Cellular membranes have long served as an inspiration for nanomaterial research. The preparation of ultrathin polydopamine (PDA) films with integrated protein pores containing phospholipids and an ...embedded domain of a membrane protein glycophorin A as simplified cell membrane mimics is reported. Large area, ultrathin PDA films are obtained by electropolymerization on gold surfaces with 10–18 nm thickness and dimensions of up to 2.5 cm2. The films are transferred from gold to various other substrates such as nylon mesh, silicon, or substrates containing holes in the micrometer range, and they remain intact even after transfer. The novel transfer technique gives access to freestanding PDA films that remain stable even at the air interfaces with elastic moduli of ≈6–12 GPa, which are higher than any other PDA films reported before. As the PDA film thickness is within the range of cellular membranes, monodisperse protein nanopores, so‐called “nanodiscs,” are integrated as functional entities. These nanodisc‐containing PDA films can serve as semi‐permeable films, in which the embedded pores control material transport. In the future, these simplified cell membrane mimics may offer structural investigations of the embedded membrane proteins to receive an improved understanding of protein‐mediated transport processes in cellular membranes.
Electropolymerization of large area, ultrathin polydopamine films with dimensions up to 2.5 cm2 and their transfer to various substrates is realized. Phospholipid nanodiscs containing a model protein are introduced into the polymer film. Such hybrid films with different incorporated membrane proteins give a platform to obtain a better understanding of how transport in a cell functions.
SELEX (Systematic Evolution of Ligands by Exponential enrichment) processes aim on the evolution of high-affinity aptamers as binding entities in diagnostics and biosensing. Aptamers can represent ...game-changers as constituents of diagnostic assays for the management of instantly occurring infectious diseases or other health threats. Without in-process quality control measures SELEX suffers from low overall success rates. We present a quantitative PCR method for fast and easy quantification of aptamers bound to their targets. Simultaneous determination of melting temperatures (
T
m
) of each SELEX round delivers information on the evolutionary success via the correlation of increasing GC content and
T
m
alone with a round-wise increase of aptamer affinity to the respective target. Based on nine successful and published previous SELEX processes, in which the evolution/selection of aptamer affinity/specificity was demonstrated, we here show the functionality of the IMPATIENT-qPCR for polyclonal aptamer libraries and resulting individual aptamers. Based on the ease of this new evolution quality control, we hope to introduce it as a valuable tool to accelerate SELEX processes in general.
IMPATIENT-qPCR SELEX success monitoring. Selection and evolution of high-affinity aptamers using SELEX technology with direct aptamer evolution monitoring using melting curve shifting analyses to higher
T
m
by quantitative PCR with fluorescence dye SYBR Green I.
Key points
• Fast and easy analysis.
• Universal applicability shown for a series of real successful projects.
Graphical Abstract
Throughout life, the body is subjected to various mechanical forces on the organ, tissue, and cellular level. Mechanical stimuli are essential for organ development and function. One organ whose ...function depends on the tightly connected interplay between mechanical cell properties, biochemical signaling, and external forces is the lung. However, altered mechanical properties or excessive mechanical forces can also drive the onset and progression of severe pulmonary diseases. Characterizing the mechanical properties and forces that affect cell and tissue function is therefore necessary for understanding physiological and pathophysiological mechanisms. In recent years, multiple methods have been developed for cellular force measurements at multiple length scales, from subcellular forces to measuring the collective behavior of heterogeneous cellular networks. In this short review, we give a brief overview of the mechanical forces at play on the cellular level in the lung. We then focus on the technological aspects of measuring cellular forces at many length scales. We describe tools with a subcellular resolution and elaborate measurement techniques for collective multicellular units. Many of the technologies described are by no means restricted to lung research and have already been applied successfully to cells from various other tissues. However, integrating the knowledge gained from these multi-scale measurements in a unifying framework is still a major future challenge.
The weakness of senescent dermal fibroblasts Rebehn, Lydia; Khalaji, Samira; KleinJan, Fenneke ...
Proceedings of the National Academy of Sciences - PNAS,
08/2023, Letnik:
120, Številka:
34
Journal Article
Recenzirano
Odprti dostop
Skin is the largest human organ with easily noticeable biophysical manifestations of aging. As human tissues age, there is chronological accumulation of biophysical changes due to internal and ...environmental factors. Skin aging leads to decreased elasticity and the loss of dermal matrix integrity via degradation. The mechanical properties of the dermal matrix are maintained by fibroblasts, which undergo replicative aging and may reach senescence. While the secretory phenotype of senescent fibroblasts is well studied, little is known about changes in the fibroblasts biophysical phenotype. Therefore, we compare biophysical properties of young versus proliferatively aged primary fibroblasts via fluorescence and traction force microscopy, single-cell atomic force spectroscopy, microfluidics, and microrheology of the cytoskeleton. Results show senescent fibroblasts have decreased cytoskeletal tension and myosin II regulatory light chain phosphorylation, in addition to significant loss of traction force. The alteration of cellular forces is harmful to extracellular matrix homeostasis, while decreased cytoskeletal tension can amplify epigenetic changes involved in senescence. Further exploration and detection of these mechanical phenomena provide possibilities for previously unexplored pharmaceutical targets against aging.
Summary
Dopamine offers the possibility to build polymer films on any substrates via an oxidative process. This redox process cannot only be used for self‐oxidation in air, but also in an ...electrochemical deposition process using cyclic voltammetry. Electropolymerization provides the opportunity to customize film thicknesses on surfaces detectable by X‐ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The topography and nanomechanical properties of the films have been characterized in liquid via AFM. Electrochemical quartz crystal microbalance (EQCM) allows the determination of the deposited polymer quantities during the polymerization process and the ultra‐thin films have been detected by attenuated total reflection Fourier transform infrared spectroscopy (ATR‐FTIR).
Predation and habitat deterioration are the main reasons for the strong decline of ground‐nesting farmland birds such as the grey partridge Perdix perdix in Europe. Grey partridge nests and ...incubating females are especially vulnerable to predation. We have previously demonstrated that predator activity is much lower inside flower blocks (agri‐environment schemes sown with a flower seed mix) than at their edges and that predator activity in flower blocks depends on the surrounding landscape. Here, we investigate whether these differences in predator activity translate into differences in grey partridge nest predation and assess predation patterns relative to landscape and nest site characteristics. We recorded the success of 56 nests of radio‐tagged grey partridges between 2009 and 2017 in an agricultural landscape in Central Germany. We used Bayesian logistic regression to analyse the effects of nest site and landscape characteristics on nest predation on a subset of 46 nests (21 nests successful, 25 predated). Distance to the edge of the nesting habitat was the most important predictor, reducing predation probability from 66.8% at the edge to 18.5% at 85.5 m. Predation probability decreased with increasing length of habitat borders, habitat diversity and the area of permanent grasslands and fallows. Predation probability was higher further from settlements and increased with increasing woodland area in the agricultural matrix. When considering linear landscape structures, nest predation patterns matched the patterns of predator activity from our previous studies. Results suggest that the distance to the edge of the nesting habitat is most important and that nest predation may be reduced by providing sufficiently broad nesting habitats. Nest predation may further be minimized by increasing habitat diversity and coverage of extensive vegetation types and by establishing conservation measures for grey partridges further away from woodlands. These measures may also benefit other ground‐nesting farmland birds.
High nest predation is a serious threat to grey partridges Perdix perdix and other ground‐nesting farmland birds. We analysed nest predation in radio‐tagged grey partridges and found that nest site size and location strongly influenced predation rates. Our results highlight the importance of large nesting habitats and confirm that a landscape‐based approach can help reduce predation risk.
Heparin-induced thrombocytopenia (HIT) is the most frequent drug-induced immune reaction affecting blood cells. Its antigen is formed when the chemokine platelet factor 4 (PF4) complexes with ...polyanions. By assessing polyanions of varying length and degree of sulfation using immunoassay and circular dichroism (CD)-spectroscopy, we show that PF4 structural changes resulting in antiparallel β-sheet content >30% make PF4/polyanion complexes antigenic. Further, we found that polyphosphates (polyP-55) induce antigenic changes on PF4, whereas fondaparinux does not. We provide a model suggesting that conformational changes exposing antigens on PF4/polyanion complexes occur in the hairpin involving AA 32-38, which form together with C-terminal AA (66-70) of the adjacent PF4 monomer a continuous patch on the PF4 tetramer surface, explaining why only tetrameric PF4 molecules express "HIT antigens". The correlation of antibody binding in immunoassays with PF4 structural changes provides the intriguing possibility that CD-spectroscopy could become the first antibody-independent, in vitro method to predict potential immunogenicity of drugs. CD-spectroscopy could identify compounds during preclinical drug development that induce PF4 structural changes correlated with antigenicity. The clinical relevance can then be specifically addressed during clinical trials. Whether these findings can be transferred to other endogenous proteins requires further studies.
Integrins are pivotal proteins in cell–cell adhesion, signaling and apoptosis. These properties render them attractive targets for drugs, especially those involved in cancer treatment. Recently, the ...structures of the extracellular domains of one of the integrin subtypes was solved with X‐ray crystallography in the free form as well as bound to a ligand. These structures in combination with NMR spectroscopic data, electron microscopy images, and molecular modeling provide deeper insight into the mechanism of integrin‐mediated signal transduction. The structures make structure‐based rational drug design possible and are certainly hallmarks in integrin research.
A milestone in integrin research was the determination of the structure of the extracellular domain of αvβ3 integrin both in the free and complexed forms (an integrin complex with a cyclic RGD peptide is shown). Calculations on the interactions of the transmembrane α and β helices, NMR studies of the intracellular domain, and electron microsopic investigations provide a new insight into the mode of action of the integrin as well as a plausible model for signal transduction through the cell membrane.
Processing of the amyloid precursor protein (APP) by β‐ and γ‐secretases leads to the generation of amyloid‐β (Aβ) peptides with varying lengths. Particularly Aβ42 contributes to cytotoxicity and ...amyloid accumulation in Alzheimer's disease (AD). However, the precise molecular mechanism of Aβ42 generation has remained unclear. Here, we show that an amino‐acid motif GxxxG within the APP transmembrane sequence (TMS) has regulatory impact on the Aβ species produced. In a neuronal cell system, mutations of glycine residues G29 and G33 of the GxxxG motif gradually attenuate the TMS dimerization strength, specifically reduce the formation of Aβ42, leave the level of Aβ40 unaffected, but increase Aβ38 and shorter Aβ species. We show that glycine residues G29 and G33 are part of a dimerization site within the TMS, but do not impair oligomerization of the APP ectodomain. We conclude that γ‐secretase cleavages of APP are intimately linked to the dimerization strength of the substrate TMS. The results demonstrate that dimerization of APP TMS is a risk factor for AD due to facilitating Aβ42 production.
Working memory is a core cognitive function and its deficits is one of the most common cognitive impairments. Reduced working memory capacity manifests as reduced accuracy in memory recall and ...prolonged speed of memory retrieval in older adults. Currently, the relationship between healthy older individuals' age-related changes in resting brain oscillations and their working memory capacity is not clear. Eyes-closed resting electroencephalogram (rEEG) is gaining momentum as a potential neuromarker of mild cognitive impairments. Wearable and wireless EEG headset measuring key electrophysiological brain signals during rest and a working memory task was utilized. This research's central hypothesis is that rEEG (e.g., eyes closed for 90 s) frequency and network features are surrogate markers for working memory capacity in healthy older adults. Forty-three older adults' memory performance (accuracy and reaction times), brain oscillations during rest, and inter-channel magnitude-squared coherence during rest were analyzed. We report that individuals with a lower memory retrieval accuracy showed significantly increased alpha and beta oscillations over the right parietal site. Yet, faster working memory retrieval was significantly correlated with increased delta and theta band powers over the left parietal sites. In addition, significantly increased coherence between the left parietal site and the right frontal area is correlated with the faster speed in memory retrieval. The frontal and parietal dynamics of resting EEG is associated with the "
" during working memory in healthy older adults. Our results suggest that rEEG brain oscillations at local and distant neural circuits are surrogates of working memory retrieval's accuracy and processing speed. Our current findings further indicate that rEEG frequency and coherence features recorded by wearable headsets and a brief resting and task protocol are potential biomarkers for working memory capacity. Additionally, wearable headsets are useful for fast screening of cognitive impairment risk.
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