Quantitative and structural changes of blood platelet cytoskeleton proteins in multiple sclerosis (MS).

Epidemiological studies show that cardiovascular events related to platelet hyperactivity remain the leading causes of death among multiple sclerosis (MS) patients. Quantitative or structural changes of platelet cytoskeleton alter their morphology and function. Here, we demonstrated, for the first time, the structural changes in MS platelets that may be related to their hyperactivity. MS platelets were found to form large aggregates compared to control platelets. In contrast to the control, the images of overactivated, irregularly shaped MS platelets show changes in the cytoskeleton architecture, fragmented microtubule rings. Furthermore, MS platelets have long and numerous pseudopodia rich in actin filaments. We showed that MS platelets and megakaryocytes, overexpress ß1-tubulin and ß-actin mRNAs and proteins and have altered post-translational modification patterns. Moreover, we identified two previously undisclosed mutations in the gene encoding ß1-tubulin in MS. We propose that the demonstrated structural changes of platelet cytoskeleton enhance their ability to adhere, aggregate, and degranulate fueling the risk of adverse cardiovascular events in MS.

Activity of Lysosomal ABCC3, ABCC5 and ABCC10 is Responsible for Lysosomal Sequestration of Doxorubicin and Paclitaxel-Oregongreen488 in Paclitaxel-Resistant Cancer Cell Lines.

BACKGROUND/AIMS: Cancer cell multidrug resistance induced by paclitaxel contributes to the high failure rates of chemotherapy and relapse of the disease. Several mechanisms have been described that underlie the observed resistance, including the overexpression of ABCB1 (P-glycoprotein), which represents an ATP-binding cassette (ABC) transmembrane protein, and its functional occurrence in lysosomal membranes is linked to drug accumulation in these organelles. METHODS: Using clinically-relevant models of paclitaxel-resistant triple-negative breast cancer and non-small cell lung cancer cell lines, we provide evidence for the role of ABCC subfamily members in the lysosomal sequestration of drugs in multidrug resistant phenotypes. Proteins expression level and its cellular localisation was measured using Western Blot and confocal microscopy. Drug accumulation was analysed by confocal microscopy and flow cytometry. Drug cytotoxicity was tested using resasurin assay and anexin V propidium iodide staining. RESULTS: Regardless of the alteration in gene expression, paclitaxel induced the intracellular redistribution of ABCC3, ABCC5 and ABCC10 and their enrichment in lysosomes. The use of ABCC inhibitors and transient silencing of these three genes limited the accumulation of doxorubicin and paclitaxel-OregonGreen488 in lysosomes, while having little impact on the total drug level inside cells. The cancer cells were also sensitized to various structurally unrelated chemotherapeutics of differing acidity. CONCLUSION: The results suggest that lysosome membranes anchored ABCC proteins which remained functionally active and were capable to load chemotherapeutics into lysosomes in paclitaxel-resistant cancer cells. Therefore, targeting of lysosomal ABCC transporters may help to overcome paclitaxel-induced resistance by reducing the accumulation of drugs in lysosomes.

Ruthenium metallodendrimer against triple-negative breast cancer in mice.

Carbosilane metallodendrimers, based on the arene Ru(II) complex (CRD13) and integrated to imino-pyridine surface groups have been investigated as an anticancer agent in a mouse model with triple-negative breast cancer. The dendrimer entered into the cells efficiently, and exhibited selective toxicity for 4T1 cells. In vivo investigations proved that a local injection of CRD13 caused a reduction of tumour mass and was non-toxic. ICP analyses indicated that Ru(II) accumulated in all tested tissues with a greater content detected in the tumour.

Combination of Copper Metallodendrimers with Conventional Antitumor Drugs to Combat Cancer in In Vitro Models.

Copper carbosilane metallodendrimers containing chloride ligands and nitrate ligands were mixed with commercially available conventional anticancer drugs, doxorubicin, methotrexate and 5-fluorouracil, for a possible therapeutic system. To verify the hypothesis that copper metallodendrimers can form conjugates with anticancer drugs, their complexes were biophysically characterized using zeta potential and zeta size methods. Next, to confirm the existence of a synergetic effect of dendrimers and drugs, in vitro studies were performed. The combination therapy has been applied in two cancer cell lines: MCF-7 (human breast cancer cell line) and HepG2 (human liver carcinoma cell line). The doxorubicin (DOX), methotrexate (MTX) and 5-fluorouracil (5-FU) were more effective against cancer cells when conjugated with copper metallodendrimers. Such combination significantly decreased cancer cell viability when compared to noncomplexed drugs or dendrimers. The incubation of cells with drug/dendrimer complexes resulted in the increase of the reactive oxygen species (ROS) levels and the depolarization of mitochondrial membranes. Copper ions present in the dendrimer structures enhanced the anticancer properties of the whole nanosystem and improved drug effects, inducing both the apoptosis and necrosis of MCF-7 (human breast cancer cell line) and HepG2 (human liver carcinoma cell line) cancer cells.

Controlling the Spatiotemporal Release of Nerve Growth Factor by Chitosan/Polycaprolactone Conduits for Use in Peripheral Nerve Regeneration.

Tubular polymeric structures have been recognized in the treatment of peripheral nerves as comparable to autologous grafting. The best therapeutic outcomes are obtained with conduits releasing therapeutic molecules. In this study, a new approach for the incorporation of biologically active agent-loaded microspheres into the structure of chitosan/polycaprolactone conduits was developed. The support of a polycaprolactone helix formed by 3D melt extrusion was coated with dopamine in order to adsorb nerve growth factor-loaded microspheres. The complex analysis of the influence of process factors on the coverage efficiency of polycaprolactone helix by nerve grow factor-loaded microspheres was analyzed. Thus, the PCL helix characterized with the highest adsorption of microspheres was subjected to nerve growth factor release studies, and finally incorporated into chitosan hydrogel deposit through the process of electrophoretic deposition. It was demonstrated by chemical and physical tests that the chitosan/polycaprolactone conduit meets the requirements imposed on peripheral nerve implants, particularly mimicking mechanical properties of surrounding soft tissue. Moreover, the conduit may support regrowing nerves for a prolonged period, as its structure and integrity persist upon incubation in lysozyme-contained PBS solution up to 28 days at body temperature. In vitro cytocompatibility toward mHippoE-18 embryonic hippocampal cells of the chitosan/polycaprolactone conduit was proven. Most importantly, the developed conduits stimulate axonal growth and support monocyte activation, the latter is advantageous especially at early stages of nerve regeneration. It was demonstrated that, through the described approach for controlling spatiotemporal release of nerve growth factors, these biocompatible structures adjusted to the specific peripheral nerve injury case can be manufactured.

Dendronized Gold Nanoparticles as Carriers for gp160 (HIV-1) Peptides: Biophysical Insight into Complex Formation.

The unavailability of effective and safe human immunodeficiency virus (HIV) vaccines incites several approaches for development of the efficient antigen/adjuvant vaccination composite. In this study, three different dendronized gold nanoparticles (AuNPs 13-15) were investigated for a complexation ability with gp160 synthetic peptides derived from an HIV envelope. It has been shown that HIV peptides interacted with nanoparticles as evident from the changes in their secondary structures, restricted the mobility of the attached fluorescence dye, and enhanced peptide helicity confirmed by the fluorescence polarization and circular dichroism results. Transmission electron microscopy visualized complexes as cloud-like structures with attached nanoparticles. AuNP 13-15 nanoparticles bind negatively charged peptides depending on the number of functional groups; the fastest saturation and peptide retardation were observed for the most dendronized nanoparticle as indicated from dynamic light scattering, laser Doppler velocimetry, and agarose gel electrophoresis experiments. Dendronized gold nanoparticles can be considered one of the potential HIV peptide-based vaccination platforms.

The Effect of Photoperiod on Necrosis Development, Photosynthetic Efficiency and 'Green Islands' Formation in Brassica juncea Infected with Alternaria brassicicola.

The main goal of growing plants under various photoperiods is to optimize photosynthesis for using the effect of day length that often acts on plants in combination with biotic and/or abiotic stresses. In this study, Brassica juncea plants were grown under four different day-length regimes, namely., 8 h day/16 h night, 12 h day/12 h night, 16 h day/8 h night, and continuous light, and were infected with a necrotrophic fungus Alternaria brassicicola. The development of necroses on B. juncea leaves was strongly influenced by leaf position and day length. The largest necroses were formed on plants grown under a 16 h day/8 h night photoperiod at 72 h post-inoculation (hpi). The implemented day-length regimes had a great impact on leaf morphology in response to A. brassicicola infection. They also influenced the chlorophyll and carotenoid contents and photosynthesis efficiency. Both the 1st (the oldest) and 3rd infected leaves showed significantly higher minimal fluorescence (F0) compared to the control leaves. Significantly lower values of other investigated chlorophyll a fluorescence parameters, e.g., maximum quantum yield of photosystem II (Fv/Fm) and non-photochemical quenching (NPQ), were observed in both infected leaves compared to the control, especially at 72 hpi. The oldest infected leaf, of approximately 30% of the B. juncea plants, grown under long-day and continuous light conditions showed a 'green island' phenotype in the form of a green ring surrounding an area of necrosis at 48 hpi. This phenomenon was also reflected in changes in the chloroplast's ultrastructure and accelerated senescence (yellowing) in the form of expanding chlorosis. Further research should investigate the mechanism and physiological aspects of 'green islands' formation in this pathosystem.

Interaction of Cationic Carbosilane Dendrimers and Their siRNA Complexes with MCF-7 Cells.

The application of siRNA in gene therapy is mainly limited because of the problems with its transport into cells. Utilization of cationic dendrimers as siRNA carriers seems to be a promising solution in overcoming these issues, due to their positive charge and ability to penetrate cell membranes. The following two types of carbosilane dendrimers were examined: CBD-1 and CBD-2. Dendrimers were complexed with pro-apoptotic siRNA (Mcl-1 and Bcl-2) and the complexes were characterized by measuring their zeta potential, circular dichroism and fluorescence of ethidium bromide associated with dendrimers. CBD-2/siRNA complexes were also examined by agarose gel electrophoresis. Both dendrimers form complexes with siRNA. Moreover, the cellular uptake and influence on the cell viability of the dendrimers and dendriplexes were evaluated using microscopic methods and XTT assay on MCF-7 cells. Microscopy showed that both dendrimers can transport siRNA into cells; however, a cytotoxicity assay showed differences in the toxicity of these dendrimers.

The Effect of Pore Size Distribution and l-Lysine Modified Apatite Whiskers (HAP) on Osteoblasts Response in PLLA/HAP Foam Scaffolds Obtained in the Thermally Induced Phase Separation Process.

In this research, we prepared foam scaffolds based on poly(l-lactide) (PLLA) and apatite whiskers (HAP) using thermally induced phase separation technique supported by the salt leaching process (TIPS-SL). Using sodium chloride having a size of (a) 150-315 µm, (b) 315-400 µm, and (c) 500-600 µm, three types of foams with different pore sizes have been obtained. Internal structure of the obtained materials has been investigated using SEM as well as µCT. The materials have been studied by means of porosity, density, and compression tests. As the most promising, the composite prepared with salt size of 500-600 µm was prepared also with the l-lysine modified apatite. The osteoblast hFOB 1.19 cell response for the scaffolds was also investigated by means of cell viability, proliferation, adhesion/penetration, and biomineralization. Direct contact cytotoxicity assay showed the cytocompatibility of the scaffolds. All types of foam scaffolds containing HAP whiskers, regardless the pore size or l-lysine modification induced significant stimulatory effect on the cal-cium deposits formation in osteoblasts. The PLLA/HAP scaffolds modified with l-lysine stimulated hFOB 1.19 osteoblasts proliferation. Compared to the scaffolds with smaller pores (150-315 µm and 315-400 µm), the PLLA/HAP foams with large pores (500-600 µm) promoted more effective ad-hesion of osteoblasts to the surface of the biomaterial.

A Multiparametric Study of Internalization of Fullerenol C60(OH)36 Nanoparticles into Peripheral Blood Mononuclear Cells: Cytotoxicity in Oxidative Stress Induced by Ionizing Radiation.

The aim of this study was to investigate the uptake and accumulation of fullerenol C60(OH)36 into peripheral blood mononuclear cells (PBMCs). Some additional studies were also performed: measurement of fullerenol nanoparticle size, zeta potential, and the influence of fullerenol on the ionizing radiation-induced damage to PMBCs. Fullerenol C60(OH)36 demonstrated an ability to accumulate in PBMCs. The accumulation of fullerenol in those cells did not have a significant effect on cell survival, nor on the distribution of phosphatidylserine in the plasma membrane. However, fullerenol-induced depolarization of the mitochondrial membrane proportional to the compound level in the medium was observed. Results also indicated that increased fullerenol level in the medium was associated with its enhanced transport into cells, corresponding to its influence on the mitochondrial membrane. The obtained results clearly showed the ability of C60(OH)36 to enter cells and its effect on PBMC mitochondrial membrane potential. However, we did not observe radioprotective properties of fullerenol under the conditions used in our study.

Ruthenium Dendrimers against Human Lymphoblastic Leukemia 1301 Cells.

Ruthenium atoms located in the surfaces of carbosilane dendrimers markedly increase their anti-tumor properties. Carbosilane dendrimers have been widely studied as carriers of drugs and genes owing to such characteristic features as monodispersity, stability, and multivalence. The presence of ruthenium in the dendrimer structure enhances their successful use in anti-cancer therapy. In this paper, the activity of dendrimers of generation 1 and 2 against 1301 cells was evaluated using Transmission Electron Microscopy, comet assay and Real Time PCR techniques. Additionally, the level of reactive oxygen species (ROS) and changes of mitochondrial potential values were assessed. The results of the present study show that ruthenium dendrimers significantly decrease the viability of leukemia cells (1301) but show low toxicity to non-cancer cells (peripheral blood mononuclear cells-PBMCs). The in vitro test results indicate that the dendrimers injure the 1301 leukemia cells via the apoptosis pathway.

Dendrimers complexed with HIV-1 peptides interact with liposomes and lipid monolayers.

AIMS: We have investigated the effect of surface charge of model lipid membranes on their interactions with dendriplexes formed by HIV-derived peptides and 2 types of positively charged carbosilane dendrimers (CBD). METHODS: Interaction of dendriplexes with lipid membranes was measured by fluorescence anisotropy, dynamic light scattering and Langmuir-Blodgett techniques. The morphology of the complexes was examined by transmission electron microscopy. RESULTS: All dendriplexes independent of the type of peptide interacted with model lipid membranes. Negatively charged vesicles composed of a mixture of DMPC/DPPG interacted more strongly, and it was accompanied by an increase in anisotropy of the fluorescent probe localized in polar domain of lipid bilayers. There was also an increase in surface pressure of the lipid monolayers. Mixing negatively charged liposomes with dendriplexes increased liposome size and made their surface charges more positive. CONCLUSIONS: HIV-peptide/dendrimer complexes interact with model lipid membranes depending on their surface charge. Carbosilane dendrimers can be useful as non-viral carriers for delivering HIV-peptides into cells.

Physiological and antioxidant responses of two accessions of Arabidopsis thaliana in different light and temperature conditions.

During their lifetime, plants need to adapt to a changing environment, including light and temperature. To understand how these factors influence plant growth, we investigated the physiological and antioxidant responses of two Arabidopsis accessions, Shahdara (Sha) from the Shahdara valley (Tajikistan, Central Asia) in a mountainous area and Lovvik-5 (Lov-5) from northern Sweden to different light and temperature conditions. These accessions originate from different latitudes and have different life strategies, both of which are known to be influenced by light and temperature. We showed that both accessions grew better in high-light and at a lower temperature (16°C) than in low light and at 23°C. Interestingly, Sha had a lower chlorophyll content but more efficient non-photochemical quenching than Lov-5. Sha, also showed a higher expression of vitamin E biosynthetic genes. We did not observe any difference in the antioxidant prenyllipid level under these conditions. Our results suggest that the mechanisms that keep the plastoquinone (PQ)-pool in more oxidized state could play a role in the adaptation of these accessions to their local climatic conditions.

Human lung vascular endothelium may limit infection with HRV16 via IFN-ß-dependent mechanisms.

Human rhinovirus 16 (HRV16) may induce inflammatory and antiviral responses in the human lung vascular endothelium (ECs) and impair its barrier functions after infection. However, ECs may regain barrier and metabolic functions. Mechanisms of limitation of HRV16 infection in the lung vascular endothelium are unknown. Human lung vascular endothelium (HMVEC-L) was infected with HRV16. IFN-ß, OAS-1, and PKR expression was assessed by real-time PCR, flow cytometry, and confocal microscope. To prove the significance of IFN-ß in the limitation of HRV16 replication, HMVEC-Ls were preincubated with anti-IFN-ß Abs. To prove the involvement of OAS-1 and PKR in the IFN-dependent limitation of HRV16 replication, HMVEC-Ls were transfected with respective siRNA. HRV16 stimulated IFN-ß production and activated intracellular mechanisms of antiviral immunity based on OAS-1 and PKR activation. Blocking of IFN-ß contributed to the inhibition of intracellular mechanisms of antiviral immunity (OAS-1, PKR) and boosted replication of HRV16. Effective OAS-1 silencing by siRNA caused the increase of HRV16 copy numbers after HRV16 infection. siRNA upregulated the other genes related to the antiviral response. The infected lung vascular endothelium may limit the HRV16 infection. This limitation may be associated with the induction of IFN-ß-dependent intracellular mechanisms based on OAS-1 and PKR activity.

Photosynthesis governed by nanoparticulate titanium dioxide. The Pisum sativum L. case study.

Wide availability of anthropogenic TiO2 nanoparticles facilitates their penetration into environment and prompts interactions with plants. They alter plants growth and change their nutritional status. In particular, metabolic processes are affected. In this work the effect of nanometric TiO2 on photosynthesis efficiency in green pea (Pisum sativum L.) was studied. Hydroponic cultivations with three Ti levels (10; 50 and 100 mg L-1) were applied. At all concentrations nanoparticles penetrated into plant tissues and were detected by the single particle ICP-MS/MS method. Nanoparticles altered the CO2 assimilation rate and gas exchange parameters (i.e. transpiration, stomatal conductance, sub-stomatal CO2 concentration). The most pronounced effects were observed for Ti 50 mg L-1 cultivation where photosynthesis efficiency, transpiration and stomatal conductance were increased by 14.69%, 4.58% and 8.92%, respectively. They were further confirmed by high maximum ribulose 1,5-bisphosphate carboxylation rate (27.40% increase), maximum electron transport rate (21.51% increase) and the lowest CO2 compensation point (45.19% decrease). Furthermore, concentrations of Cu, Mn, Zn, Fe, Mg, Ca, K and P were examined with the most pronounced changes observed for elements directly involved in photosynthesis (Cu, Zn, Mn, and Fe). The Cu concentrations in roots, stems and leaves for Ti 50 mg L-1 cultivation were below the control by 33.15%, 38.28% and 10.76%, respectively. The Zn content in analogous treatment and organs decreased by 30.24%, 26.69% and 13.35%. The Mn and Fe levels in leaves were increased by 72.22% and 50.32%, respectively. Our results indicated that plant defence mechanisms which restrain the water uptake have been overcome in pea by photocatalytic activity of nanoparticulate TiO2 which stimulated photosynthesis. On the contrary to the substantial stomatal conductance, the transpiration has been reduced because exceptional part of water flow was already consumed in chloroplasts and could not have been freed to the atmosphere.

Polyphenolic dendrimers as carriers of anticancer siRNA.

Dendrimers have emerged as an important group of nanoparticles to transport drugs, DNA, or RNA into target cells in cancer and other diseases. Various functional modifications can be imposed on dendrimers to increase the efficacy and specificity in delivering their cargo to the target cells and decrease their toxicity. In the present work, we evaluated the potential of carbosilane polyphenolic dendrimers modified with caffeic acid (CA) and polyethylene glycol (PEG) to deliver proapoptotic Mcl-1 and Bcl-2 siRNAs to A549 cancer cells. Dendrimers formed stable complexes with siRNAs as assessed by transmission electron microscopy and gel electrophoresis. Modification of dendrimers with PEG reduced the size and the zeta potential of dendrimer/siRNA complexes. The presence of PEG caused a red shift of the CD spectrum, and this effect was the more pronounced, the higher the dendrimer/siRNA ratio was. The nanocomplexes were internalized by A549. All studied dendrimer/siRNA formulations inhibited tumor cell migration and adhesion and caused an increase in the population of early apoptotic cells. Among four tested dendrimers, the polyphenolic compound containing two caffeic acid moieties complexed with siRNA demonstrated the lowest polydispersity index and showed an excellent transfection profile. In conclusion, this dendrimer are a promising candidate for the delivery of siRNA into cancer cells in further in vivo studies.

Histone H3 posttranslational modified enzymes defined neutrophil plasticity and their vulnerability to IL-10 in the course of the inflammation.

BACKGROUND: Neutrophils are a heterogeneous population capable of antimicrobial functions associated with pre-activation/activation and tissue regeneration. The specific polarisation of immune cells is mediated by the modification of 'chromatin landscapes', which enables differentiated access and activity of regulatory elements that guarantee their plasticity during inflammation No specific pattern within histone posttranslational modifications (PTMs) controlling this plasticity has been identified. METHODS: Using the in vitro model of inflammation, reflecting different states of neutrophils from resting, pre-activated cells to activated and reducing tissue regeneration, we have analysed 11 different histone posttranslational modifications (PTMs), PTM enzymes associated with remodelling neutrophil chromatin, and H3K4me3 ChIP-Seq Gene Ontology analysis focusing on the processes related to histone PTMs. These findings were verified by extrapolation to adequate clinical status, using neutrophils derived from the patients with sepsis (systemic septic inflammation with LPS-stimulated neutrophils), neuromyelitis optical spectrum disorders (aseptic inflammation with pre-activated neutrophils) and periodontitis (local self-limiting septic inflammation with IL-10-positive neutrophils). RESULTS: Physiological activation of neutrophils comprises a pre-activation characterised by histone H3K27ac and H3K4me1, which position enhancers; direct LPS exposure is induced explicitly by H3K4me3 which marked Transcription Start Site (TSS) regions and low-level of H3K9me3, H3K79me2 and H3K27me3 which, in turn, marked repressed genes. Contrary to antimicrobial action, IL-10 positively induced levels of H3S10p and negatively H3K9me3, which characterised processes related to the activation of genes within heterochromatin mediated by CHD1 and H3K9me3 specific demethylase JMJD2A. IL-10 protects changes within histone PTMs induced by TNF or LPS that affected H3K4me3-specific methyltransferase SETD1A and MLL1. Neutrophils previously exposed to inflammatory factors become unvulnerable to IL-10 because previous LPS stimulation interrupts TSS regions marked by H3K4me3 of CHD1 and JMJD2A genes. Therefore, LPS-activated neutrophils are disabled to induce CHD1/JMJD2A enzymes by IL-10, making this process irreversible. Because transcription of JMJD2A and CHD1 also depends on TSS positioning by H3K4me3, neutrophils before LPS stimulation become insensitive to IL-10. CONCLUSION: Neutrophils, once pre-activated by TNF or directly stimulated by LPS, become insensitive to the anti-inflammatory effects of IL-10, and vice versa; IL-10 protects neutrophils against these proinflammatory stimuli. This phenomenon is responsible for disturbing the natural process of resolving inflammation and tissue regeneration.