Mechanochemical consequences of myopathy-linked mutations in Tpm2.2 on striated muscle contractility.

Tropomyosin (Tpm) is an actin-binding protein central to muscle contraction regulation. The Tpm sequence consists of periodic repeats corresponding to seven actin-binding sites, further divided in two functionally distinct halves. To clarify the importance of the first and second halves of the actin-binding periods in regulating the interaction of myosin with actin, we introduced hypercontractile mutations D20H, E181K located in the N-terminal halves of periods 1 and 5 and hypocontractile mutations E41K, N202K located in the C-terminal halves of periods 1 and 5 of the skeletal muscle Tpm isoform Tpm2.2. Wild-type and mutant Tpms displayed similar actin-binding properties, however, as revealed by FRET experiments, the hypercontractile mutations affected the binding geometry and orientation of Tpm2.2 on actin, causing a stimulation of myosin motor performance. Contrary, the hypocontractile mutations led to an inhibition of both, actin activation of the myosin ATPase and motor activity, that was more pronounced than with wild-type Tpm2.2. Single ATP turnover kinetic experiments indicate that the introduced mutations have opposite effects on product release kinetics. While the hypercontractile Tpm2.2 mutants accelerated product release, the hypocontractile mutants decelerated product release from myosin, thus having either an activating or inhibitory influence on myosin motor performance, which agrees with the muscle disease phenotypes caused by these mutations.

A Novel Variant in TPM3 Causing Muscle Weakness and Concomitant Hypercontractile Phenotype.

A novel variant of unknown significance c.8A > G (p.Glu3Gly) in TPM3 was detected in two unrelated families. TPM3 encodes the transcript variant Tpm3.12 (NM_152263.4), the tropomyosin isoform specifically expressed in slow skeletal muscle fibers. The patients presented with slowly progressive muscle weakness associated with Achilles tendon contractures of early childhood onset. Histopathology revealed features consistent with a nemaline rod myopathy. Biochemical in vitro assays performed with reconstituted thin filaments revealed defects in the assembly of the thin filament and regulation of actin-myosin interactions. The substitution p.Glu3Gly increased polymerization of Tpm3.12, but did not significantly change its affinity to actin alone. Affinity of Tpm3.12 to actin in the presence of troponin ± Ca2+ was decreased by the mutation, which was due to reduced interactions with troponin. Altered molecular interactions affected Ca2+-dependent regulation of the thin filament interactions with myosin, resulting in increased Ca2+ sensitivity and decreased relaxation of the actin-activated myosin ATPase activity. The hypercontractile molecular phenotype probably explains the distal joint contractions observed in the patients, but additional research is needed to explain the relatively mild severity of the contractures. The slowly progressive muscle weakness is most likely caused by the lack of relaxation and prolonged contractions which cause muscle wasting. This work provides evidence for the pathogenicity of the TPM3 c.8A > G variant, which allows for its classification as (likely) pathogenic.

Effects of chronic exposure to cadmium and copper on the proteome profile of hemolymph in false widow spider Steatoda grossa (Theridiidae).

The aim of this study was to evaluate the quantitative and qualitative changes in the proteome of the hemolymph of female Steatoda grossa spiders (Theridiidae) that were chronically exposed to cadmium and copper in food and were additionally immunostimulated (phorbol 12-myristate 13-acetate (PMA); bacterial suspensions: Staphylococcus aureus (G+), Pseudomonas fluorescens (G-). It was found that the expression of nearly 90 proteins was altered in cadmium-intoxicated spiders and more than 60 in copper-exposed individuals. Regardless of the type of metal used, these proteins were mainly overexpressed in the hemolymph of the exposed spiders. On the other hand, immunostimulation did not significantly change the number of proteins with altered expression in metal-intoxicated individuals. Hemocyanin (Hc) was found to be the most abundant of the proteins identified with altered expression. In copper-intoxicated spiders, immunostimulation increased the expression of A-, E-, F-, and G-chain-containing proteins, while in the case of cadmium-intoxicates spiders, it decreased the expression of E- and A-chain-containing Hc and increased the expression of G-chain-containing Hc. Regardless of the type of metal and immunostimulant used, there was an increase in the expression of actin. In addition, cadmium increased the expression of cullin, vimentin, and ceruloplasmin. The changes observed in the expression of hemolymph proteins indicate their protective function in S. grossa (Theridiidae) spiders under conditions of metal exposure.

Diclofenac Diminished the Unfolded Protein Response (UPR) Induced by Tunicamycin in Human Endothelial Cells.

Diclofenac belongs to the class of nonsteroidal anti-inflammatory drugs (NSAIDs), which are amongst the most frequently prescribed drugs to treat fever, pain and inflammation. Despite the presence of NSAIDs on the pharmaceutical market for several decades, epidemiological studies have shown new clinical applications of NSAIDs, and new mechanisms of their action were discovered. The unfolded protein response (UPR) activated under endoplasmic reticulum (ER) stress is involved in the pathophysiology of many diseases and may become a drug target, therefore, the study evaluated the effects of diclofenac on the tunicamycin-induced UPR pathways in endothelial cells. RT PCR analysis showed that diclofenac significantly inhibited activation of ER stress-responsive genes, i.e., CHOP/DITT3, GRP78/HSPA5 and DNAJB9. Additionally, the drug diminished the significant upregulation and release of the GRP78 protein, as evaluated using the ELISA assay, which was likely to be involved in the mechanism of the UPR activation resulting in apoptosis induction in endothelial cells. These results suggest the value of diclofenac as a factor capable of restoring the ER homeostasis in endothelial cells by diminishing the UPR.

The Importance of Endoplasmic Reticulum Stress as a Novel Antidepressant Drug Target and Its Potential Impact on CNS Disorders.

Many central nervous system (CNS) diseases, including major depressive disorder (MDD), are underpinned by the unfolded protein response (UPR) activated under endoplasmic reticulum (ER) stress. New, more efficient, therapeutic options for MDD are needed to avoid adverse effects and drug resistance. Therefore, the aim of the work was to determine whether UPR signalling pathway activation in astrocytes may serve as a novel target for antidepressant drugs. Among the tested antidepressants (escitalopram, amitriptyline, S-ketamine and R-ketamine), only S-ketamine, and to a lesser extent R-ketamine, induced the expression of most ER stress-responsive genes in astrocytes. Furthermore, cell viability and apoptosis measuring assays showed that (R-)S-ketamine did not affect cell survival under ER stress. Under normal conditions, S-ketamine played the key role in increasing the release of brain-derived neurotrophic factor (BDNF), indicating that the drug has a complex mechanism of action in astrocytes, which may contribute to its therapeutic effects. Our findings are the first to shed light on the relationship between old astrocyte specifically induced substance (OASIS) stabilized by ER stress and (R-)S-ketamine; however, the possible involvement of OASIS in the mechanism of therapeutic ketamine action requires further study.

Osteosarcoma cells in early and late stages as cancer in vitro progression model for assessing the responsiveness of cells to silver nanoparticles.

Understanding of biology of osteosarcoma malignant progression is indispensable for enhancement of conventional chemotherapy by the use of silver nanoparticles (AgNPs). We presented an in vitro model of cancer progression closely resembling processes occurring in vivo in terms of protein profile. A comparison of cytotoxic and genotoxic potential of AgNPs in Saos-2 cells in early stages of cancerous progression (early passages) with the cells in advanced stages (late passages) demonstrated significantly reduced responsiveness of the late passage cells to nanoparticles toxicity. It was also confirmed by proteome analysis as we identified considerably higher number of differentially expressed proteins in Saos-2 cells in early passages compared to the late passage cells. Our studies showed that the ability of AgNPs as potential drug carriers to deliver a medication and/or to evoke toxic effects might be significantly diminished in advanced stages of cancer progression.

Dysfunction of endothelial cells exposed to nanomaterials assessed by atomic force spectroscopy.

The study of the impact of nanomaterials on endothelial cell elasticity with the atomic force spectroscopy (AFS) can be a significant model for assessing nanomaterials toxic effects in vitro. The mechanical properties of cells exposed to nanostructures can provide information not only about cellular nano and micro-structure, but also about cell physiology. The toxicity of nanostructures is an important issue which must be carefully considered when the optimal nanomaterial is defined. There are no universal properties characterizing such a nanomaterial, i.e. depending on the intended use, the requirements can be diverse. For example, for biomedical use a nanomaterial should not negatively affect the cells or should cause the expected therapeutic or diagnostic effects in justified cases. The present study was devoted to the effects of silver nanoparticles (SNPs), multi-walled carbon nanotubes (MWCNTs) and poly(amidoamine) (PAMAM) dendrimers of 4th generation on functioning of endothelial cells. Immortalized endothelial cells were exposed for 24 h to the tested nanomaterials used in concentrations reducing cellular viability to the levels of 90 % and 75 %. The innovative nature of our work is the comparison of cell elasticity performed with various AFS probes, which enabled detection of local and global elasticity alteration caused by the nanostructures. The obtained results demonstrated changes in elasticity of endothelial cell induced by the nanostructures, which were closely correlated with the level of cellular viability, forming of actin stress fibres and elevated levels of reactive oxygen species. Trend of changes in local and global elasticity of cells exposed to nanostructures was similar, but the magnitude of the response was dependent on the selected probe. SNPs and MWCNTs evoked cells stiffening, which was correlated with changes in production levels of reactive oxygen species (ROS) and the cytoskeletal alteration. Softening of cells exposed to PAMAM dendrimers correlated with increased number of apoptotic cells and ROS production levels. Based on the obtained results we conclude, that the structure and the type of nanostructure (nanoparticle) is essential for their localization inside the cells and for the toxic effect on the endothelial cells.

Comprehensive Biological Evaluation of Biomaterials Used in Spinal and Orthopedic Surgery.

Biological acceptance is one of the most important aspects of a biomaterial and forms the basis for its clinical use. The aim of this study was a comprehensive biological evaluation (cytotoxicity test, bacterial colonization test, blood platelets adhesion test and transcriptome and proteome analysis of Saos-2 cells after contact with surface of the biomaterial) of biomaterials used in spinal and orthopedic surgery, namely, Ti6Al4V ELI (Extra Low Interstitials), its modified version obtained as a result of melting by electron beam technology (Ti6Al4V ELI-EBT), polyether ether ketone (PEEK) and polished medical steel American Iron and Steel Institute (AISI) 316L (the reference material). Biological tests were carried out using the osteoblasts-like cells (Saos-2, ATCC HTB-85) and bacteria Escherichia coli (DH5α). Results showed lack of cytotoxicity of all materials and the surfaces of both Ti6Al4V ELI and PEEK exhibit a significantly higher resistance to colonization with E. coli cells, while the more porous surface of the same titanium alloy produced by electron beam technology (EBT) is more susceptible to microbial colonization than the control surface of polished medical steel. None of the tested materials showed high toxicity in relation to E. coli cells. Susceptibility to platelet adhesion was very high for polished medical steel AISI 316L, whilst much lower for the other biomaterials and can be ranked from the lowest to the highest as follows: PEEK < Ti6Al4V ELI < Ti6Al4V ELI-EBT. The number of expressed genes in Saos-2 cells exposed to contact with the examined biomaterials reached 9463 genes in total (ranging from 8455 genes expressed in cells exposed to ELI to 9160 genes in cells exposed to PEEK). Whereas the number of differentially expressed proteins detected on two-dimensional electrophoresis gels in Saos-2 cells after contact with the examined biomaterials was 141 for PEEK, 223 for Ti6Al4V ELI and 133 for Ti6Al4V ELI-EBT. Finally, 14 proteins with altered expression were identified by mass spectrometry. In conclusion, none of the tested biomaterials showed unsatisfactory levels of cytotoxicity. The gene and protein expression analysis, that represents a completely new approach towards characterization of these biomaterials, showed that the polymer PEEK causes much more intense changes in gene and protein expression and thus influences cell metabolism.

Designing laser-modified surface structures on titanium alloy custom medical implants using a hybrid manufacturing technology.

The hybrid technology combines an efficient material-removal process and implant surface treatment by the laser reducing time of manufacture process compared to currently used machining technologies. It also permits precise structuring of the implant material surface. Six structures of the Ti6Al4V ELI surface were designed and studied how the structure topography prepared with the hybrid technology affected the Escherichia coli adhesion to the surface and viability, as well as the growth, adhesion, and viability of human osteogenic Saos-2 cells cultured on the investigated surfaces. Results have confirmed that the microtopography of medical titanium alloy plays a beneficial role in bacterial adhesion and viability (number of bacteria found on reference surface: [5.9 ± 0.44] × 106 CFU/ml, sample no. 3: [8.8 ± 0.93] × 104 CFU/ml, and sample no. 5: [1.2 ± 0.23] × 107 CFU/ml; CFU - Colony Forming Unit). All tested structured surfaces enabled good cell attachment and proliferation of Saos-2 cells (viability of Saos-2 cells [% of control] for reference surface: 81.93%; sample no. 3: 75% and sample no. 5: 100%). Transcriptome analysis of genes commonly expressed in the process of osseointegration demonstrated that the use of hybrid technology allows designing structures that enhance osseointegration but it should be coupled with other methods of preventing bacterial growth, or with a different strategy to limit microbial colonization with the satisfactory osseointegration potential.

Simultaneous transcriptome and proteome analysis of EA.hy926 cells under stress conditions induced by nanomaterials.

Today, the extensive and constantly growing number of applications in the field of nanotechnology poses a lot of questions about the potential toxicity of nanomaterials (NMs) toward cells of different origins. In our work we employed the tools of molecular biology to evaluate changes that occur in human endothelial cells at the transcriptomic and proteomic level, following 24 h of exposure to three different classes of NMs. Using microarray technology, we demonstrated that 24 h of exposure to silver nanoparticles (SNPs), multiwalled carbon nanotubes (MWCNTs) and polyamidoamine dendrimers (PAMAMs) leads to changes in 299, 1271, and 431 genes, respectively, influencing specific molecular pathways. The 2D-DIGE and mass spectrometry analysis revealed that differentially expressed proteins were involved in numerous cellular processes, for example, cytoskeletal reorganization, cell growth and proliferation, or response to stress. Both, transcriptome and proteome alterations indicate reorganization of mechanism regulating cell functioning. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1024-1034, 2019.

Toxicity of silver nanoparticles, multiwalled carbon nanotubes, and dendrimers assessed with multicellular organism Caenorhabditis elegans.

Nematode Caenorhabditis elegans (C. elegans) was used to investigate the impact of silver nanoparticles (SNP), multiwalled carbon nanotubes (MWCNT), and polyamidoamine dendrimers (PAMAM) used in concentration of 1010 particle/mL. Population-based observations and gene expression analysis were employed in this study. SNP and PAMAM caused decrease in the number of live nematodes and their body length, but MWCNT did not affect the population of nematodes. Gene expression analysis revealed significant changes caused by the presence of all studied nanomaterials, and the results strongly suggest a specific metabolic response of the nematode organism to exposure to various nanomaterials. It was shown that C. elegans is a very sensitive organism capable to respond specifically to the exposure to some nanomaterials and therefore could be considered as a possible biosensor for early warning of presence of some nanoparticles.

Human brain endothelial barrier cells are distinctly less vulnerable to silver nanoparticles toxicity than human blood vessel cells: A cell-specific mechanism of the brain barrier?

The blood-brain barrier (BBB) constitutes a distinctive and tightly regulated interface between the brain and the peripheral circulation. The objective of studies was to compare responses of human endothelial cells representing the model of blood vessels - EA.hy926 and HUVEC cells and the model of the brain endothelial barrier - HBEC5i cells to silver nanoparticles (SNPs). A contact of SNPs with endothelial cells resulted in a formation of SNP agglomerates. Consequently, the SNPs uptake by endothelial cells affected cell viability and membrane integrity however observed responses were different. Brain endothelial barrier HBEC5i cells were much less vulnerable to SNPs toxicity comparing to EA.hy926 and HUVEC cells. It can be ascribed to the presence of specialized cellular components of the brain barrier, protecting HBEC5i cells against toxic SNPs. Fundamental understanding of SNPs inducing the BBB dysfunction may initiate engineering novel SNPs which are safe for the BBB and thereby safe for the brain.