of the eventual presence of proteins with amyloid properties in plants, that could potentially play a role in defense through aggregation properties.

Rubber particle proteins REF1 and SRPP1 interact differently with native lipids extracted from Hevea brasiliensis latex.

Rubber particle membranes from the Hevea latex contain predominantly two proteins, REF1 and SRPP1 involved in poly(cis-1,4-isoprene) synthesis or rubber quality. The repartition of both proteins on the small or large rubber particles seems to differ, but their role in the irreversible coagulation of the rubber particle is still unknown. In this study we highlighted the different modes of interactions of both recombinant proteins with different classes of lipids extracted from Hevea brasiliensis latex, and defined as phospholipids (PL), glycolipids (GL) and neutral lipids (NL). We combined two biophysical methods, polarization modulated-infrared reflection adsorption spectroscopy (PM-IRRAS) and ellipsometry to elucidate their interactions with monolayers of each class of lipids. REF1 and SRPP1 interactions with native lipids are clearly different; SRPP1 interacts mostly in surface with PL, GL or NL, without modification of its structure. In contrast REF1 inserts deeply in the lipid monolayers with all lipid classes. With NL, REF1 is even able to switch from α-helice conformation to ß-sheet structure, as in its aggregated form (amyloid form). Interaction between REF1 and NL may therefore have a specific role in the irreversible coagulation of rubber particles.

Synthetic toxic Aß1-42 oligomers can assemble in different morphologies.

BACKGROUND: Alzheimer's disease is the most common neurodegenerative disease associated with aggregation of Aß peptides. Aß toxicity is mostly related to the capacity of intermediate oligomers to disrupt membrane integrity. We previously expressed Aß1-42 in a eukaryotic cellular system and selected synthetic variants on their sole toxicity. The most toxic mutant G37C forms stable oligomers. METHODS: Different biophysical methods (Fluorescence spectroscopy, cross-linking, mass spectrometry (MS), Small Angle X-ray Scattering (SAXS), Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), calcein leakage) were used. RESULTS: The oligomers are mostly populated by a 14mers resulting from the packing of homodimers. These homodimers come from the formation of a disulfide bridge between two monomers. This link stabilizes the multimers and prevents the assembly into amyloid fibrils. These oligomers affect the membrane integrity. The reduction of disulfide bonds leads to a rearrangement and redirects assembly of Aß amyloid fibrils. CONCLUSION: The toxic synthetic AßG37C mutant can assemble into an amyloid of unusual morphology through the formation of anti-parallel ß-sheets. This pathway involves the formation of oligomers resulting from the arrangement of Aß dimers linked by covalent di-sulfide link, being these oligomers harmful for the membranes. GENERAL SIGNIFICANCE: The capacity to produce large amount of stable oligomers without additional detergents or extrinsic cross-linkers allow further structural and biophysical studies to understand their capacity to assemble and disrupt the membranes, a key event in Alzheimer's disease.

Tip-Enhanced Raman Spectroscopy to Distinguish Toxic Oligomers from Aß1-42 Fibrils at the Nanometer Scale.

For the first time, natural Aß1-42 fibrils (WT) implicated in Alzheimer's disease, as well as two synthetic mutants forming less toxic amyloid fibrils (L34T) and highly toxic oligomers (oG37C), are chemically characterized at the scale of a single structure using tip-enhanced Raman spectroscopy (TERS). While the proportion of TERS features associated with amino acid residues is similar for the three peptides, a careful examination of amide I and amide III bands allows us to clearly distinguish WT and L34T fibers organized in parallel ß-sheets from the small and more toxic oG37C oligomers organized in anti-parallel ß-sheets.