Search

The concentration of extracellular vesicles (EVs) is an essential attribute of biofluids and EV preparations. EV concentration in body fluids was correlated with health status. The abundance of EV secreted by cultured cells into growth medium is vital in signaling studies, tissue and disease models, and biomanufacturing of acellular therapeutic secretome. A limited number of physical principles sensitive to EV concertation have been discovered so far. Particle-by-particle counting methods enumerate individual particles scattering light, modulating the Coulter current, or appearing in EM images. The available ensemble techniques in current use rely on the concentration-dependent signal intensity, as in the case of ELISA. In this study, we propose for the first-time the ensemble-based characterization of EV concentration by dynamic surface tension (DST) probe and demonstrate its implementation. We show that DST measurements agree with the widely used NTA measurements of EV concertation. The proposed method is low-cost and requires only basic laboratory equipment for implementation.

Gas-liquid interfaces are reaching a particular interest in biomedicine. Microbubbles, ultrasound contrast agents of clinical routine, gained increasing attention as theranostic platforms due to the preserved acoustic response, drug conjugation capabilities, and applicability in biological barrier opening. A combination of microbubbles and photodynamic therapy agents can enhance the photodynamic effect, yet the evaluation of agent conjugation on microbubble stabilization and photodynamic effect is needed. Hence, two commercially available phthalocyanine photosensitizers - Holosens® (ZnPc) and Photosens® (AlPc) - were coupled with bovine serum albumin before microbubble synthesis. We demonstrated an albumin: phthalocyanine ratio of 1:1 and covalent attachment for ZnPc, a ratio of 1:3 with electrostatic binding for AlPc. Submicron-sized microbubbles (air- and SF

A novel asymmetric depth filtration (DF) approach for isolation of extracellular vesicles (EVs) from biological fluids is presented, and its performance is compared with established methods. The developed workflow is simple, inexpensive, and relatively fast. Compared with ultracentrifugation and size-exclusion chromatography, the developed method isolates EVs with higher purity and yield. Only standard laboratory equipment is needed for its implementation, which makes it suitable for low-resource locations. The described implementation of the method is suitable for EV isolation from small biological samples in diagnostic and treatment guidance applications. Following the scale-up routes adopted in the biomanufacturing of therapeutics, which routinely rely on DF as one of the product purification steps, the developed method may be scaled up to harvesting therapeutic EVs from large volumes of growth medium.

Simple Summary For the first time, we demonstrated that the significant decrease in p63/p73 expression together with the absence of functional p53 could underlie an increase in the fraction of polyploid cells, transformation rates, and the glycolytic NAD(P)H production in multifraction X-ray radiation exposure (MFR)-surviving cancer cells, providing conditions for radioresistance associated with epithelial–mesenchymal transition (EMT)-like process activation. During radiation therapy (RT), the treatment dose, fractionation, and dose limits for organs at risk (OARs) do not change between patients and are still prescribed mainly based on the Tumor, Node, Metastasis (TNM) stage, performance status, and comorbidities, taking no account of the tumor biology. Our data once again emphasize that non-small cell lung cancer (NSCLC) therapy approaches should become more personalized according to RT regimen, tumor histology, and molecular status of critical proteins. Abstract Radiotherapy is a primary treatment modality for patients with unresectable non-small cell lung cancer (NSCLC). Tumor heterogeneity still poses the central question of cancer radioresistance, whether the presence of a particular cell population inside a tumor undergoing a selective outgrowth during radio- and chemotherapy give rise to metastasis and tumor recurrence. In this study, we examined the impact of two different multifraction X-ray radiation exposure (MFR) regimens, fraction dose escalation (FDE) in the split course and the conventional hypofractionation (HF), on the phenotypic and molecular signatures of four MFR-surviving NSCLC cell sublines derived from parental A549 (p53 wild-type) and H1299 (p53-null) cells, namely A549FR/A549HR, H1299FR/H1299HR cells. We demonstrate that sublines surviving different MFR regimens in a total dose of 60 Gy significantly diverge in their molecular traits related to irradiation regimen and p53 status. The observed changes regarding radiosensitivity, transformation, proliferation, metabolic activity, partial epithelial-to-mesenchymal transition (EMT) program activation and 1D confined migratory behavior (wound healing). For the first time, we demonstrated that MFR exposure led to the significant decrease in the expression of p63 and p73, the p53-family members, in p53null cells, which correlated with the increase in cell polyploidy. We could not find significant differences in FRA1 expression between parental cells and their sublines that survived after any MFR regimen regardless of p53 status. In our study, the FDE regimen probably causes partial EMT program activation in MFR-survived NSCLC cells through either Vimentin upregulation in p53null or an aberrant N-cadherin upregulation in p53wt cells. The HF regimen likely less influences the EMT activation irrespectively of the p53 status of MFR-survived NSCLC cells. Our data highlight that both MFR regimens caused overall higher cell transformation of p53null H1299FR and H1299HR cells than their parental H1299 cells. Moreover, our results indicate that the FDE regimen raised the radioresistance and transformation of MFR-surviving NSCLC cells irrespectively of their p53 status, though the HF regimen demonstrated a similar effect on p53null NSCLC cells only. Our data once again emphasize that NSCLC therapy approaches should become more personalized according to radiation therapy (RT) regimen, tumor histology, and molecular status of critical proteins.

Ionizing radiation (IR) is used for patients diagnosed with unresectable non-small cell lung cancer (NSCLC). However, radiotherapy remains largely palliative due to the survival of specific cell subpopulations. In the present study, the sublines of NSCLC cells, A549IR (p53wt) and H1299IR (p53null) survived multifraction X-ray radiation exposure (MFR) at a total dose of 60 Gy were investigated three weeks after the MFR course. We compared radiosensitivity (colony formation), expression of epithelial-mesenchymal transition (EMT) markers, migration activity, autophagy, and HR-dependent DNA double-strand break (DSB) repair in the bulk and entire CD44high/CD166high CSC-like populations of both parental and MFR survived NSCLC cells. We demonstrated that the p53 status affected: the pattern of expression of N-cadherin, E-cadherin, Vimentin, witnessing the appearance of EMT-like phenotype of MFR-surviving sublines, 1D confined migratory behavior (wound healing), the capability of an irradiated cell to continue to divide and form a colony of NSCLC cells before and after MFR, influencing the CD44/CD166 expression level in MFR-surviving NSCLC cells after additional single irradiation. Our data further emphasize the impact of p53 status on the decay of γH2AX foci and the associated efficacy of the DSB repair in NSCLC cells survived after MFR. We revealed that Rad51 protein might play a principal role in MFR-surviving of p53 null NSCLC cells promoting DNA DSB repair by homologous recombination (HR) pathway. The proportion of Rad51 + cells elevated in CD44high/CD166high population in MFR-surviving p53wt and p53null sublines and their parental cells. The p53wt ensures DNA-PK-mediated DSB repair for both parental and MFR-surviving cells irrespectively of a subsequent additional single irradiation. Whereas in the absence of p53, a dose-dependent increase of DNA-PK-mediated non-homologous end joining (NHEJ) occurred as an early post-irradiation response is more intensive in the CSC-like population MFR-surviving H1299IR, compared to their parental H1299 cells. Our study strictly observed a significantly higher content of LC3 + cells in the CD44high/CD166high populations of p53wt MFR-surviving cells, which enriched the CSC-like cells in contrast to their p53null counterparts. The additional 2 Gy and 5 Gy X-ray exposure leads to the dose-dependent increase in the proportion of LC3 + cells in CD44high/CD166high population of both parental p53wt and p53null, but not MFR-surviving NSCLC sublines. Our data indicated that autophagy is not necessarily associated with CSC-like cells’ radiosensitivity, emphasizing that careful assessment of other milestone processes (such as senescence and autophagy-p53-Zeb1 axis) of primary radiation responses may provide new potential targets modulated for therapeutic benefit through radiosensitizing cancer cells while rescuing normal tissue. Our findings also shed light on the intricate crosstalk between autophagy and the p53-related EMT, by which MFR-surviving cells might obtain an invasive phenotype and metastatic potential.

There has been growing interest in recent years in developing multifunctional materials for studying the structure interface in biological systems. In this regard, the multimodal systems, which possess activity in the near-infrared (NIR) region, become even more critical for the possibility of improving examined biotissue depth and, eventually, data analysis. Herein, we engineered bi-modal contrast agents by integrating carbon nanotubes (CNT) and gold nanoparticles (AuNP) around silica microspheres using the Layer-by-Layer self-assembly method. The experimental studies revealed that microspheres with CNT sandwiched between AuNP exhibit strong absorption in the visible and NIR regions and high optoacoustic contrast (OA, also called photoacoustics) and Raman scattering when illuminated with 532 nm and 785 nm lasers, respectively. The developed microspheres demonstrated amplification of the signal in the OA flow cytometry at the laser wavelength of 1064 nm. This finding was further validated with ex vivo brain tissue using a portable Raman spectrometer and imaging with the Raster-scanning OA mesoscopy technique. The obtained data suggest that the developed contrast agents can be promising in applications of localization OA tomography (LOT), OA flow cytometry, and multiplex SERS detection.

Radiation therapy is one of the main methods of treating patients with non-small cell lung cancer (NSCLC). However, the resistance of tumor cells to exposure remains the main factor that limits successful therapeutic outcome. To study the molecular/cellular mechanisms of increased resistance of NSCLC to ionizing radiation (IR) exposure, we compared A549 (p53 wild-type) and H1299 (p53-deficient) cells, the two NSCLC cell lines. Using fractionated X-ray irradiation of these cells at a total dose of 60 Gy, we obtained the survived populations and named them A549IR and H1299IR, respectively. Further characterization of these cells showed multiple alterations compared to parental NSCLC cells. The additional 2 Gy exposure led to significant changes in the kinetics of &gamma, H2AX and phosphorylated ataxia telangiectasia mutated (pATM) foci numbers in A549IR and H1299IR compared to parental NSCLC cells. Whereas A549, A549IR, and H1299 cells demonstrated clear two-component kinetics of DNA double-strand break (DSB) repair, H1299IR showed slower kinetics of &gamma, H2AX foci disappearance with the presence of around 50% of the foci 8 h post-IR. The character of H2AX phosphorylation in these cells was pATM-independent. A decrease of residual &gamma, H2AX/53BP1 foci number was observed in both A549IR and H1299IR compared to parental cells post-IR at extra doses of 2, 4, and 6 Gy. This process was accompanied with the changes in the proliferation, cell cycle, apoptosis, and the expression of ATP-binding cassette sub-family G member 2 (ABCG2, also designated as CDw338 and the breast cancer resistance protein (BCRP)) protein. Our study provides strong evidence that different DNA repair mechanisms are activated by multifraction radiotherapy (MFR), as well as single-dose IR, and that the enhanced cellular survival after MFR is reliant on both p53 and 53BP1 signaling along with non-homologous end-joining (NHEJ). Our results are of clinical significance as they can guide the choice of the most effective IR regimen by analyzing the expression status of the p53&ndash, 53BP1 pathway in tumors and thereby maximize therapeutic benefits for the patients while minimizing collateral damage to normal tissue.

A novel asymmetric depth filtration (DF) approach for isolation of extracellular vesicles (EVs) from biological fluids is presented, and its performance is compared with established methods. The developed workflow is simple, inexpensive, and relatively fast. Compared with ultracentrifugation and size-exclusion chromatography, the developed method isolates EVs with higher purity and yield. Only standard laboratory equipment is needed for its implementation, which makes it suitable for low-resource locations. The described implementation of the method is suitable for EV isolation from small biological samples in diagnostic and treatment guidance applications. Following the scale-up routes adopted in the biomanufacturing of therapeutics, which routinely rely on DF as one of the product purification steps, the developed method may be scaled up to harvesting therapeutic EVs from large volumes of growth medium.

Proteins can aggregate in response to stresses, including hyperosmotic shock. Formation and disassembly of aggregates is a relatively slow process. We describe a novel instant response of the cell to hyperosmosis, during which chaperones and other proteins form numerous foci with properties uncharacteristic of classical aggregates. These foci appeared/disappeared seconds after shock onset/removal, in close correlation with cell volume changes. Genome-wide and targeted testing revealed chaperones, metabolic enzymes, P-body components and amyloidogenic proteins in the foci. Most of these proteins can form large assemblies and for some, the assembled state was pre-requisite for participation in foci. A genome-wide screen failed to identify genes whose absence prevented foci participation by Hsp70. Shapes of and interconnections between foci, revealed by super-resolution microscopy, indicated that the foci were compressed between other entities. Based on our findings, we suggest a new model of cytosol architecture as a collection of numerous gel-like regions suspended in a liquid network. This network is reduced in volume in response to hyperosmosis and forms small pockets between the gel-like regions., Summary: We describe a novel cellular response to hyperosmotic shock – rapid reversible formation of foci by various proteins. Data on foci behavior suggest a novel ‘beads in liquid’ cytosolic structure.

Proteins can aggregate in response to stresses, including hyperosmotic shock. Formation and disassembly of aggregates is a relatively slow process. We describe a novel instant response of the cell to hyperosmosis, during which chaperones and other proteins form numerous foci with properties uncharacteristic of classical aggregates. These foci appeared/disappeared seconds after shock onset/removal, in close correlation with cell volume changes. Genome-wide and targeted testing revealed chaperones, metabolic enzymes, P-body components and amyloidogenic proteins in the foci. Most of these proteins can form large assemblies and for some, the assembled state was pre-requisite for participation in foci. A genome-wide screen failed to identify genes whose absence prevented foci participation by Hsp70. Shapes of and interconnections between foci revealed by super-resolution microscopy indicated that the foci were compressed between other entities. Based on our findings, we propose a new model of the cytosol architecture as a collection of numerous of gel-like regions suspended in a liquid network. This network is reduced in volume in response to hyperosmosis and forms small pockets between the gel-like regions.

Indibulin (D-24851) derivatives with bisphosphonate fragment connected to the N1 atom of imidazole ring were synthesized by alkylation of (indolyl-3)methylglyoxylates with ethylenebisphosphonate. Biological evaluation of targeted compounds 4a–d using the phenotypic sea urchin embryo assay provided evidence that replacing of p-chlorobenzene ring in indibulin by bisphosphonate group did not eliminate antimitotic microtubule destabilizing activity. The most active molecule, tetraacid 5a, at physiological pH formed tetrasodium salt 6a with aqueous solubility value of at least 10 mg/mL. Molecule 5a was more potent in the sea urchin embryo assay than the parent indibulin. This compound also exhibited pronounced cytotoxicity against A549 lung carcinoma and A375 melanoma cell lines.

An enormous technological progress has resulted in an explosive growth in the amount of biological and chemical data that is typically multivariate and tangled in structure. Therefore, several computational approaches have mainly focused on dimensionality reduction and convenient representation of high-dimensional datasets to elucidate the relationships between the observed activity (or effect) and calculated parameters commonly expressed in terms of molecular descriptors. We have collected the experimental data available in patent and scientific publications as well as specific databases for various agrochemicals. The resulting dataset was then thoroughly analyzed using Kohonen-based self-organizing technique. The overall aim of the presented study is to investigate whether the developed in silico model can be applied to predict the agrochemical activity of small molecule compounds and, at the same time, to offer further insights into the distinctive features of different agrochemical categories. The preliminary external validation with several plant growth regulators demonstrated a relatively high prediction power (67%) of the constructed model. This study is, actually, the first example of a large-scale modeling in the field of agrochemistry.

Chemical genomics attracts considerable attention by offering crucial tools for plant biology to regulate plant growth and development. However, most chemical screens are time consuming and laborious, and require a high input of resources. Here we describe a broadly applicable method for the ultrarapid high-content phenotypic screening of small chemical compound libraries for new plant growth regulators. The assay is based on determination of pollen tube growth and can be completed in less than 8 h. Using this method, we identified novel inhibitors and modulators of plant growth and showed that compounds selected using a Nicotiana tabacum-based assay were biologically active in other plants, such as Arabidopsis thaliana.

A concise six-step protocol for the synthesis of isoflavone glaziovianin A (GVA) and its alkoxyphenyl derivatives 9 starting with readily available plant metabolites from dill and parsley seeds was developed. The reaction sequence involved an efficient conversion of the key intermediate epoxides 7 into the respective β-ketoaldehydes 8 followed by their Cu(I)-mediated cyclization into the target series 9. The biological activity of GVA and its derivatives was evaluated using a panel of seven human cancer cell lines and an in vivo sea urchin embryo assay. Both screening platforms confirmed the antimitotic effect of the parent GVA (9cg) and its alkoxy derivatives. Structure-activity relationship studies suggested that compounds 9cd and 9cf substituted with trimethoxy- and dillapiol-derived B-rings, respectively, were less active than the parent 9cg. Of the evaluated human cancer cell lines, the A375 melanoma cell line was the most sensitive to the tested molecules. Notably, the target compounds were not cytotoxic against human peripheral blood mononuclear cells up to 10 μM concentration. Phenotypic readouts from the sea urchin assay unequivocally suggest a direct microtubule-destabilizing effect of isoflavones 9cg, 9cd, and 9cf.