Probing Red Blood Cell Membrane Microviscosity Using Fluorescence Anisotropy Decay Curves of the Lipophilic Dye PKH26

Red blood cell (RBC) aggregation and deformation are governed by the molecular processes occurring on the membrane. Since several social important diseases are accompanied by alterations in RBC aggregation and deformability, it is important to develop a diagnostic parameter of RBC membrane structural integrity and stability. In this work, we propose membrane microviscosity assessed by time-resolved fluorescence anisotropy of the lipophilic PKH26 fluorescent probe as a diagnostic parameter. We measured the fluorescence decay curves of the PKH26 probe in the RBC membrane to establish the optimal parameters of the developed fluorescence assay. We observed a complex biphasic profile of the fluorescence anisotropy decay characterized by two correlation times corresponding to the rotational diffusion of free PKH26, and membrane-bounded molecules of the probe. The developed assay allowed us to estimate membrane microviscosity ηm in the range of 100–500 cP depending on the temperature, which paves the way for assessing RBC membrane properties in clinical applications as predictors of blood microrheological abnormalities.     

Dose-Dependent Cytotoxicity of Polypropylene Microplastics (PP-MPs) in Two Freshwater Fishes

The massive accumulation of plastics over the decades in the aquatic environment has led to the dispersion of plastic components in aquatic ecosystems, invading the food webs. Plastics fragmented into microplastics can be bioaccumulated by fishes via different exposure routes, causing several adverse effects. In the present study, the dose-dependent cytotoxicity of 8–10 μm polypropylene microplastics (PP-MPs), at concentrations of 1 mg/g (low dose) and 10 mg/g dry food (high dose), was evaluated in the liver and gill tissues of two fish species, the zebrafish (Danio rerio) and the freshwater perch (Perca fluviatilis). According to our results, the inclusion of PP-MPs in the feed of D. rerio and P. fluviatilis hampered the cellular function of the gills and hepatic cells by lipid peroxidation, DNA damage, protein ubiquitination, apoptosis, autophagy, and changes in metabolite concentration, providing evidence that the toxicity of PP-MPs is dose dependent. With regard to the individual assays tested in the present study, the biggest impact was observed in DNA damage, which exhibited a maximum increase of 18.34-fold in the liver of D. rerio. The sensitivity of the two fish species studied differed, while no clear tissue specificity in both fish species was observed. The metabolome of both tissues was altered in both treatments, while tryptophan and nicotinic acid exhibited the greatest decrease among all metabolites in all treatments in comparison to the control. The battery of biomarkers used in the present study as well as metabolomic changes could be suggested as early-warning signals for the assessment of the aquatic environment quality against MPs. In addition, our results contribute to the elucidation of the mechanism induced by nanomaterials on tissues of aquatic organisms, since comprehending the magnitude of their impact on aquatic ecosystems is of great importance.     

Mitochondrial DNA as a Candidate Marker of Multiple Organ Failure after Cardiac Surgery

Assess the level of mitochondrial DNA depending on the presence of multiple organ failure in patients after heart surgery. The study included 60 patients who underwent surgical treatment of valvular heart disease using cardiopulmonary bypass. Uncomplicated patients were included in the 1st group (n = 30), patients with complications and multiple organ failure (MOF) were included in the 2nd group (n = 30). Serum mtDNA levels were determined by quantitative real-time polymerase chain reaction with fluorescent dyes. Mitochondrial DNA gene expression did not differ between group before surgery. Immediately after the intervention, cytochrome B gene expression was higher in the group with MOF, and it remained high during entire follow-up period. A similar trend was observed in cytochrome oxidase gene expression. Increased NADH levels of gene expressions during the first postoperative day were noted in both groups, the expression showed tendency to increase on the third postoperative day. mtDNA gene expression in the “MOF present” group remained at a higher level compared with the group without complications. A positive correlation was reveled between the severity of MOF according to SOFA score and the level of mtDNA (r = 0.45; p = 0.028) for the end-point “First day”. The ROC analysis showed that mtDNA circulating in plasma (AUC = 0.605) can be a predictor of MOF development. The level of mtDNA significantly increases in case of MOF, irrespective of its cause. (2) The expression of mtDNA genes correlates with the level of MOF severity on the SOFA score.     

Mlig-SKP1 Gene Is Required for Spermatogenesis in the Flatworm Macrostomum lignano

In a free-living flatworm, Macrostomum lignano, an S-phase kinase-associated protein 1 (SKP1) homologous gene was identified as enriched in proliferating cells, suggesting that it can function in the regulation of stem cells or germline cells since these are the only two types of proliferating cells in flatworms. SKP1 is a conserved protein that plays a role in ubiquitination processes as a part of the Skp1-Cullin 1-F-box (SCF) ubiquitin ligase complex. However, the exact role of Mlig-SKP1 in M. lignano was not established. Here, we demonstrate that Mlig-SKP1 is neither involved in stem cell regulation during homeostasis, nor in regeneration, but is required for spermatogenesis. Mlig-SKP1(RNAi) animals have increased testes size and decreased fertility as a result of the aberrant maturation of sperm cells. Our findings reinforce the role of ubiquitination pathways in germ cell regulation and demonstrate the conserved role of SKP1 in spermatogenesis.     

Inactivation of PTEN and ZFHX3 in Mammary Epithelial Cells Alters Patterns of Collective Cell Migration

Whole exome sequencing of invasive mammary carcinomas revealed the association of mutations in PTEN and ZFHX3 tumor suppressor genes (TSGs). We generated single and combined PTEN and ZFHX3 knock-outs (KOs) in the immortalized mammary epithelial cell line MCF10A to study the role of these genes and their potential synergy in migration regulation. Inactivation of PTEN, but not ZFHX3, induced the formation of large colonies in soft agar. ZFHX3 inactivation in PTEN KO, however, increased colony numbers and normalized their size. Cell migration was affected in different ways upon PTEN and ZFHX3 KO. Inactivation of PTEN enhanced coordinated cell motility and thus, the collective migration of epithelial islets and wound healing. In contrast, ZFHX3 knockout resulted in the acquisition of uncoordinated cell movement associated with the appearance of immature adhesive junctions (AJs) and the increased expression of the mesenchymal marker vimentin. Inactivation of the two TSGs thus induces different stages of partial epithelial-to-mesenchymal transitions (EMT). Upon double KO (DKO), cells displayed still another motile state, characterized by a decreased coordination in collective migration and high levels of vimentin but a restoration of mature linear AJs. This study illustrates the plasticity of migration modes of mammary cells transformed by a combination of cancer-associated genes.     

Peripheral Inflammatory Markers TNF-α and CCL2 Revisited: Association with Parkinson’s Disease Severity

One of the major mediators of neuroinflammation in PD is tumour necrosis factor alpha (TNF-α), which, similar to other cytokines, is produced by activated microglia and astrocytes. Although TNF-α can be neuroprotective in the brain, long-term neuroinflammation and TNF release can be harmful, having a neurotoxic role that leads to death of oligodendrocytes, astrocytes, and neurons and, therefore, is associated with neurodegeneration. Apart from cytokines, a wide family of molecules with homologous structures, namely chemokines, play a key role in neuro-inflammation by drawing cytotoxic T-lymphocytes and activating microglia. The objective of the current study was to examine the levels of the serum TNF-α and CCL2 (Chemokine (C-C motif) ligand 2), also known as MCP-1 (Monocyte Chemoattractant Protein-1), in PD patients compared with healthy controls. We also investigated the associations between the serum levels of these two inflammatory mediators and a number of clinical symptoms, in particular, disease severity and cognition. Such an assessment may point to their prognostic value and provide some treatment hints. PD patients with advanced stage on the Hoehn–Yahr scale showed an increase in TNF-α levels compared with PD patients with stages 1 and 2 (p = 0.01). Additionally, the UPDRS score was significantly associated with TNF-α levels. CCL2 levels, however, showed no significant associations.     

Molecular Characteristics,Receptor Specificity,and Pathogenicity of Avian Influenza Viruses Isolated from Wild Ducks in Russia

Avian influenza viruses (AIV) of wild ducks are known to be able to sporadically infect domestic birds and spread along poultry. Regular surveillance of AIV in the wild is needed to prepare for potential outbreaks. During long-year monitoring, 46 strains of AIV were isolated from gulls and mallards in Moscow ponds and completely sequenced. Amino acid positions that affect the pathogenicity of influenza viruses in different hosts were tested. The binding affinity of the virus for receptors analogs typical for different hosts and the pathogenicity of viruses for mice and chickens were investigated. Moscow isolates did not contain well-known markers of pathogenicity and/or adaptation to mammals, so as a polybasic cleavage site in HA, substitutions of 226Q and 228G amino acids in the receptor-binding region of HA, and substitutions of 627E and 701D amino acids in the PB2. The PDZ-domain ligand in the NS protein of all studied viruses contains the ESEV or ESEI sequence. Although several viruses had the N66S substitution in the PB1-F2 protein, all Moscow isolates were apathogenic for both mice and chickens. This demonstrates that the phenotypic manifestation of pathogenicity factors is not absolute but depends on the genome context.     

The Interplay between Aquaporin-1 and the Hypoxia-Inducible Factor 1α in a Lipopolysaccharide-Induced Lung Injury Model in Human Pulmonary Microvascular Endothelial Cells

Aquaporin-1 (AQP1), a water channel, and the hypoxia-inducible factor 1α (HIF1A) are implicated in acute lung injury responses, modulating among others pulmonary vascular leakage. We hypothesized that the AQP1 and HIF1A systems interact, affecting mRNA, protein levels and function of AQP1 in human pulmonary microvascular endothelial cells (HPMECs) exposed to lipopolysaccharide (LPS). Moreover, the role of AQP1 in apoptosis and wound healing progression was examined. Both AQP1 mRNA and protein expression levels were higher in HPMECs exposed to LPS compared to untreated HPMECs. However, in the LPS-exposed HIF1A-silenced cells, the mRNA and protein expression levels of AQP1 remained unaltered. In the permeability experiments, a statistically significant volume increase was observed at the 360 s time-point in the LPS-exposed HPMECs, while LPS-exposed HIF1A-silenced HPMECs did not exhibit cell swelling, implying a dysfunctional AQP1. AQP1 did not seem to affect cell apoptosis yet could interfere with endothelial migration and/or proliferation. Based on our results, it seems that HIF1A silencing negatively affects AQP1 mRNA and protein expression, as well as AQP1 function, in the setting of lung injury.     

Effects of Drying and Extraction Methods on the Quality and Antioxidant Activity of Sea Buckthorn (Hippophae rhamnoides) Berries and Leaves

Sea buckthorn is a promising source of bioactive compounds. However, there is limited information on the effect of post harvest drying and extraction of sea buckthorn on its antioxidant capacity. The effect of freeze, air, and solar dryingon the extraction yield of sea buckthorn berries and leaves was evaluated with respect to their bioactive content. Sequential extraction with solvents of different polarity and solvent-free microwave-assisted extraction were applied. Freeze-drying has better performance in the berries’ extracts, while air-drying has better performance in the leaves’ extracts. All data were analyzed by Multi Factor Analysis of Variance (ANOVA) and Duncan's post-hoc tests at a level of α = .05. Although the extraction yield in the sequential extraction of sea buckthorn berries is significantly better, the leaves’ extracts exhibit superior radical scavenging ability. Accelerated solar drying appears to preserve sea buckthorn leaves without degrading their antioxidant content. The components responsible for the high antioxidant activity of leaves’ extracts were found to be several flavonoids and polyphenols. The extract obtained by the microwave extraction of fresh berries exhibits moderate antioxidant activity compared to the polar extracts of freeze-dried berries.