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  • 36 Reads
Recent progress in molecular recognition imaging of protein systems at the nanoscale level

Identification of proteins has received considerable attention in recent years due to the increasing interest to resolve individual biomolecules under physiological relevant conditions. In this framework, atomic force microscopy (AFM), has shown its great potential to acquire a variety of biomolecular physico-chemical properties at the single molecule level [1]. Particularly, force spectroscopy based on AFM (AFM-FS) allows to determine the intermolecular interactions between two biomolecules, requiring being one covalently immobilized on a flat surface and the other linked onto the AFM tip. Previous work was developed in this field by simultaneous topography and recognition imaging (TREC) [2] and tuning-fork-based transverse dynamic force microscopy (TDFM) [3] albeit both methods lack of providing quantitive information. To overcome the aforementioned limitations force-volume (F-V) [4] emerged as promising alternative but the extremely large data acquisition times can lead drifting effects during the image record. Here, we present the intermittent jumping force mode (JM) as suitable approach to gather quantitative high-resolution force maps at local areas of the scanned sample with fast-acquisition times. Using this mode applying very low forces under respulsive regime conditions simultaneous maps of topography and specific rupture forces corresponding to the unbinding of the protein:ligand complexes are obtained. Two different protein systems are employed to illustrate the capabilities of the built up methodological improvements. First, the flavoenzyme system formed between flavodoxin NADP+ reductase (FNR) and its redox partners, ferredoxin and flavodoxin [5, 6] and second, the strongest non-covalent complexes observed in nature between avidin and streptavidin and biotin [7]. In the first case, the results were optimized when an oriented immobilization procedure was designed. In the second work, discrimination between avidin and streptavidin molecules in a hybrid sample was achieved with an unique sensor ligand. The most relevant scientific outcomes can serve as proof-of-principle stage to design diagnostic devices with ultra-sensitivity detection signal for drug screening applications.

[1] Müller, D.J.; Dumitru, A.C.; Lo Giudice, C.; Gaub, H.E.; Hinterdorfer, P.; Hummer, G.; De Yoreo, J.J.; Alsteens, D. Atomic Force Microscopy-Based Force Spectroscopy and Multiparametric Imaging of Biomolecular and Cellular Systems. Chem. Rev. 2020, 121, 11701-11725.
[2] Stroh, C.M.; Ebner, A.; Geretschlager, M.; Freudenthale, G.; Kienberger, F.; Kamruzzahan, A.S.M.; Smith-Gill, S.J.; Gruber, H.J.; Hinterdorfer, P. Simultaneous topography and recognition imaging using force microscopy. Biophys. J. 2004, 87, 1981-1990.
[3] Hofer, M.; Adamsmaier, S.; van Zanten, T.S.; Chtcheglova, L.A.; Manzo, C.; Duman, M.; Mayer, B.; Ebner, A.; Moertelmaier, M.; Kada, G.; et al. Molecular recognition imaging using tuning-fork based transverse dynamic force microscopy. Ultramicroscopy 2010, 110, 605-611.
[4] Ludwig, M.; Dettmann, W.; Gaub, H.E. Atomic force microscope imaging contrast based on molecular recognition. Biophys. J. 1997, 72, 445-448.
[5] Marcuello, C.; De Miguel R.; Gómez-Moreno C.; Martínez-Júlvez, M.; Lostao, A. An efficient method for enzyme immobilization evidenced by atomic force microscopy. Protein Eng. Des. Sel. 2012, 25, 715-723.
[6] Marcuello, C.; de Miguel, R.; Martínez-Júlvez, M.; Gómez-Moreno, C.; Lostao, A. Mechanostability of the Single-Electron-Transfer Complexes of Anabaena Ferredoxin-NADP+ Reductase. ChemPhysChem. 2015, 16, 3161-3169.
[7] Marcuello, C.; De Miguel R.; Lostao, A. Molecular Recognition of Proteins through Quantitative Force Maps at Single Molecule Level. Biomolecules 2022, 12, 594.

  • Open access
  • 31 Reads
Role of APOE Gene Polymorphisms in Coronary Artery Disease

Coronary artery disease (CAD) is one of the major causes of global mortality and morbidity. It is a multifactorial disease as it involves a complex relationship between environmental, biochemical, and genetic factors. Single nucleotide polymorphisms (SNPs) are the most promising and commonly used genetic markers for disease risk association. Apolipoprotein E (APOE) is listed among the top genes that play a crucial role in coronary artery disease pathophysiology. Our current meta-analysis aims to find the association of two extensively studied variants, including rs429385 and rs7412, with coronary artery disease. Eligible articles were retrieved using different MeSH terms, keywords, and databases, including PubMed, Ovid, Cochrane Library, web of science, and Embase. Our study was conducted according to the Preferred Reporting Items for Systematic Reviews, and Meta-Analyses2009 (PRISMA 2009) guidelines. Our pooled analysis suggests the ε4 allele of APOE was significantly associated with CAD (odds ratio 2.00; 95% and CI, 1.48–2.71). In addition, meta-analysis results, including the shape of funnel plots, showed no publication bias. However, we recommend more studies with larger cohort sizes, should be conducted in different ethnic groups that may provide more conclusive findings.

  • Open access
  • 34 Reads
Natural Diversity of Telomeric DNA Sequences in Bryophytes

Telomeres - the most important specialized nucleoprotein structures at the ends of linear eukaryotic chromosomes involved in protecting of DNA from damage. The functional characteristic of telomeres is the length. Changes in telomere structure is of enormous evolutionary importance as they can affect whole-genome stability. However, plants remain a poorly studied group in the light of telomere biology. Bryophytes are the prospective plant group for the understanding of the protection and evolution mechanisms of plant telomeres. The aim of the work was to determine natural variability of telomere lengths in different bryophyte species and ecotypes. We used axenic cultures of moss Physcomitrium patens ecotypes (Gransden, Reute, Villersexel, Kaskaskia), moss Ceratodon purpureus (male R40 and female GG1), as well as natural isolates Sphagnum fallax, S. girgensohnii and Sphagnum sp. The analysis of the telomeres was carried out by the TRF method. It was found that different ecotypes of P. patens plants have different telomere lengths in the range from 1000 to 1500 bp. The mean telomere length in a female plant C. purpureus was 480-500 bp, in a male plant 900-1000 bp. The telomere lengths of S. fallax MW and S. girgensohnii were about mean ~2000 bp. However, telomeres were slightly shorter in isolate of S. sp, whose telomere length ranged from 1100 to 1500 bp. In addition, it was found that all studied Bryophytes had differences in the location of specific telomeric sequences. Thus, we have shown that the telomere lengths of bryophytes can vary both between species and within one species. Also, interstitial telomeric DNA is a distinguishing characteristic of bryophyte genomes.