Fabrication of plasmonic probes for reproducible nanospectroscopic investigation of lipid monolayers - The electrochemical etching with DC-pulsed voltage.

Tip-enhanced Raman spectroscopy (TERS) is a label-free analytical technique that characterizes molecular systems, potentially even with a nanometric resolution. In principle, the metallic plasmonic probe is illuminated with a laser beam generating the localized surface plasmons, which induce a strong local electric field enhancement in close proximity to the probe. Such field enhancement improves the Raman scattering cross-section from the sample volume localized near the probe apex. TERS provides a high spatial resolution and a great sensitivity, however, it is rather rarely used due to technical limitations causing unstable enhancement and the relative lack of data reproducibility. Despite many scientific efforts for the fabrication of effective TER probes providing robust TER enhancement still requires further investigations. In this work, we explore new possibilities based on preparation of scanning tunnelling microscopy (STM) plasmonic probes, since by nature of the tunnelling effect, they potentially could offer a very high spatial resolution in STM guided TERS experiments. Here we compare two methods of STM-TERS probe preparation for effective spectra acquisition. Our results strongly indicate that an application of square pulse voltage upon the etching procedure significantly improves the quality of the TER data over those obtained with a constant voltage one. To demonstrate the efficiency of our probes we present the results of hyperspectral TER mapping of the 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) monolayer deposited on an ultra-pure and atomically flat gold substrate.

Tip-enhanced Raman spectroscopy reveals the structural rearrangements of tau protein aggregates at the growth phase.

Tau protein aggregates inside neurons in the course of Alzheimer's disease (AD). Because of the enormous number of people suffering from AD, this disease has become one of the world's major health and social problems. The presence of tau lesions clearly correlates with cognitive impairments in AD patients, thus, tau is the target of potential treatments for AD, next to amyloid-ß. The exact mechanism of tau aggregation has not been understood in detail so far; especially little is known about the structural rearrangements of tau aggregates at the growth phase. The research into tau conformation at each step of the aggregation pathway will contribute to the design of effective therapeutic approaches. To follow the secondary structure of individual tau aggregates at the growth phase, we applied tip-enhanced Raman spectroscopy (TERS). The nanospectroscopic approach enabled us to follow the structure of individual aggregates occurring in the subsequent phases of tau aggregation. We applied multivariate data analysis to extract the spectral differences for tau aggregates at different aggregation phases. Moreover, atomic force microscopy (AFM) allowed the tracking of the morphological alterations for species occurring with the progression of tau aggregation.

Molecular alterations in metaphase chromosomes induced by bleomycin.

Chromosomes are intranuclear structures, their main function is to store and transmit genetic information during cell division. They are composed of tightly packed DNA in the form of chromatin, which is constantly exposed to various damaging factors. The resulting changes in DNA can have serious consequences (e.g. mutations) if they are not repaired or repaired incorrectly. In this article, we studied chromosomes isolated from human cervical cancer cells (HeLa) exposed to a genotoxic drug causing both single- and double-strand breaks. Specifically, we used bleomycin to induce DNA damage. We followed morphological and chemical changes in chromosomes upon damage induction. Atomic force microscopy was used to visualize the morphology of chromosomes, while Raman microspectroscopy enabled the detection of changes in the chemical structure of chromatin with the resolution close to the diffraction limit. Additionally, we extracted spectra corresponding to chromosome I or chromatin from hyperspectral Raman maps with convolutional neural networks (CNN), which were further analysed with the principal component analysis (PCA) algorithm to reveal molecular markers of DNA damage in chromosomes. The applied multimodal approach revealed simultaneous morphological and molecular changes, including chromosomal aberrations, alterations in DNA conformation, methylation pattern, and increased protein expression upon the bleomycin treatment at the level of the single chromosome.

Lithocholic acid-based oligomers as drug delivery candidates targeting model of lipid raft.

This study presents a new approach to designing a lithocholic acid functionalized oligomer (OLithocholicAA-X) that can be used as a drug carrier with additional, beneficial activity. Namely, this novel oligomer can incorporate an anti-cancer drug due to the application of an effective backbone as its component (lithocholic acid) alone is known to have anticancer activity. The oligomer was synthesized and characterized in detail by nuclear magnetic resonance, attenuated total reflectance Fourier-transform infrared spectroscopy, ultraviolet-visible spectroscopy, thermal analysis, and mass spectrometry analysis. We selected lipid rafts as potential drug carrier-membrane binding sites. In this respect, we investigated the effects of OLithocholicAA-X on model lipid raft of normal and altered composition, containing an increased amount of cholesterol (Chol) or sphingomyelin (SM), using Langmuir monolayers and liposomes. The surface topography of the studied monolayers was additionally investigated by atomic force microscopy (AFM). The obtained results showed that the investigated oligomer has affinity for a system that mimics a normal lipid raft (SM:Chol 2:1). On the other hand, for systems with an excess of SM or Chol, thermodynamically unfavorable fluidization of the films occurs. Moreover, AFM topographies showed that the amount of SM determines the bioavailability of the oligomer, causing fragmentation of its lattice.

Surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS) in label-free characterization of erythrocyte membranes and extracellular vesicles at the nano-scale and molecular level.

The manuscript presents the potential of surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS) for label-free characterization of extracellular microvesicles (EVs) and their isolated membranes derived from red blood cells (RBCs) at the nanoscale and at the single-molecule level, providing detection of a few individual amino acids, protein and lipid membrane compartments. The study shows future directions for research, such as investigating the use of the mentioned techniques for the detection and diagnosis of diseases. We demonstrate that SERS and TERS are powerful techniques for identifying the biochemical composition of EVs and their membranes, allowing the detection of small molecules, lipids, and proteins. Furthermore, extracellular vesicles released from red blood cells (REVs) can be broadly classified into exosomes, microvesicles, and apoptotic bodies, based on their size and biogenesis pathways. Our study specifically focuses on microvesicles that range from 100 to 1000 nanometres in diameter, as presented in AFM images. Using SERS and TERS spectra obtained for REVs and their membranes, we were able to characterize the chemical and structural properties of microvesicle membranes with high sensitivity and specificity. This information may help better distinguish and categorize different types of EVs, leading to a better understanding of their functions and potential biomedical applications.

Combined analytical approach empowers precise spectroscopic interpretation of subcellular components of pancreatic cancer cells.

The lack of specific and sensitive early diagnostic options for pancreatic cancer (PC) results in patients being largely diagnosed with late-stage disease, thus inoperable and burdened with high mortality. Molecular spectroscopic methodologies, such as Raman or infrared spectroscopies, show promise in becoming a leader in screening for early-stage cancer diseases, including PC. However, should such technology be introduced, the identification of differentiating spectral features between various cancer types is required. This would not be possible without the precise extraction of spectra without the contamination by necrosis, inflammation, desmoplasia, or extracellular fluids such as mucous that surround tumor cells. Moreover, an efficient methodology for their interpretation has not been well defined. In this study, we compared different methods of spectral analysis to find the best for investigating the biomolecular composition of PC cells cytoplasm and nuclei separately. Sixteen PC tissue samples of main PC subtypes (ductal adenocarcinoma, intraductal papillary mucinous carcinoma, and ampulla of Vater carcinoma) were collected with Raman hyperspectral mapping, resulting in 191,355 Raman spectra and analyzed with comparative methodologies, specifically, hierarchical cluster analysis, non-negative matrix factorization, T-distributed stochastic neighbor embedding, principal components analysis (PCA), and convolutional neural networks (CNN). As a result, we propose an innovative approach to spectra classification by CNN, combined with PCA for molecular characterization. The CNN-based spectra classification achieved over 98% successful validation rate. Subsequent analyses of spectral features revealed differences among PC subtypes and between the cytoplasm and nuclei of their cells. Our study establishes an optimal methodology for cancer tissue spectral data classification and interpretation that allows precise and cognitive studies of cancer cells and their subcellular components, without mixing the results with cancer-surrounding tissue. As a proof of concept, we describe findings that add to the spectroscopic understanding of PC.

Nanoscale insights into the local structural rearrangements of amyloid-ß induced by bexarotene.

A better understanding of the abnormal protein aggregation and the effect of anti-aggregation agents on the fibrillation pathways and the secondary structure of aggregates can determine strategies for the early treatment of dementia. Herein, we present a combination of experimental and theoretical studies providing new insights into the influence of the anti-aggregation drug bexarotene on the secondary structure of individual amyloid-ß aggregates and its primary aggregation. The molecular rearrangements and the spatial distribution of ß-sheets within individual aggregates were monitored at the nanoscale with infrared nanospectroscopy. We observed that bexarotene limits the parallel ß-sheets formation, known to be highly abundant in fibrils at later phases of the amyloid-ß aggregation composed of in-register cross-ß structure. Moreover, we applied molecular dynamics to provide molecular-level insights into the investigated system. Both theoretical and experimental results revealed that bexarotene slows down the protein aggregation process via steric effects, largely prohibiting the antiparallel to parallel ß-sheet rearrangement. We also found that bexarotene interacts not only via the single hydrogen bond formation with the peptide backbone but also with the amino acid side residue via a hydrophobic effect. The studied model of the drug-amyloid-ß interaction contributes to a better understanding of the inhibition mechanism of the amyloid-ß aggregation by the small molecule drugs. However, our nanoscale findings need to meet in vivo research requiring different analytical approaches.

Variabilities in global DNA methylation and ß-sheet richness establish spectroscopic landscapes among subtypes of pancreatic cancer.

PURPOSE: Knowledge about pancreatic cancer (PC) biology has been growing rapidly in recent decades. Nevertheless, the survival of PC patients has not greatly improved. The development of a novel methodology suitable for deep investigation of the nature of PC tumors is of great importance. Molecular imaging techniques, such as Fourier transform infrared (FTIR) spectroscopy and Raman hyperspectral mapping (RHM) combined with advanced multivariate data analysis, were useful in studying the biochemical composition of PC tissue. METHODS: Here, we evaluated the potential of molecular imaging in differentiating three groups of PC tumors, which originate from different precursor lesions. Specifically, we comprehensively investigated adenocarcinomas (ACs): conventional ductal AC, intraductal papillary mucinous carcinoma, and ampulla of Vater AC. FTIR microspectroscopy and RHM maps of 24 PC tissue slides were obtained, and comprehensive advanced statistical analyses, such as hierarchical clustering and nonnegative matrix factorization, were performed on a total of 211,355 Raman spectra. Additionally, we employed deep learning technology for the same task of PC subtyping to enable automation. The so-called convolutional neural network (CNN) was trained to recognize spectra specific to each PC group and then employed to generate CNN-prediction-based tissue maps. To identify the DNA methylation spectral markers, we used differently methylated, isolated DNA and compared the observed spectral differences with the results obtained from cellular nuclei regions of PC tissues. RESULTS: The results showed significant differences among cancer tissues of the studied PC groups. The main findings are the varying content of ß-sheet-rich proteins within the PC cells and alterations in the relative DNA methylation level. Our CNN model efficiently differentiated PC groups with 94% accuracy. The usage of CNN in the classification task did not require Raman spectral data preprocessing and eliminated the need for extensive knowledge of statistical methodologies. CONCLUSIONS: Molecular spectroscopy combined with CNN technology is a powerful tool for PC detection and subtyping. The molecular fingerprint of DNA methylation and ß-sheet cytoplasmic proteins established by our results is different for the main PC groups and allowed the subtyping of pancreatic tumors, which can improve patient management and increase their survival. Our observations are of key importance in understanding the variability of PC and allow translation of the methodology into clinical practice by utilizing liquid biopsy testing.

Vibrational spectroscopy - are we close to finding a solution for early pancreatic cancer diagnosis?

Pancreatic cancer (PC) is an aggressive and lethal neoplasm, ranking seventh in the world for cancer deaths, with an overall 5-year survival rate of below 10%. The knowledge about PC pathogenesis is rapidly expanding. New aspects of tumor biology, including its molecular and morphological heterogeneity, have been reported to explain the complicated "cross-talk" that occurs between the cancer cells and the tumor stroma or the nature of pancreatic ductal adenocarcinoma-associated neural remodeling. Nevertheless, currently, there are no specific and sensitive diagnosis options for PC. Vibrational spectroscopy (VS) shows a promising role in the development of early diagnosis technology. In this review, we summarize recent reports about improvements in spectroscopic methodologies, briefly explain and highlight the drawbacks of each of them, and discuss available solutions. The important aspects of spectroscopic data evaluation with multivariate analysis and a convolutional neural network methodology are depicted. We conclude by presenting a study design for systemic verification of the VS-based methods in the diagnosis of PC.

Optimization of tip-enhanced Raman spectroscopy for probing the chemical structure of DNA.

Tip-enhanced Raman (TER) spectroscopy combines the nanometric spatial resolution of atomic force microscopy (AFM) and the chemical sensitivity of Raman spectroscopy. Thus, it provides a unique possibility to obtain spectroscopic information on individual, nanometre-size molecules. The enhancement of Raman scattering cross-section requires modification of the AFM tip apex with a plasmonic nanostructure. Despite numerous advances of TERS research, attaining good reproducibility and stable enhancement is still challenging mainly due to the lack of optimized probes and sample preparation procedures. Moreover, current nanospectroscopic standard samples - carbon nanotubes (CNTs) have relatively simple chemical structure, and therefore, they are far from real-life analytes, especially biological samples. In this work we focus on the optimization of TERS technique for efficient DNA measurements, including: a preparation of atomically-flat gold substrates, fixative free deposition of DNA and optimization of TERS probe preparation. Here we demonstrate a comprehensive comparison of the efficacy of several types of TERS probes. Applying the systematic approach, we obtained reliable and reproducible TER spectra of DNA. Thus, we provide preparation procedures of a new standard TERS sample, TERS substrates and TERS probes. Our research provides a solid foundation for further research on DNA and its interaction with other biomolecules upon biologically significant processes such as DNA damage and repair.

Plasmonic hot spots reveal local conformational transitions induced by DNA double-strand breaks.

DNA double-strand breaks (DSBs) are typical DNA lesions that can lead to cell death, translocations, and cancer-driving mutations. The repair process of DSBs is crucial to the maintenance of genomic integrity in all forms of life. However, the limitations of sensitivity and special resolution of analytical techniques make it difficult to investigate the local effects of chemotherapeutic drugs on DNA molecular structure. In this work, we exposed DNA to the anticancer antibiotic bleomycin (BLM), a damaging factor known to induce DSBs. We applied a multimodal approach combining (i) atomic force microscopy (AFM) for direct visualization of DSBs, (ii) surface-enhanced Raman spectroscopy (SERS) to monitor local conformational transitions induced by DSBs, and (iii) multivariate statistical analysis to correlate the AFM and SERS results. On the basis of SERS results, we identified that bands at 1050 cm-1 and 730 cm-1 associated with backbone and nucleobase vibrations shifted and changed their intensities, indicating conformational modifications and strand ruptures. Based on averaged SERS spectra, the PLS regressions for the number of DSBs caused by corresponding molar concentrations of bleomycin were calculated. The strong correlation (R2 = 0.92 for LV = 2) between the predicted and observed number of DSBs indicates, that the model can not only predict the number of DSBs from the spectra but also detect the spectroscopic markers of DNA damage and the associated conformational changes.

Current Pathology Model of Pancreatic Cancer.

Pancreatic cancer (PC) is one of the most aggressive and lethal malignant neoplasms, ranking in seventh place in the world in terms of the incidence of death, with overall 5-year survival rates still below 10%. The knowledge about PC pathomechanisms is rapidly expanding. Daily reports reveal new aspects of tumor biology, including its molecular and morphological heterogeneity, explain complicated "cross-talk" that happens between the cancer cells and tumor stroma, or the nature of the PC-associated neural remodeling (PANR). Staying up-to-date is hard and crucial at the same time. In this review, we are focusing on a comprehensive summary of PC aspects that are important in pathologic reporting, impact patients' outcomes, and bring meaningful information for clinicians. Finally, we show promising new trends in diagnostic technologies that might bring a difference in PC early diagnosis.

Raman Research on Bleomycin-Induced DNA Strand Breaks and Repair Processes in Living Cells.

Even several thousands of DNA lesions are induced in one cell within one day. DNA damage may lead to mutations, formation of chromosomal aberrations, or cellular death. A particularly cytotoxic type of DNA damage is single- and double-strand breaks (SSBs and DSBs, respectively). In this work, we followed DNA conformational transitions induced by the disruption of DNA backbone. Conformational changes of chromatin in living cells were induced by a bleomycin (BLM), an anticancer drug, which generates SSBs and DSBs. Raman micro-spectroscopy enabled to observe chemical changes at the level of single cell and to collect hyperspectral images of molecular structure and composition with sub-micrometer resolution. We applied multivariate data analysis methods to extract key information from registered data, particularly to probe DNA conformational changes. Applied methodology enabled to track conformational transition from B-DNA to A-DNA upon cellular response to BLM treatment. Additionally, increased expression of proteins within the cell nucleus resulting from the activation of repair processes was demonstrated. The ongoing DNA repair process under the BLM action was also confirmed with confocal laser scanning fluorescent microscopy.

Oxysterols can act antiviral through modification of lipid membrane properties - The Langmuir monolayer study.

In this paper we tested how oxysterols influence on fusion process between viral lipid envelope and host cells membranes. For this purpose, the Zika virus was selected, while dendritic cell (DC) and neural cell (NC) membranes were chosen as target membranes. The investigated systems were modeled as multicomponent Langmuir monolayers and characterized using surface manometry and imaging in micro- (Brewster angle microscopy, BAM) and nanoscale (Atomic Force Microscopy, AFM) to monitor local heterogeneity. The fusion process was conducted by mixing viral and host cell membranes devoid and in the presence of oxysterols: 25-hydroxycholesterol (25-OH) and 7ß-hydroxycholesterol (7ß-OH) as representatives of chain- and ring-oxidized oxysterols, respectively. Our results show that oxysterols hinder the fusion with host cell membranes by modifying their biophysical properties. Moreover, oxysterols applied to an already infected membrane reverse the changes caused by the infection. It could therefore be concluded that oxysterols may display antiviral activity in two ways: they prevent the healthy membrane from viral infection by blocking the fusion process; and protect already infected membrane from pathological changes induced by the virus.

Revealing local molecular distribution, orientation, phase separation, and formation of domains in artificial lipid layers: Towards comprehensive characterization of biological membranes.

Lipids, together with molecules such as DNA and proteins, are one of the most relevant systems responsible for the existence of life. Selected lipids are able to assembly into various organized structures, such as lipid membranes. The unique properties of lipid membranes determine their complex functions, not only to separate biological environments, but also to participate in regulatory functions, absorption of nutrients, cell-cell communication, endocytosis, cell signaling, and many others. Despite numerous scientific efforts, still little is known about the reason underlying the variability within lipid membranes, and its biochemical significance. In this review, we discuss the structural complexity of lipid membranes, as well as the importance to simplify studied systems in order to understand phenomena occurring in natural, complex membranes. Such systems require a model interface to be analyzed. Therefore, here we focused on analytical studies of artificial systems at various interfaces. The molecular structure of lipid membranes, specifically the nanometric thickens of molecular bilayer, limits in a major extent the choice of highly sensitive methods suitable to study such structures. Therefore, we focused on methods that combine high sensitivity, and/or chemical selectivity, and/or nanometric spatial resolution, such as atomic force microscopy, nanospectroscopy (tip-enhanced Raman spectroscopy, infrared nanospectroscopy), phase modulation infrared reflection-absorption spectroscopy, sum-frequency generation spectroscopy. We summarized experimental and theoretical approaches providing information about molecular structure and composition, lipid spatial distribution (phase separation), organization (domain shape, molecular orientation) of lipid membranes, and real-time visualization of the influence of various molecules (proteins, drugs) on their integrity. An integral part of this review discusses the latest achievements in the field of lipid layer-based biosensors.

Revealing DNA Structure at Liquid/Solid Interfaces by AFM-Based High-Resolution Imaging and Molecular Spectroscopy.

DNA covers the genetic information in all living organisms. Numerous intrinsic and extrinsic factors may influence the local structure of the DNA molecule or compromise its integrity. Detailed understanding of structural modifications of DNA resulting from interactions with other molecules and surrounding environment is of central importance for the future development of medicine and pharmacology. In this paper, we review the recent achievements in research on DNA structure at nanoscale. In particular, we focused on the molecular structure of DNA revealed by high-resolution AFM (Atomic Force Microscopy) imaging at liquid/solid interfaces. Such detailed structural studies were driven by the technical developments made in SPM (Scanning Probe Microscopy) techniques. Therefore, we describe here the working principles of AFM modes allowing high-resolution visualization of DNA structure under native (liquid) environment. While AFM provides well-resolved structure of molecules at nanoscale, it does not reveal the chemical structure and composition of studied samples. The simultaneous information combining the structural and chemical details of studied analyte allows achieve a comprehensive picture of investigated phenomenon. Therefore, we also summarize recent molecular spectroscopy studies, including Tip-Enhanced Raman Spectroscopy (TERS), on the DNA structure and its structural rearrangements.

25-hydroxycholesterol interacts differently with lipids of the inner and outer membrane leaflet - The Langmuir monolayer study complemented with theoretical calculations.

25-hydroxycholesterol (25-OH), a molecule with unusual behavior at the air/water interface, being anchored to the water surface alternatively with a hydroxyl group at C(3) or C(25), has been investigated in mixtures with main membrane phospholipids (phosphatidylcholines - PCs, and phosphatidylethanolamines - PEs), characteristic of the outer and inner membrane leaflet, respectively. To achieve this goal, the classical Langmuir monolayer approach based on thermodynamic analysis of interactions was conducted in addition to microscopic imaging of films (in situ with BAM and after transfer onto mica with AFM), surface-sensitive spectroscopy (PM-IRRAS), as well as theoretical calculations. Our results show that the strength of interactions is primarily determined by the kind of polar group (strong, attractive interactions leading to surface complexes formation were found to occur with PCs while weak or repulsive ones with PEs). Subsequently, the saturation of phosphatidylcholines apolar chain(s) was found to be crucial for the structure of the formed complexes. Namely, saturated PC (DPPC) does not have preferences regarding the orientation of 25-OH molecule in surface complexes (which results in the two possible 25-OH arrangements), while unsaturated PC (DOPC) enforces one specific orientation of oxysterol (with C(3)-OH group). Our findings suggest that the transport of 25-OH between inner and outer membrane leaflet can proceed without orientation changes, which is thermodynamically advantageous. This explains results found in real systems showing significant differences in the rate of transmembrane transport of 25-OH and the other chain-oxidized oxysterols compared to their ring-oxidized analogues or cholesterol.

Nanoscale Hyperspectral Imaging of Amyloid Secondary Structures in Liquid.

Abnormal aggregation of amyloid-ß is a very complex and heterogeneous process. Owing to methodological limitations, the aggregation pathway is still not fully understood. Herein a new approach is presented in which the secondary structure of single amyloid-ß aggregates is investigated with tip-enhanced Raman spectroscopy (TERS) in a liquid environment. Clearly resolved TERS signatures of the amide I and amide III bands enabled a detailed analysis of the molecular structure of single aggregates at each phase of the primary aggregation of amyloid-ß and also of small species on the surface of fibrils attributed to secondary nucleation. Notably, a ß-sheet rearrangement from antiparallel in protofibrils to parallel in fibrils is observed. This study allows better understanding of Alzheimer's disease etiology and the methodology can be applied in studies of other neurodegenerative disorders.

Stearoyl-CoA Desaturase 1 Activity Determines the Maintenance of DNMT1-Mediated DNA Methylation Patterns in Pancreatic ß-Cells.

Metabolic stress, such as lipotoxicity, affects the DNA methylation profile in pancreatic ß-cells and thus contributes to ß-cell failure and the progression of type 2 diabetes (T2D). Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme that is involved in monounsaturated fatty acid synthesis, which protects pancreatic ß-cells against lipotoxicity. The present study found that SCD1 is also required for the establishment and maintenance of DNA methylation patterns in ß-cells. We showed that SCD1 inhibition/deficiency caused DNA hypomethylation and changed the methyl group distribution within chromosomes in ß-cells. Lower levels of DNA methylation in SCD1-deficient ß-cells were followed by lower levels of DNA methyltransferase 1 (DNMT1). We also found that the downregulation of SCD1 in pancreatic ß-cells led to the activation of adenosine monophosphate-activated protein kinase (AMPK) and an increase in the activity of the NAD-dependent deacetylase sirtuin-1 (SIRT1). Furthermore, the physical association between DNMT1 and SIRT1 stimulated the deacetylation of DNMT1 under conditions of SCD1 inhibition/downregulation, suggesting a mechanism by which SCD1 exerts control over DNMT1. We also found that SCD1-deficient ß-cells that were treated with compound c, an inhibitor of AMPK, were characterized by higher levels of both global DNA methylation and DNMT1 protein expression compared with untreated cells. Therefore, we found that activation of the AMPK/SIRT1 signaling pathway mediates the effect of SCD1 inhibition/deficiency on DNA methylation status in pancreatic ß-cells. Altogether, these findings suggest that SCD1 is a gatekeeper that protects ß-cells against the lipid-derived loss of DNA methylation and provide mechanistic insights into the mechanism by which SCD1 regulates DNA methylation patterns in ß-cells and T2D-relevant tissues.

Molecular insight into neurodegeneration - Langmuir monolayer study on the influence of oxysterols on model myelin sheath.

Systematic studies on the influence of selected ring-oxidized (7α-hydroxycholesterol, 7α-OH; 7ß-hydroxycholesterol, 7ß-OH; 7-ketocholesterol, 7-K) and chain-oxidized (25-OH) sterols on lipid layer of myelin were performed. Myelin sheath was modeled as five-component Langmuir monolayer (Chol:PE:SM:PS:PC 50:20:12:9:9). Particular oxysterols have been incorporated into the model myelin sheath by replacing cholesterol totally or partially (1:1). The effect of oxysterol incorporation was characterized with surface pressure and electric surface potential - area isotherms and visualized with Brewster angle microscopy (BAM) and atomic force microscopy (AFM). It has been noticed that model myelin loses its homogeneous structure (due to the appearance of domains) at physiological bilayer conditions (30-35 mN/m). In the presence of oxysterols, the fluidity of myelin model increases and the organization of lipids is altered, which is reflected in the decrease of electric surface potential changes (ΔV). The strongest myelin/oxysterol interactions have been observed for 7-K and 25-OH, being the most cytotoxic oxysterols found in biological tests.