Forces of RBC interaction with single endothelial cells in stationary conditions: Measurements with laser tweezers

Red blood cells (RBCs) are able to interact and communicate with endothelial cells (ECs). Under some pathological or even normal conditions, the adhesion of RBCs to the endothelium can be observed. Presently, the mechanisms and many aspects of the interaction between RBCs and ECs are not fully understood. In this work, we considered the interaction of single RBCs with single ECs in vitro aiming to quantitatively determine the force of this interaction using laser tweezers. Measurements were performed under different concentrations of proaggregant macromolecules and in the presence or absence of tumor necrosis factor (TNF-[Formula: see text]) activating the ECs. We have shown that the strength of interaction depends on the concentration of fibrinogen or dextran proaggregant macromolecules in the environment. A nonlinear increase in the force of cells interaction (from 0.4 pN to 21 pN) was observed along with an increase in the fibrinogen concentration (from 3[Formula: see text]mg/mL to 9[Formula: see text]mg/mL) in blood plasma, as well as with the addition of dextran macromolecules (from 10[Formula: see text]mg/mL to 60[Formula: see text]mg/mL). Dextran with a higher molecular mass (500[Formula: see text]kDa) enhances the adhesion of RBCs to ECs greater compared to the dextran with a lower molecular mass (70[Formula: see text]kDa). With the preliminary activation of ECs with TNF-[Formula: see text], the force of interaction increases. Also, the adhesion of echinocytes to EC compared to discocytes is significantly higher. These results may help to better understand the process of interaction between RBCs and ECs.

Synthetic NAC 71-82 Peptides Designed to Produce Fibrils with Different Protofilament Interface Contacts

Alpha-synucleinopathies are featured by fibrillar inclusions in brain cells. Although α-synuclein fibrils display structural diversity, the origin of this diversity is not fully understood. We used molecular dynamics simulations to design synthetic peptides, based on the NAC 71-82 amino acid fragment of α-synuclein, that govern protofilament contacts and generation of twisted fibrillar polymorphs. Four peptides with structures based on either single or double fragments and capped or non-capped ends were selected for further analysis. We determined the fibrillar yield and the structures from these peptides found in the solution after fibrillisation using protein concentration determination assay and circular dichroism spectroscopy. In addition, we characterised secondary structures formed by individual fibrillar complexes using laser-tweezers Raman spectroscopy. Results suggest less mature fibrils, based on the lower relative β-sheet content for double- than single-fragment peptide fibrils. We confirmed this structural difference by TEM analysis which revealed, in addition to short protofibrils, more elongated, twisted and rod-like fibril structures in non-capped and capped double-fragment peptide systems, respectively. Finally, time-correlated single-photon counting demonstrated a difference in the Thioflavin T fluorescence lifetime profiles upon fibril binding. It could be proposed that this difference originated from morphological differences in the fibril samples. Altogether, these results highlight the potential of using peptide models for the generation of fibrils that share morphological features relevant for disease, e.g., twisted and rod-like polymorphs.

EXPRESS: Characterization of Drug Resistance in Chronic Myeloid Leukemia Cells Based on Laser Tweezers Raman Spectroscopy

Multidrug resistance (MDR) is highly associated with poor prognosis of chronic myeloid leukemia (CML). This work aims to explore whether the laser tweezers Raman spectroscopy (LTRS) could be practical in separating adriamycin (ADR) resistance CML cells K562/ADR from its parental cells K562, and to explore the potential mechanisms. Detection of LTRS initially reflected the spectral differences caused by chemoresistance including bands assigned to carbohydrates, amino acid, protein, lipids and nucleic acid. In addition, principal components analysis (PCA) as well as the classification and regression trees (CRT) algorithms showed that the specificity and sensitivity were above 90%. Moreover, the band data-based CRT model and receiver operating characteristic (ROC) curve further determined some important bands and band intensity ratios to be reliable indexes in discriminating K562 chemoresistance status. Finally, we highlighted three metabolism pathways correlated with chemoresistance. This work demonstrates that the label-free LTRS analysis combined with multivariate statistical analyses have great potential to be a novel analytical strategy at the single-cell level for rapid evaluation the chemoresistance status of K562 cells.

Effect of Red Blood Cell Aging In Vivo on Their Aggregation Properties In Vitro: Measurements with Laser Tweezers

Red blood cell (RBC) aggregation highly influences hemorheology and blood microcirculation in the human body. The aggregation properties of RBCs can vary due to numerous factors, including RBC age. The aim of this work was to estimate in vitro the differences in the RBC aggregation properties of different RBC age populations in single-cell experiments using laser tweezers. RBCs from five healthy volunteers were separated into four subpopulations by Percoll density gradient centrifugation. Each subpopulation of the RBC was separately resuspended in autologous plasma or dextran 70 kDa (50 mg/mL). The aggregation force between the single cells was measured with holographic laser tweezers. The obtained data demonstrated an enhancement of RBC aggregation force in doublets with age: the older the cells, the higher the aggregation force. The obtained data revealed the differences between the aggregation and aggregability of RBC in dependence of the RBC in vivo age.

Disinfection chemicals mode of action on the bacterial spore structure and their Raman spectra

Contamination of toxic spore-forming bacteria is problematic since spores can survive a plethora of disinfection chemicals. It is also problematic to rapidly detect if the disinfection chemical was active, leaving spores dead. Robust decontamination strategies, as well as reliable detection methods to identify dead from viable spores, are thus critical. Vibrational detection methods such as Raman spectroscopy has been suggested for rapid diagnostics and differentiation of live and dead spores. We investigate in this work, using laser tweezers Raman spectroscopy, the changes in Raman spectra of Bacillus thuringiensis spores treated with sporicidal agents such as chlorine dioxide, peracetic acid, and sodium hypochlorite. We also imaged treated spores using SEM and TEM to verify if any changes to the spore structure can be correlated to the Raman spectra. We found that chlorine dioxide did not change the Raman spectrum or the spore structure; peracetic acid shows a time-dependent decrease in the characteristic DNA/DPA peaks and ∼20 % of the spores were degraded and collapsed; spores treated with sodium hypochlorite show an abrupt drop in DNA and DPA peaks within 20 minutes all though the spore structure was overall intact, however, the exosporium layer was reduced. Structural changes appeared over several minutes, compared to the inactivation time of the spores, which is less than a minute. We conclude that vibrational spectroscopy provides powerful means to detect changes in spores but it might be problematic to identify if spores are live or dead after a decontamination procedure.