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G. mellonella is a promising species for use in the biodegradation of plastics. It is easy to breed and has high resistance to diverse climatic conditions, which is particularly valuable when considering its potential application in the decomposition of plastics. Although it does not pose a direct threat to humans, the greater wax moth is a parasitic species in bee hives, contributing to the population decline of these insects. This species has demonstrated the capacity for biodegradation of the most common types of plastics, such as polyethylene and polypropylene (PP). The microbiome inhabiting the digestive system is responsible for this degradation. However, there reports on whether consumed plastics or their decomposition products will adversely affect the structure and functioning of the internal organs are rather poor. The aim of these studies was to determine whether the consumption of PP by a larger wax moth (G. mellonella) larvae caused any ultrastructural changes in the organs of the animal's body and to evaluate the survival rate of the animals and describe their reproduction. Thus, this study provided a preliminary understanding of histological and ultrastructural changes caused, or not caused, by the PP diet. We investigated whether any degenerative changes appeared in cells of selected organs – midgut, silk gland, and fat body – under PP consumption by G. mellonella caterpillars (7th instar larvae). We also examined whether there was an increase in levels of reactive oxygen species (ROS) in selected organs, as well as the ability of larvae to survive and undergo metamorphosis. The animals were divided into four groups: G0-C, G0-S, G0-24, and G0-48. The research was carried out using transmission electron microscopy (TEM), confocal microscopy, and flow cytometry. Our study on G. mellonella larvae fed with PP bags showed that a diet containing such plastic did not affect internal organs at the ultrastructural level. Cells in the analyzed organs – midgut, silk gland, and fat body – showed no degenerative changes. An increase in the intensity of autophagy and cell vacuolization was noted, but they probably act as a survival pathway. These observations suggest that the final larval stage of the greater wax moth can potentially be applied in PP biodegradation.

The syncytial groups of germ cells (germ-line cysts) forming in ovaries of clitellate annelids are an attractive model to study mitochondrial stage-specific changes. Using transmission electron microscopy, serial block-face scanning electron microscopy, and fluorescent microscopy, we analyzed the mitochondria distribution and morphology and the state of membrane potential in female cysts in Enchytraeus albidus. We visualized in 3D at the ultrastructural level mitochondria in cysts at successive stages: 2-celled, 4-celled, 16-celled cysts, and cyst in advanced oogenesis. We found that mitochondria form extensive aggregates—they are fused and connected into large and branched mitochondrial networks. The most extensive networks are formed with up to 10 000 fused mitochondria, whereas individual organelles represent up to 2% of the total mitochondrial volume. We classify such a morphology of mitochondria as a dynamic hyperfusion state and suggest that this can maintain their high activity and intensify the process of cellular respiration within the syncytial cysts. We found some individual mitochondria undergoing degradation, which implies that damaged mitochondria are removed from networks for their final elimination. As growing oocytes were shown to possess less active mitochondria than the nurse cells, the high activity of mitochondria in the nurse cells and their dynamic hyperfusion state are attributed to serve the needs of the growing oocyte. In addition, we measured by calorimetry the total antioxidant capacity of germ-line cysts in comparison with somatic tissue, and it suggests that antioxidative defense systems, together with mitochondrial networks, can effectively protect germ-line mitochondria from damage.

The aim of this work is to analyze the usefulness of Sr3(PO4)2 as a potential antibacterial agent in fast-degradable polymer coatings. The Ti implant was anodized and then covered by a poly(adipic anhydride) layer blended with Sr3(PO4)2 particles by dip coating. The average size of the particles was 450 nm ± 53 nm (Zetasizer analysis), and phase analysis (X-ray diffractometer) showed mainly crystalline phases. Surface morphology and surface roughness were analyzed by scanning electron microscopy with energy dispersive X-ray (EDX). The oxide layer thickness on the implant surface was between 4.57 μm and 8.09 μm. The polymer layer with Sr3(PO4)2 particles was determined by confocal microscopy, and the average thickness was 25 μm. The contact angle of the anodized Ti implant alone was 37.3° ± 9.9° and decreased with time up to 20° after 1900s. After 1 h of implant immersion in Ringer solution, 42.15% of the Sr compound loaded in the polymer layer was released (determined by inductively coupled plasma atomic emission spectrometry (ICP-AES)). Fast degradation of the polymer caused 65.21% of the loaded Sr compound to be released from the implant surface after 24 h. The concentration of Sr3(PO4)2 was enough to significantly inhibit the adhesion of Staphylococcus aureus ATCC 25923 bacteria on the implant surface. The implant surface is cytocompatible with osteoblast-like MG-63 cells. The preliminary results of hybrid coating analysis showed that Sr3(PO4)2 may be used as an alternative ceramic material for the formation of a functional layer for orthopedic applications.

Adult specimens or larvae of invertebrates used as food for vertebrates are often maintained close to gluten so they might become vectors for cereal proteins. However, the tissues and internal organs can respond differently in animals with different feeding habits. The midgut epithelium might be a first and sufficient barrier preventing uptake and effects of gluten on the whole body, while the fat body is the main organ that accumulates different xenobiotics. Good models for such research are animals that do not feed on gluten-rich products in their natural environment. The project's goal was to investigate alterations in the midgut epithelium and fat body of the herbivorous millipede Telodeinopus aoutii (Diplopoda) and analyze cell death processes activated by gluten. It enabled us to determine whether changes were intensified or reversed by adaptive mechanisms. Adult specimens were divided into control and experimental animals fed with mushrooms supplemented with gluten and analyzed using transmission electron microscopy, flow cytometry, and confocal microscopy. Two organs were isolated for the qualitative and quantitative analysis: the midgut and the fat body. Our study of the herbivorous T. aoutii which does not naturally feed on gluten containing diet showed that continuous and prolonged gluten feeding activates repair processes that inhibit the processes of cell death (apoptosis and necrosis) and induce an increase in cell viability.

To date, hundreds of millions tons of plastics has been produced worldwide. Their production and disposal are associated with high pollution and carbon release into the atmosphere. A more environmentally friendly alternative is bioplastics, and the most popular is polyhydroxybutyrate (PHB) polymer. Large amounts of PHB can be obtained from activated sludge where used cooking oil or other industrial waste can be used as potential substrates. In this work, efficient bioplastic production strategies are studied, and the considered substrate is a mixture of oil and peptone. Pseudomonas fluorescens bacteria are used to accumulate PHB, and the cultivation of microorganisms is carried out in batch and continuous-flow bioreactors. Microscopic observations and laboratory essays are performed to confirm presence of PHB and other key parameters. The obtained results allow us to determine the optimal feeding strategy.

The data from independent gene expression sources may be integrated for the purpose of molecular diagnostics of cancer. So far, multiple approaches were described. Here, we investigated the impacts of different data fusion strategies on classification accuracy and feature selection stability, which allow the costs of diagnostic tests to be reduced. We used molecular features (gene expression) combined with a feature extracted from the independent clinical data describing a patient’s sample. We considered the dependencies between selected features in two data fusion strategies (early fusion and late fusion) compared to classification models based on molecular features only. We compared the best accuracy classification models in terms of the number of features, which is connected to the potential cost reduction of the diagnostic classifier. We show that for thyroid cancer, the extracted clinical feature is correlated with (but not redundant to) the molecular data. The usage of data fusion allows a model to be obtained with similar or even higher classification quality (with a statistically significant accuracy improvement, a p-value below 0.05) and with a reduction in molecular dimensionality of the feature space from 15 to 3–8 (depending on the feature selection method). Both strategies give comparable quality results, but the early fusion method provides better feature selection stability.

The multifaceted nature of subarachnoid hemorrhage (SAH) pathogenesis is poorly understood, to date, no pharmacological agent has been found to be efficacious for the prevention of brain injury when used for acute SAH intervention. This study was undertaken to evaluate the beneficial effects of low-dose neuroprotective agent minocycline on brain microvascular ultrastructures which are not studied in detail. We studied SAH brain injury using in vivo pre-chiasmatic subarachnoid hemorrhage rodent model. We analyzed the qualitative and quantitative ultrastructural morphology of capillaries and surrounding neuropil in the rodent brains with SAH and/or minocycline administration. Here, we report that low-dose minocycline (1 mg/kg) displayed protective effects on capillaries and surrounding cells from significant SAH-induced changes. Ultrastructural morphology analysis revealed also that the minocycline stopped endothelial cells from abnormal production of vacuoles and vesicles, that compromise BBB transcellular transport. The reported ultrastructural abnormalities as well as neuroprotective effects of minocycline during SAH were not directly mediated by inhibition of MMP-2, MMP-9, or EMMPRIN. However, SAH brain tissue treated with minocycline was protected from development of other morphological features associated with oxidative stress and the presence of immune cells in the perivascular space. These data advance the knowledge on the SAH effect on the ultrastructure of brain tissue in the SAH rodent model and the neuroprotective effect of minocycline when administered in low-doses.

The multifaceted nature of subarachnoid hemorrhage (SAH) pathogenesis is poorly understood. To date, no pharmacological agent has been found to be efficacious for the prevention of brain injury when used for acute SAH intervention. This study was undertaken to evaluate the beneficial effects of low-dose neuroprotective agent minocycline on brain microvascular ultrastructures that have not been studied in detail. We studied SAH brain injury using an in vivo prechiasmatic subarachnoid hemorrhage rodent model. We analyzed the qualitative and quantitative ultrastructural morphology of capillaries and surrounding neuropil in the rodent brains with SAH and/or minocycline administration. Here, we report that low-dose minocycline (1 mg/kg) displayed protective effects on capillaries and surrounding cells from significant SAH-induced changes. Ultrastructural morphology analysis revealed also that minocycline stopped endothelial cells from abnormal production of vacuoles and vesicles that compromise blood–brain barrier (BBB) transcellular transport. The reported ultrastructural abnormalities as well as neuroprotective effects of minocycline during SAH were not directly mediated by inhibition of MMP-2, MMP-9, or EMMPRIN. However, SAH brain tissue treated with minocycline was protected from development of other morphological features associated with oxidative stress and the presence of immune cells in the perivascular space. These data advance the knowledge on the effect of SAH on brain tissue ultrastructure in an SAH rodent model and the neuroprotective effect of minocycline when administered in low doses.

The suppressive activity of monocyte chemoattractant protein 1-induced protein 1 (MCPIP1) an inflammation-related ribonuclease, has been described in a few cancer types but has yet to be assessed in the most common subtype of skin cancer: melanoma. Here, we have evaluated the MCPIP1 expression in melanoma tissues by reanalysis of publicly available transcriptome data from 89 melanoma samples, and immunohistochemical staining of 21 primary and 81 metastatic melanomas. Our data implicated decreased MCPIP1 expression in melanoma tumors compared to normal tissues, and positive correlation between high ribonuclease expression and melanoma-specific survival of patients. To investigate the ribonuclease activity in melanoma cells, MCPIP1 was ectopically expressed in the MV3 human melanoma cell line. Following the transcriptome, proteome, and intracellular signaling of MCPIP1-overexpressing MV3 cells was assessed via real-time quantitative polymerase chain reaction, Western blot analysis, and RNAseq. MV3 cells overexpressing MCPIP1 exhibited a broad range of alterations in the transcriptome and proteome, as well as in the phosphorylation status of a number of proteins, strongly indicating MCPIP1-dependent cell cycle arrest and inhibition of Akt/mTOR signaling in these cells. Moreover, we have shown, that MCPIP1 overexpression downregulates miRNA-193a-3p expression in MV3 cells. Furthermore, the majority of the described effects were dependent on the ribonucleolytic activity of the protein. The presented body of data strongly suggests a potential tumor suppressor role and possible future application as a positive prognostic marker of MCPIP1 protein in melanoma.

Mitochondria are organelles that play a crucial role in cell physiology, cell death, and aging. They are among the first responders to different stressors that originate from the environment. Cadmium as a heavy metal affects different levels of body organization: from organs through tissues and cells to organelles. Based on our previous research results, we decided to check how the exposure to cadmium affects the functioning of mitochondria in different organs of soil living centipede Lithobius forficatus. The activity of mitochondria in somatic and germ cells has been analyzed using transmission electron microscopy (TEM), confocal microscopy, and flow cytometry. Changes in the mitochondrial membrane potential and mitochondrial dismutase (MnSOD) activity in relation to the accumulation of reactive oxygen species (ROS) caused by cadmium exposure have been studied. Individuals were divided into 3 experimental groups depending on cadmium concentration in soil. Changes in mitochondrial ultrastructure caused by cadmium are tissue-dependent and associated with an increase of ROS levels. The system of ROS and MnSOD activation works more efficiently in the case of gonads than in the digestive system. While the short-term cadmium exposure alters the fine structure of both the somatic and germ-line cells in gonads, the long-term cadmium exposure causes mitochondrial ultrastructure regeneration.

The relevance of diabetes mellitus (DM) to the efficacy of mechanical thrombectomy (MT) has been the subject of few studies and with only inconclusive results.This study aimed to evaluate the effect of DM and admission hyperglycaemia on the efficacy and safety of MT in stroke patients.This retrospective study analysis focused on the relevance of admission hyperglyacemia and DM to the functional status of patients treated with MT at the Upper Silesian Medical Centre of the Silesian Medical University in Katowice, Poland.417 stroke patients (median age 70 years) were qualified for the study. There were 103 patients (24.70%) with DM. Admission hyperglycaemia ≥ 140 mg% was found in 91 patients (21.82%), of whom 69 were diagnosed with DM before or during hospitalisation. The parameters with the strongest effect on the functional status on days 7, 90 and 365 were: age, and neurological status according to the National Institutes of Health Stroke Scale (NIHSS) on the first day of ischaemic stroke before MT. The angiographic effect indirect after MT and patient functional status on days 7, 90 and 365 were comparable between the groups, regardless of the DM burden. The frequency of symptomatic intracranial bleeding 24 hours after MT was comparable between patients with and patients without DM (p = 0.092). Model based on parameters were age, NIHSS on the first day of ischaemic stroke, an when score in Thrombolysis In Cerebral Infarct (TICI) showed good predictive attributes for the functional status of patients in the acute period (day 7). Age, a lack of admission hyperglycaemia, and the neurological state on day 1 of ischaemic stroke (before MT) were the key parameters for a favourable outcome (≤ 2 points on the modified Rankin Scale, mRS) on day 90. Admission hyperglycaemia ≥ 140 mg/dL, regardless of the presence or absence of DM, had a negative effect on achieving a good functional status one week after stroke onset.Diabetes mellitus has a neutral effect on the angiographic and clinical outcomes of mechanical thrombectomy in stroke patients. It does not increase the risk of intracranial haemorrhage after instrumental therapy. It is admission hyperglycaemia, rather than diabetes mellitus, that is a predictor of poor functional status in patients treated with thrombectomy. According to our results, the patient's neurological status, age, and the outcome of thrombectomy are relevant to the functional status in the acute ischaemic stroke period.

Background and Purpose: The multifaceted nature of Subarachnoid hemorrhage (SAH) pathogenesis is poorly understood. Despite the high morbidity and disability rate associated with SAH, to date, no pharmacological agent has been found to be efficacious for the prevention of brain injury when used for acute SAH intervention.Methods: We studied SAH brain injury using in vivo pre-chiasmatic subarachnoid hemorrhage rodent model. We analyzed the qualitative and quantitative ultrastructural morphology of capillaries and surrounding neuropil in the rodent brains with SAH and/or minocycline administration. We also performed vast analysis of mRNA, protein and in situ expression of tight junctions proteins, claudin-5 and occludin and matrix metalloproteinases, MMP-2, MMP-9 and their upstream activator EMMPRIN. Lastly, we tested minocycline effect on functional outcomes in tested rats. Results: Here, we report that 1 mg/kg minocycline displayed neuroprotective effects on neurovascular morphology. Using ultrastructural analyses, we found that minocycline protected brain capillaries and surrounding cells from significant SAH-induced changes. Further analysis revealed that the blood-brain barrier in SAH brains is compromised by abnormal vacuole and vesicle production, rather than by tight junction disruptions, which were found to be generally intact. Additionally, our findings suggested that the reported ultrastructural abnormalities as well as minocycline neuroprotective effect during SAH were not directly mediated by the inhibition of MMP-2, MMP-9, or EMMPRIN. However, SAH brain tissue treated with minocycline was protected from development of other kind of morphological features associated with oxidative stress and the presence of immune cells in the perivascular space. Conclusions: These data expand knowledge on the SAH effect on brain tissue as well as functional outcome in the SAH rodent model and minocycline neuroprotective effect when administered in low-doses. Additionally, we showed the multifactorial neuroprotective mechanism of minocycline, as is the pathology associated with SAH.

This paper describes a formation of hybrid coatings on a Ti–2Ta–3Zr–36Nb surface. This is accomplished by plasma electrolytic oxidation and a dip-coating technique with poly(adipic anhydride) ((C6H8O3)n) that is loaded with drugs: amoxicillin (C16H19N3O5S), cefazolin (C14H14N8O4S3) or vancomycin (C66H75Cl2N9O24 · xHCl). The characteristic microstructure of the polymer was evaluated using scanning electron microscopy and confocal microscopy. Depending on the surface treatment, the surface roughness varied (between 1.53 μm and 2.06 μm), and the wettability was change with the over of time. X-ray photoelectron spectroscopy analysis showed that the oxide layer did not affect the polymer layer or loaded drugs. However, the drugs lose their stability in a phosphate-buffered saline solution after 6.5 h of exposure, and its decrease was greater than 7% (HPLC analysis). The stability, drug release and concentration of the drug loaded into the material were precisely analyzed by high-performance liquid chromatography. The results correlated with the degradation of the polymer in which the addition of drugs caused the percent of degraded polymer to be between 35.5% and 49.4% after 1 h of material immersion, depending on the mass of the loaded drug and various biological responses that were obtained. However, all of the coatings were cytocompatible with MG-63 osteoblast-like cells. The drug concentrations released from the coatings were sufficient to inhibit adhesion of reference and clinical bacterial strains (S. aureus). The coatings with amoxicillin showed the best results in the bacterial inhibition zone, whereas coatings with cefazolin inhibited adhesion of the above bacteria on the surface., Graphical abstract Image 1, Highlights • Hybrid layers containing fast degradable poly(adipic anhydride) (PADA) were performed. • Drugs stability, concentration of released and loaded drugs were evaluated using HPLC. • Adhesion of S. aureus clinical and reference strains confirmed the antibaterial properties. • Performed hybrid layers were cytocompatible (MG-63 tests).

Oxide-polymer coatings were formed on the surface of the vanadium-free Ti–15Mo titanium alloy. The Ti alloy surface was modified by the plasma electrolytic oxidation process, and then, the polymer layer of a poly (D, l-lactide-co-glycolide) with doxycycline was formed. The polymer evenly covered the porous oxide layer and filled some of the pores. However, the microstructure of the polymer surface was completely different from that of the PEO layer. The surface morphology, roughness and microstructure of the polymer layer were examined by scanning electron microscopy (SEM) and a confocal microscope. The results confirmed the effectiveness of polymer and doxycycline deposition in their stable chemical forms. The drug analysis was performed by high-performance liquid chromatography. The 1H NMR technique was used to monitor the course of hydrolytic degradation of PLGA. It was shown that the PLGA layer is hydrolysed within a few weeks, and the polyglycolidyl part of the copolymer is hydrolysed to glycolic acid as first and much faster than the polylactide one to lactic acid. This paper presents influence of different microstructures on the biological properties of modified titanium alloys. Cytocompatibility and bacterial adhesion tests were evaluated using osteoblast-like MG-63 cells and using the reference S. aureus and S. epidermidis strains. The results showed that the optimum concentration of doxycycline was found to inhibit the growth of the bacteria and that the layer is still cytocompatible., Graphical abstract Image 1, Highlights • Formation of the oxide-polymer layer containing doxycycline is presented. • Changes in the doxycycline structure and the evaluation of their stability was analyzed using the HPLC. • Thickness of the polymer layer was determined using the confocal microscopy. • The coatings showed the antibacterial properties and were cytocompatible with osteoblast-ike MG-63 cells.

The role of the inflammation-silencing ribonuclease, MCPIP1 (monocyte chemoattractant protein-induced protein 1), in neoplasia continuous to emerge. The ribonuclease can cleave not only inflammation-related transcripts but also some microRNAs (miRNAs) and viral RNAs. The suppressive effect of the protein has been hitherto suggested in breast cancer, clear cell renal cell carcinoma, osteosarcoma, and neuroblastoma. Our previous results have demonstrated a reduced levels of several oncogenes, as well as inhibited growth of neuroblastoma cells upon MCPIP1 overexpression. Here, we investigate the mechanisms underlying the suppression of MYCN proto-oncogene, bHLH transcription factor (MYCN)-amplified neuroblastoma cells overexpressing the MCPIP1 protein. We showed that the levels of several transcripts involved in cell cycle progression decreased in BE(2)-C and KELLY cells overexpressing MCPIP1 in a ribonucleolytic activity-dependent manner. However, RNA immunoprecipitation indicated that only AURKA mRNA (encoding for Aurora A kinase) interacts with the ribonuclease. Furthermore, the application of a luciferase assay suggested MCPIP1-dependent destabilization of the transcript. Further analyses demonstrated that the entire conserved region of AURKA seems to be indispensable for the interaction with the MCPIP1 protein. Additionally, we examined the effect of the ribonuclease overexpression on the miRNA expression profile in MYCN-amplified neuroblastoma cells. However, no significant alterations were observed. Our data indicate a key role of the binding and cleavage of the AURKA transcript in an MCPIP1-dependent suppressive effect on neuroblastoma cells.

Cadmium is a heavy metal that is treated as an environmental pollutant (air, water, soil). In order to understand the potential effects of cadmium in soil and soil invertebrates, it is important to describe all alterations which appear at different levels in organisms. The main aim of this study was to investigate, analyze and describe the alterations caused by cadmium short- and long-term intoxication at different levels in the organisms: from tissues to cells and organelles. In addition, the activation of cell death mechanisms that take part in homeostasis maintenance according to cadmium has been studied. Therefore, as the species for this project, a terrestrial and well-known widespread European species – the centipede Lithobius forficatus (Myriapoda, Chilopoda, Lithobiomorpha) – was chosen. This omnivorous species lives under upper layers of soil, under stones, litter, rocks, and leaves, and it is also commonly found in human habitats. The animals were divided into three groups: C – the control group, animals cultured in a horticultural soil; Cd1 – animals cultured in a horticultural soil supplemented with 80 mg/kg (dry weight) of CdCl2, 12 days – short-term exposure; Cd2 – animals cultured in a horticultural soil supplemented with 80 mg/kg (dry weight) of CdCl2, 45 days – long-term exposure. The midgut was isolated from each specimen and it was prepared for analysis using some histological, histochemical and immunohistochemical methods. Our studies showed that short-term intoxication causes intensification of autophagy and digestion of reserve material, while long-term exposure to this heavy metal causes activation of cell death processes together with inhibition of autophagy connected with the lack of reserve material. Additionally, we can infer that autophagy and cell death are nutrient deprivation-induced processes. Finally, we can conclude that short- and long-term exposure of soil centipede to cadmium affects different mechanisms and processes of cell death.

Background Molecularly targeted therapy has revolutionized the treatment of advanced melanoma. However, despite its high efficiency, a majority of patients experience relapse within 1 year of treatment because of acquired resistance, and approximately 10–25% patients gain no benefit from these agents owing to intrinsic resistance. This is mainly caused by the genetic heterogeneity of melanoma cells. Objective We aimed to validate the predictive significance of selected genes in advanced melanoma patients before treatment with BRAF/MEK inhibitors. Patients and Methods Archival DNA derived from 37 formalin-fixed paraffin-embedded pre-treatment advanced melanoma samples of patients treated with targeted therapy was used for next-generation sequencing analysis using the Ion Torrent platform. The AmpliSeq Custom Panel comprised coding sequences or hot spots of 23 melanoma genes: ATM, BRAF, CDK4, CDKN2A, CTNNB1, EGFR, HOXD8, HRAS, IDH1, KIT, KRAS, MAP3K8, MAP2K1, MAP2K2, MITF, MYC, NF1, NRAS, PAX5, PIK3R1, PTEN, RAC1, and RB1. The sequences were evaluated for genomic alterations and further validated using Sanger sequencing. Results Our analysis revealed non-BRAF genetic alterations in 28 out of 37 samples (75.7%). Genetic changes were identified in PTEN, CDK4, CDKN2A, CTNNB1, EGFR, HOXD8, HRAS, KIT, MAP2K1, MAP2K2, MITF, MYC, NF1, PAX5, RAC1, and RB1. Fifteen known pathogenic mutations (single nucleotide variants or indels) and 11 variants of unknown significance were detected. Statistical analysis revealed an association between the presence of pathogenic mutations and time to progression during treatment with combination therapy. Conclusions Pathogenic mutations identified by gene panel sequencing have potential predictive value for targeted therapy of melanoma and are worth further validation in a larger series of cases. The role of some known mutations (e.g. CDK4R24, PTEN c.801 + 1G > A, CTNNB1S45F) as well as variants of unknown significance identified in this study (e.g. MITFR316K, KITG498S) in the generation of resistance to BRAF/MEK inhibitors should be further investigated. Electronic supplementary material The online version of this article (10.1007/s11523-020-00695-0) contains supplementary material, which is available to authorized users.

MicroRNAs (miRNAs) have great therapeutic potential; however, their delivery still faces huge challenges, especially given the short half-life of naked miRNAs due to rapid hydrolysis or inactivation by abundant nucleases in the systemic circulation. Therefore, the search for reliable miRNA delivery systems is crucial. Nanogels are one of the more effective nanocarriers because they are biocompatible and have a high drug-loading capacity. In this study, acrylamide-based nanogels containing cationic groups and redox-sensitive crosslinkers were developed for cellular delivery of anti-miR21 (a-miR21). To achieve this, post-polymerization loading of a-miR21 oligonucleotides into nanogels was performed by utilizing the electrostatic interaction between positively charged nanogels and negatively charged oligonucleotides. Different molar ratios of the amine groups (N) on the cationic nanogel and phosphate groups (P) on the miRNA were investigated. An N/P ratio of 2 allowed high miRNA loading capacity (MLC, 6.7% w/w) and miRNA loading efficiency (MLE, 99.7% w/w). Successful miRNA loading was confirmed by dynamic light scattering (DLS) and electrophoretic light scattering (ELS) measurements. miRNA-loaded nanogels (NG/miRNA) formed stable dispersions in biological media and showed an enhanced miRNA release profile in the presence of glutathione (GSH). Moreover, the addition of heparin to dissociate the miRNA from the cationic nanogels resulted in the complete release of miRNA. Lastly, a cell uptake study indicated that NG/miRNA could be easily taken up by cancer cells.

Background: The objective of this study was to evaluate the impact of weather factors on stroke parameters. Methods: This retrospective study analyzed the records of stroke patients concerning the influence of meteorological conditions and moon phases on stroke parameters. Results: The study group consisted of 402 patients aged between 20 and 102, women constituted 49.8% of the subjects. Ischaemic stroke was diagnosed in 90.5% of patients and hemorrhagic stroke was diagnosed in 9.5% of patients. The highest number of hospitalizations due to stroke was observed in January (48 events), the lowest number was observed in July (23 events). There was no statistically significant correlation between the meteorological parameters on the day of onset and the preceding day of stroke and the neurological status (NIHSS) of patients. Mean air temperature on the day of stroke and the day preceding stroke was significantly lower in the group of patients discharged with a very good functional status (≤2 points in modified Rankin scale (mRS)) compared to the patients with a bad functional status (&gt, 2 points in mRS), respectively: 7.98 ± 8.01 vs. 9.63 ± 7.78, p = 0.041 and 8.13 ± 7.72 vs. 9.70 ± 7.50, p = 0.048). Humidity above 75% on the day of stroke was found to be a factor for excellent functional state (RR 1.61, p = 0.016). The total anterior circulation infarcts (in comparison with stroke in the other localization) were more frequent (70%) during a third quarter moon (p = 0.011). The following parameters had a significant influence on the number of stroke cases in relation to autumn having the lowest number of onsets: mean temperature (OR 1.019 95% CI 1.014–1.024, p &lt, 0.000), humidity (OR 1.028, CI 1.023–1.034, p &lt, 0.0001), wind speed (OR 0.923, 95% CI 0.909–0.937, p &lt, 0.0001), insolation (OR 0.885, 95% CI 0.869–0.902, p &lt, 0.0001), precipitation (OR 0.914, 95% CI 0.884–0.946, p &lt, 0.0001). Conclusion: Air humidity and air temperature on the day of stroke onset as well as air temperature on the day preceding stroke are important for the functional status of patients in the acute disease period. A combination of the following meteorological parameters: lowered mean temperature and low sunshine, high humidity and high wind speed all increase the risk of stroke during the winter period. High humidity combined with high precipitation, low wind speed and low sunshine in the autumn period are associated with the lowest stroke incidence risk. A possible relationship between phases of the moon and the incidence requires further investigation.

Background and Objective: In recent years, progress in microfabrication technologies has attracted the attention of researchers across disciplines. Microfluidic devices have the potential to be developed into powerful tools that can elucidate the biophysical behavior of blood flow in microvessels. Such devices can also be used to separate the suspended physiological fluid from whole in vitro blood, which includes cells. Therefore, it is essential to acquire a detailed description of the complex interaction between erythrocytes (red blood cells; RBCs) and plasma. RBCs tend to undergo axial migration caused by occurrence of the Fåhræus-Lindqvist effect. These dynamics result in a cell-free layer (CFL), or a low volume fraction of cells, near the vessel wall. The aim of the paper is to develop a numerical model capable of reproducing the behavior of multiphase flow in a microchannel obtained under laboratory conditions and to compare two multiphase modelling techniques Euler-Euler and Euler-Lagrange. Methods: In this work, we employed a numerical Computational Fluid Dynamics (CFD) model of the blood flow within microchannels with two hyperbolic contraction shapes. The simulation was used to reproduce the blood flow behavior in a microchannel under laboratory conditions, where the CFL formation is visible downstream of the hyperbolic contraction. The multiphase numerical model was developed using Euler-Euler and hybrid Euler-Lagrange approaches. The hybrid CFD simulation of the RBC transport model was performed using a Discrete Phase Model. Blood was assumed to be a nonhomogeneous mixture of two components: dextran, whose properties are consistent with plasma, and RBCs, at a hematocrit of 5% (percent by volume of RBCs). Results: The results show a 5 μm thick CFL in a microchannel with a broader contraction and a 35 μm thick CFL in a microchannel with a narrower contraction. The RBC volume fraction in the CFL is less than 2%, compared to 7–8% in the core flow. The results are consistent for both multiphase simulation techniques used. The simulation results were then validated against the experimentally-measured CFL in each of the studied microchannel geometries. Conclusions: Reasonable agreement between experiments and simulations was achieved. A validated model such as the one tested in this study can expedite the microchannel design process by minimizing the need to prefabricate prototypes and test them under laboratory conditions., The work was partially supported by the Faculty of Energy and Environmental Engineering, Silesian University of Technology (SUT) within Ministry of Education and Science (Poland) statutory research funding scheme (MG, ZO) and by the Silesian University of Technology rector’s pro-quality grants No. 02/040/RGJ21/1011 (SS) and 08/060/RGJ21/1017 (ZO) and National Center of Science (Poland) No. 2017/27/B/ST8/01046 (BM). Rui Lima and João M. Miranda were partially funded by Portuguese national funds of FCT/MCTES (PIDDAC) through the base funding from the following research units: UIDB/00532/2020 (Transport Phenomena Research Center CEFT) and UIDB/04077/2020 (MEtRICs).

Proper reproduction depends on properly functioning gonads (ovaries and testes). Many xenobiotics, including heavy metals, can cause changes in somatic and germ line cells, thus damaging the reproductive capacity. The aim of this study was to investigate the effect of the heavy metal cadmium on the gonads, including germ line and somatic cells. It is important to determine whether cell death processes are triggered in both types of cells in the gonads, and which gonads are more sensitive to the presence of cadmium in the environment. The research was conducted on the soil-dwelling arthropod Lithobius forficatus (Myriapoda, Chilopoda), which is common for European fauna. Animals were cultured in soil supplemented with Cd for different periods (short- and long-term treatment). Gonads were isolated and prepared for qualitative and quantitative analysis, which enabled us to describe all changes which appeared after both the short- and long-term cadmium treatment. The results of our study showed that cadmium affects the structure and ultrastructure of both gonads in soil-dwelling organisms including the activation of cell death processes. However, the male germ line cells are more sensitive to cadmium than female germ line cells. We also observed that germ line cells are protected by the somatic cells of both gonads.

Current trends in the field of neural tissue engineering include the design of advanced biomaterials combining excellent electrochemical performance with versatile biological characteristics. The purpose of this work was to develop an antibacterial and neuroprotective coating based on a conducting polymer - poly(3,4-ethylenedioxypyrrole) (PEDOP), loaded with an antibiotic agent - tetracycline (Tc). Employing an electrochemical technique to immobilize Tc within a growing polymer matrix allowed to fabricate robust PEDOP/Tc coatings with a high charge storage capacity (63.65 ± 6.05 mC/cm

Experimental and clinical data support the neuroprotective properties of the ketogenic diet and ketone bodies, but there is still a lot to discover to comprehensively understand the underlying mechanisms. Autophagy is a key mechanism for maintaining cell homeostasis, and therefore its proper function is necessary for preventing accelerated brain aging and neurodegeneration. Due to many potential interconnections, it is possible that the stimulation of autophagy may be one of the mediators of the neuroprotection afforded by the ketogenic diet. Recent studies point to possible interconnections between ketone body metabolism and autophagy. It has been shown that autophagy is essential for hepatic and renal ketogenesis in starvation. On the other hand, exogenous ketone bodies modulate autophagy both in vitro and in vivo. Many regional differences occur between brain structures which concern i.e., metabolic responses and autophagy dynamics. The aim of the present study was to evaluate the influence of the ketogenic diet on autophagic markers and the ketone body utilizing and transporting proteins in the hippocampus and frontal cortex. C57BL/6N male mice were fed with two ketogenic chows composed of fat of either animal or plant origins for 4 weeks. Markers of autophagosome formation as well as proteins associated with ketolysis (BDH1—3-hydroxybutyrate dehydrogenase 1, SCOT/OXCT1—succinyl CoA:3-oxoacid CoA transferase), ketone transport (MCT1—monocarboxylate transporter 1) and ketogenesis (HMGCL, HMGCS2) were measured. The hippocampus showed a robust response to nutritional ketosis in both changes in the markers of autophagy as well as the levels of ketone body utilizing and transporting proteins, which was also accompanied by increased concentrations of ketone bodies in this brain structure, while subtle changes were observed in the frontal cortex. The magnitude of the effects was dependent on the type of ketogenic diet used, suggesting that plant fats may exert a more profound effect on the orchestrated upregulation of autophagy and ketone body metabolism markers. The study provides a foundation for a deeper understanding of the possible interconnections between autophagy and the neuroprotective efficacy of nutritional ketosis.

Adult specimens of the freshwater shrimp Neocaridina davidi Bouvier, 1904 (Crustacea) were starved for 7, 14, and 21 days. Specimens from the first and second experimental group were collected for the studies. The majority of animals starved for 21 days died. Additionally, some specimens from each group were refed for 4, 7, and 14 days. The epithelium of the midgut, which is composed of the intestine and hepatopancreas, was analyzed. While the epithelium of the intestine is formed by D- and R-cells, the epithelium of the hepatopancreas has R-, B-, and F-cells. Autophagy and apoptosis in the midgut epithelium were analyzed using transmission electron microscopy and immunohistochemical methods. These processes were only observed in the D-cells of the intestine and the F- and B-cells of the hepatopancreas. Starvation led to a reduction in the amount of reserve material in the B-cells. Although this process activated autophagy in both regions of the midgut, the intestine and hepatopancreas, after refeeding, the level of autophagy decreased. Starvation caused an increase in the apoptotic cells in both organs, while the refeeding caused a decrease in the number of apoptotic cells in both organs analyzed. Refeeding after periods of starvation caused an accumulation of reserve material in the hepatopancreas.

The male germ-line cysts that occur in annelids appear to be a very convenient model for spermatogenesis studies. Germ-line cysts in the studied earthworm are composed of two compartments: (1) germ cells, where each cell is connected via one intercellular bridge to (2) an anuclear central cytoplasmic mass, the cytophore. In the present paper, confocal and transmission electron microscopy were used to follow the changes in the mitochondrial activity and ultrastructure within the cysts during spermatogenesis. JC-1 was used to visualize the populations of mitochondria with a high and low membrane potential. We used the spot detection Imaris software module to obtain the quantitative data. We counted and compared the 'mitochondrial spots' - the smallest detectable signals from mitochondria. It was found that in all of the stages of cyst development, the majority of mitochondria spots showed a green fluorescence, thus indicating a low mitochondrial membrane potential (MMP). Moreover, the number of active mitochondria spots that were visualized by red JC-1 fluorescence (high MMP) drastically decreased as spermatogenesis progressed. As much as 26% of the total number of mitochondrial spots in the spermatogonial cysts showed a high MMP - 19% in the spermatocytes, 24% in the isodiametric spermatids and 3% and 6%, respectively, in the cysts that were holding early and late elongate spermatids. The mitochondria were usually thread-like and had an electron-dense matrix and lamellar cristae. Then, during spermiogenesis, the mitochondria within both the spermatids and the cytophore had a tendency to form aggregates in which the mitochondria were cemented by an electron-dense material.

Trehalose has been widely studied as a treatment for a variety of human disorders due to its ability to stimulate autophagy. Trehalose, however, is poorly adsorbed and is hydrolyzed in the intestinal mucosa, and oral delivery requires relatively high doses to induce autophagy. The parenteral injection of trehalose-releasing nanogels proposed in this study offers an alternative mode of delivery. This study aimed to develop stable colloidal dispersions of trehalose-rich nanogels that could sustainably release trehalose under physiologically relevant conditions. The nanogel design was based on the covalent incorporation of 6-O-acryloyl-trehalose within a polymer network. A series of nine trehalose-rich nanogels with highly conjugated trehalose (up to 59 % w/w) were synthesized and shown to sustainably release trehalose at a rate that is not dose dependent. The nanogels were optimized to keep colloidal stability in serum-enriched cell culture media. The stable nanogels were not cytotoxic to primary HUVECs. Two selected nanogels with opposite surface charges were subjected to extended in vitro characterization that included a cellular uptake study and a hemocompatibility assay. Both nanogels were efficiently taken up by HUVECs during a short incubation. They also proved not to be hemolytic to human RBCs in concentrations up to 2.0 mg/mL. Finally, an in vivo autophagy stimulation study employing transgenic zebrafish and Drosophila larvae demonstrated that prolonged exposure to a cationic trehalose-releasing nanogel can induce autophagic activity in in vivo systems without any detectable toxicity.

Objective Obesity is a multidimensional condition that is treatable by the restoration of a lean phenotype; however, some obesity-related outcomes may persist after weight normalization. Among the organs of the human body, the brain possesses a relatively low regenerative capacity and could retain perturbations established as a result of developmental obesity. Calorie restriction (CR) or a restricted ketogenic diet (KD) are successfully used as weight loss approaches, but their impact on obesity-related effects in the brain have not been previously evaluated. Methods We performed a series of experiments in a rat model of developmental obesity induced by a 12-week cafeteria diet, followed by CR to implement weight loss. First, we assessed the impact of obesity on neurogenesis (BrdU incorporation into the hippocampus), cognitive function (water maze), and concomitant changes in hippocampal protein expression (GC/MS-MS, western blot). Next, we repeated these experiments in a rat model of weight loss induced by CR. We also measured mitochondrial enzyme activity in rats after weight loss during the fed or fasting state. This study was extended by additional experiments with restricted KD used as a weight loss approach in order to compare the efficacy of two different nutritional interventions used in the treatment of obesity on hippocampal functions. By using a modified version of the water maze we evaluated cognitive abilities in rats subjected to weight loss by CR or a restricted KD. Results In this study, obesity affected metabolic processes, upregulated hippocampal NF-κB, and induced proteomic differences which were associated with impaired cognition and neurogenesis. Weight loss improved neurogenesis and enhanced cognition. While the expression pattern of some proteins persisted after weight loss, most of the changes appeared de novo revealing metabolic adjustment by overactivation of citrate synthase and downregulation of ATP synthase. As a consequence of fasting, the activity of these enzymes indicated hippocampal adaptation to negative energy balance during the weight loss phase of CR. Moreover, the effects on cognitive abilities measured after weight loss were negatively correlated with the animal weight measured at the final stage of weight gain. This was alleviated by KD, which improved cognition when used as a weight loss approach. Conclusions The study shows that cognition and mitochondrial metabolism in the hippocampus are affected by CR- or KD-induced weight loss.

Two highly efficient commercial organic photosensitizers—azure A (AA) and 5-(4-aminophenyl)-10,15,20-(triphenyl)porphyrin (APTPP)—were covalently attached to the glass surface to form a photoactive monolayer. The proposed straightforward strategy consists of three steps, i.e., the initial chemical grafting of 3-aminopropyltriethoxysilane (APTES) followed by two chemical postmodification steps. The chemical structure of the resulting mixed monolayer (MIX_TC_APTES@glass) was widely characterized by X-ray photoelectron (XPS) and Raman spectroscopies, while its photoactive properties were investigated in situ by UV–Vis spectroscopy with α-terpinene as a chemical trap. It was shown that both photosensitizers retain their activity toward light-activated generation of reactive oxygen species (ROS) after immobilization on the glassy surface and that the resulting nanolayer shows high stability. Thanks to the complementarity of the spectral properties of AA and APTPP, the effectiveness of the ROS photogeneration under broadband illumination can be optimized. The reported light-activated nanocoating demonstrated promising antimicrobial activity toward Escherichia coli (E. coli), by reducing the number of adhered bacteria compared to the unmodified glass surface.

The blood&ndash, brain barrier is the structure (BBB), which isolates the central nervous system from the external environmental. During a stroke, the BBB gets damaged, which is accompanied by changes in the concentrations and distributions of claudin-5, occludin, ZO-1, and other building blocks of the BBB. The aim of this study was to assess the concentrations of selected components of the BBB&mdash, occludin, claudin-5, and zonulin (ZO-1)&mdash, and to define a potential relationship between the concentrations of these three substances and the type of stroke, the location and extent of the infarct focus, the neurological/functional status in the acute phase of the disease, and the patient&rsquo, s clinical profile. Methods: In this prospective study, we qualified patients with first-in-life stroke. All patients were analyzed according to: the presence of comorbidities, type of stroke (OCSP), treatment type in the first day of hospitalization, hemorrhagic transformation of infarct focus (ECASS), neurological status on the first day of stroke (NIHSS), functional status (mRS) on the ninth day of disease. In all patients, the plasma concentrations of claudin-5, occludin, and ZO-1 on the first day of stroke were examined and next, the mean concentrations were analyzed and compared between subgroups created on the basis of demographical and clinical features. Results: The mean concentration of occludin was significantly higher in patients with partial anterior cerebral infarct (PACI) compared to patients with posterior cerebral infarct (POCI, 1.03 vs. 0.66 ng/mL, p = 0.009) and in patients with location of ischemic stroke in the carotid artery supply compared with in the vertebrobasilar supply (respectively: 1.036 vs. 0.660 ng/mL, p = 0.009). The mean concentration of claudin 5 was significantly higher in patients with PACI compared to patients with POCI (0.37 vs. 0.21 ng/mL, p = 0.011) and in patients with location of ischemic stroke in the carotid artery supply in comparison with vertebrobasilar supply (respectively: 0.373 vs. 0.249 ng/mL, p = 0.011). The differences in mean occludin and claudin 5 concentrations between female and male were statistically not significant, similarly between patients &lt, 65 years and older. A significantly higher mean concentration of zonulin was observed in patients &gt, 65 years of age compared to younger patients (0.59 vs. 0.48 ng/mL, p = 0.010) and in patients with arterial hypertension compared to patients without the disease (0.63 ng/mL vs. 0.26 ng/mL, p = 0.026). There were no statistically significant relationships between the concentration of occludin, claudin 5, and zonulin and the neurological status according to the NIHSS on the first day of stroke. Conclusions: The location of stroke in the anterior part of the brain&rsquo, s blood supply is associated with high blood levels of occludin and claudin 5 in the acute phase of stroke. The blood concentration of occludin is significantly lower in lacunar stroke comparing to this in non-lacunar stroke. Old age and arterial hypertension correlate positively with the concentration of zonulin 1 in acute stroke. There is no relationship between the blood levels of occludin, claudin 5, and zonulin 1 on the first day of stroke and the neurological and functional status in the acute phase of the disease.

Microfluidic technology is a powerful tool to precisely establish artificial microenvironments and has been used to generate numerous biomimetic devices. Here, we present a combined microenvironment platform, which consists of microchamber microfluidics filled with thermoresponsive glycomicrogels, and enables live-cell immobilization and continuous observation. Poly(N-isopropylacrylamide) microgels containing trehalose has been selected from our previous study to possess adequate physicochemical characteristics and provide potential multivalent interactions with cell surfaces. We show that the designed microplatform enables small population of cells to be trapped in individual parallel microchambers and further immobilized in an artificial extracellular matrix. We applied our platform to long-term imaging experiments and studied HeLa cell growth dynamics under continuous, diffusion-dominated medium exchange. The mathematical modeling revealed that regardless of the initial number of cells, the growth dynamic follows the exponential growth pattern over the analyzed timespan (one week). These results confirm that the presented microsystem facilitates the long-term cell culture in a cellular-mimicking microenvironment without reaching environmental constraints.

Due to its biocompatibility and advantageous electrochemical properties, platinum is commonly used in the design of biomedical devices, e.g., surgical instruments, as well as electro-medical or orthopedic implants. This article verifies the hypothesis that a thin layer of sputter-coated platinum may possess antibacterial effects. The purpose of this research was to investigate the adhesion and growth ability of a model strain of Gram-negative bacteria, Escherichia coli, on a surface of a platinum-coated glass slide. Although some previous literature reports suggests that a thin layer of platinum would inhibit the formation of bacterial biofilm, the results of this study suggest otherwise. The decrease in the number of bacterial cells attached to the platinum-coated glass, which was observed within first three hours of culturing, was found to be a short-time effect, vanishing after 24 h. Consequently, it was shown that a thin layer of sputter-coated platinum did not exhibit any antibacterial effect. For this reason, this study indicates an urgent need for the development of new methods of surface modification that could reduce bacterial surface colonization of platinum-based biomedical devices.

Obesity associates with plethora of dysfunctions including changes in the brain structures, like hippocampus which is particularly susceptible to metabolic or inflammatory alterations. The is a need to expand knowledge about the obesity related brain changes in order to enable development of new therapeutic interventions, that would support not only weight loss but also could help to restore the after-effects of obesity in the brain. In the present report we evaluated the hippocampal efficiency of obese rats. In order to induce obesity 4 weeks old male rats were fed with a chow that mimics human western diet for 12 weeks (model of diet induced obesity). Then the cognitive functions (water maze), neurogenesis (BrdU detection) and global proteomic changes (MS/MS analysis) were analyzed in separate cohorts of animals. Obese rats had worsen cognitive flexibility than normal rats, but the spatial learning and long term memory were unaffected. The number of BrdU/NeuN positive cells was lowered in obese animals suggesting decreased hippocampal neurogenesis. The analysis of proteins revealed that about 120 proteins significantly differed between hippocampi of obese and normal rats. Neuronal calcium sensor 1 (NCS-1) which participates in mechanisms involving synaptic plasticity and acquisition of spatial memory was decreased in obese rats. Neuromodulin and vinculin, required for synaptic plasticity also decreased in these animals. Impaired neurogenesis was associated with decreased level of: receptor-type tyrosine-protein phosphatase zeta and cytoplasmic dynein 1 light intermediate chain 2, which play crucial roles in controlling axonal growth and neuronal migration. Altogether the data show that proteomic changes related to obesity associate with impaired hippocampal functioning. The same set of experiments was performed on the additional cohort of obese animals subsequent to weight loss to examine if the observed brain changes persist if obesity is reversed. This research is supported by Nutricia Foundation, grant no. RG 1/2017.

Mitochondria change their morphology and distribution depending on the metabolism and functional state of a cell. Here, we analyzed the mitochondria and selected structures in female germ-line cysts in a representative of clitellate annelids – the white worm Enchytraeus albidus in which each germ cell has one cytoplasmic bridge that connects it to a common cytoplasmic mass. Using serial block-face scanning electron microscopy (SBEM), we prepared three-dimensional ultrastructural reconstructions of the entire selected compartments of a cyst at the advanced stage of oogenesis, i.e. the nurse cell, cytophore, and cytoplasmic bridges of all 16 cells (15 nurse cells and oocyte). We revealed extensive mitochondrial networks in the nurse cells, cytophore and mitochondria that pass through the cytoplasmic bridges, which indicates that a mitochondrial network can extend throughout the entire cyst. The dynamic hyperfusion state was suggested for such mitochondrial aggregations. We measured the mitochondria distribution and revealed their polarized distribution in the nurse cells and more abundant accumulation within the cytophore compared to the nurse cell. A close association of mitochondrial networks with dispersed nuage material, which seems to be the structural equivalent of a Balbiani body, not described in clitellate annelids so far, was also revealed.

Live-cell fluorescence image segmentation is an essential step in many studies, including in drug research and other contexts where keeping cells alive is crucial. Several segmentation algorithms and programs have been previously proposed; however, they do not work sufficiently well on top-down pictures with overlapping cells. Our proposed algorithm, called GRABaCELL, utilizes Graph Cut, Watershed segmentation and Hough Circular Transform to improve automatic segmentation and counting living cells. We also introduce a modified accuracy metric to determine the quality of segmentation in terms of the number of cells detected in the image. The GRABaCELL method results are vastly better in visual assessment, by both Dice index and modified accuracy metric, than all other compared methods maintaining not only a high value of these indices but also a relatively small spread.

Tardigrades are small, globally widespred invertebrates that need at least a thin layer of water to be active. There are gonochoric, hermaphroditic, and parthenogenetic species among them. The main aim of this study was to analyze the structure of the ovary, the structure of female germ cell clusters, and the course of oogenesis in the parthenogenetic species Hypsibius exemplaris, which in 2007 was recognized as a model organism. The material was analyzed using light and confocal microscopy as well as transmission and scanning electron microscopy. Histochemical and immunohistochemical methods were used. Our study showed that in the meroistic-polytrophic ovary of the examined species, branched germ cell clusters are formed in which one cell differentiates into an oocyte while the remaining cells become trophocytes. Vitellogenesis is of the mixed type: the first part of the yolk is synthesized by the oocyte (autosynthesis); the second part is synthesized by trophocytes and transported to the oocyte by cytoplasmic bridges; and the third part is synthesized outside the ovary (in storage cells) and transported to the oocyte by endocytosis. At the end of oogenesis, the trophocytes die by apoptosis. Parthenogenetic female of H. exemplaris lays from one to a dozen smooth eggs into exuviae.

To date, inconsistent results evaluating the effect of parameters on mechanical thrombectomy (MT) outcomes in stroke-patients have been published. This study aimed to identify the key parameters for functional status after MT in stroke-patients in short and long-term follow-up. Method: The study analysis focused on the relevance of selected clinical and non-clinical parameters to the functional status of the patients after MT. Results: 417 stroke-patients (mean age 67.8 ± 13.2 years) were qualified. Atrial fibrillation, and leukocytosis were significant for the neurological status on the first day of stroke (p = 0.036, and p = 0.0004, respectively). The parameters with the strongest effect on the functional status on day 10 were: age (p = 0.009), NIHSS (p = 0.002), hyperglycemia (p = 0.009), the result in TICI (p = 0.046), and first pass effect (p = 0.043). The parameters with the strongest effect on the functional status on day 365 were: age and NIHSS on the first day of stroke (p = 0.0002 and 0.002, respectively). Leukocytosis and the neurological status at baseline were key parameters associated with ICB after MT (p = 0.007 and p = 0.003, respectively). Conclusions: Age and neurological status in the ultra-acute phase of stroke are crucial for the functional status in short and long-term observations of patients treated with mechanical thrombectomy. Atrial fibrillation, hyperglycemia, and inflammatory state are relevant to the short-term post-stroke functional status. First pass effect and the degree of post-interventional reperfusion are important technical parameters to the short-term functional status. Neurological status and white blood count during the acute phase are associated with a high rate of post-procedural intracranial bleeding.

Background: The aim of the study was to assess the relevance of chronic rhinosinusitis (CRS) CT features to the efficacy of mechanical thrombectomy (MT) in patients with acute ischemic stroke. Methods: This study included 311 patients qualified for MT in whom the CRS features were assessed based on a CT scan, according to the Lund-Mackay (L-M) score. Clinical, inflammatory parameters, patients neurological (NIHSS) and functional status (mRS), and recanalisation efficacy (TICI) were compared between patients with mild lesions (L-M score 0–3 points)-group 1, and patients with more severe lesions (L–M score 4–24)-group 2. Results: There was a significant difference in the NIHSS on day seven after stroke onset-10 points in group 1 and 14 points in group 2, p = 0.02. NIHSS ≤ 6 points on day seven was found in 41.9% of patients in group 1, and in 27.5% in group 2, p = 0.042. There were no significant differences in mRS score and in the TICI score. L-M score, lipid abnormalities and CRP were factors associated with NIHSS ≥ 7 points on day 7. Conclusions: The CT features of CRS may be used as a prognostic tool for early prognosis assessment in stroke patients.

The effect of vacuum degassing of activated sludge on biological wastewater treatment over a total period of 4 years was examined. In half of the period studied, the treatment system was classic, where wastewater, after a biological reactor with nitrification-denitrification and biological dephosphatation, was directed to secondary settlers. In the second period, the treatment system was modified so that the sludge from the biological reactor was directed to the vacuum degasser, followed by the secondary settling tanks (vacuum degassing activated sludge system; VDAS). As a result of using the VDAS, a significant improvement in sludge settling ability was recorded, which allowed the plant operators to increase the amount of biomass in the biological reactor. Differences in the quantity and quality of the wastewater influent were found during both periods. However, the quality of treated wastewater and the efficiencies of nutrient removal and removal rates were usually better in the system with vacuum degassing. An improvement was achieved not only for the elimination of the different forms of nitrogen, which by definition were to be removed as ammonia and gaseous nitrogen, but also for organic compounds expressed as COD and phosphorus. As a result of the lower sludge loading, sludge production expressed in g SS per g COD removed was significantly lower (20 %) with the VDAS.

A series of thermoresponsive glycomicrogels with trehalose in the cross-links or with trehalose in the cross-links and as pending moieties was synthesized. These materials were obtained by surfactant-free precipitation copolymerization of N-isopropylacrylamide and various amounts of trehalose monomers. The resultant particles showed a spherical shape and a submicrometer hydrodynamic size with a narrow size distribution. At 25 °C, glycomicrogels in solutions with physiological ionic strength formed stable colloids, which further gelled upon heating to physiological temperature forming a macroscopic hydrogel with an interconnected porous structure. These extremely soft matrices with dynamic storage modulus in the range of 9-70 Pa were examined in 3D culture systems for HeLa cell culture in comparison to traditional 2D mode. They showed relatively low syneresis over time, especially when glycomicrogels with a high content of hydrophilic trehalose were used as building blocks. An incorporated pending trehalose composed of two α,α'-1,1'-linked d-glucose moieties was used with the intention of providing multivalent interactions with glucose transporters (GLUTs) expressed on the cell surface. A better cell viability was observed when a soft hydrogel with the highest content of trehalose and the lowest syneresis was used as a matrix compared to a 2D control assay.

The body cavity cells (storage cells, storage bodies) of four species of Parachela (hermaphroditic Isohypsibius granulifer granulifer, parthenogenetic Hypsibius dujardini, gonochoristic Xerobiotus pseudohufelandi, gonochoristic Macrobiotus polonicus) were analysed during their active life using light, confocal (laser scanning), and scanning and transmission electron microscopy. The ultrastructure of the storage cells confirmed previous studies suggesting a high level of metabolic activity. Additionally, we revealed the participation of the storage cells of H. dujardini, I. g. granulifer, and M. polonicus in the synthesis of vitellogenins. This did not seem to apply for X. pseudohufelandi. All of the species that were examined in this study accumulated polysaccharides, proteins, and lipids in their body cavity cells, but the amount of these components differed in each species. Isohypsibius g. granulifer accumulated a huge amount of polysaccharides and smaller amounts of lipids and proteins, H. dujardini and M. polonicus primarily accumulated lipids and small amounts of polysaccharides and proteins, whereas X. pseudohufelandi primarily accumulated polysaccharides and lipids, and a small amount of proteins.

In Hypsibius dujardini (Doyere, 1840), the endodermal region of the digestive system, which is called the midgut, spreads along the entire length of the body. Its wall is formed by a simple epithelium that is composed of digestive cells. In this paper, we present the first report on the presence of two groups of midgut regenerative cells that form two ‘epithelial rings’ – anterior and posterior. Additionally, we observed the proliferative abilities of the midgut regenerative cells, thus confirming the statement that they play the role of midgut stem cells. The precise ultrastructure of the digestive and regenerative cells was determined using transmission electron microscopy. Changes in the digestive and regenerative cells were correlated with the different stages of oogenesis. The process of oogenesis in H. dujardini took 4 days (at a temperature of 16 °C). Reserve material gradually accumulated in the cytoplasm of the digestive cells and histochemical staining showed that it primarily contained proteins, polysaccharides and a small quantity of lipids. The reserve material accumulated during vitellogenesis, and it began to decrease during choriogenesis. During the simplex stage, when the entire buccal–pharyngeal apparatus was expelled from the body, the stages of oogenesis were advanced, the midgut was much reduced, and the reserve material was exploited by the animal.

Thulinius ruffoi is a freshwater species that has the ability to reproduce via parthenogenesis. A meroistic polytrophic ovary is present in this species. Analyses of the germarium structure, and formation and organization of female germ-cell clusters were performed using light, confocal laser scanning, transmission electron and serial block-face scanning electron microscopy. The germarium is the small, anterior part of an ovary that contains putative germ-line stem cells. In the studied species, the female germ-cell clusters are large and branched. Only one cell in each cluster develops into an oocyte, while all the other cells become trophocytes. In this paper, we present the first report on the presence of F-actin as a component of the intercellular bridges that connect the cells in the germ-cell cluster of T. ruffoi. Moreover, our results show that the female germ-cell clusters are formed as the result of both synchronous and asynchronous divisions and that their organization can vary not only between individuals of the investigated species, but also that clusters developing simultaneously within the same ovary can have a different spatial organization.

The middle region of the digestive system, the midgut of freshwater shrimpNeocaridina davidiis composed of a tube-shaped intestine and the hepatopancreas formed by numerous caeca. Two types of cells have been distinguished in the intestine, the digestive cells (D-cells) and regenerative cells (R-cells). The hepatopancreatic tubules have three distinct zones distinguished along the length of each tubule—the distal zone with R-cells, the medial zone with differentiating cells, and the proximal zone with F-cells (fibrillar cells) and B-cells (storage cells). Fasting causes activation of cell death, a reduction in the amount of reserve material, and changes in the mitochondrial membrane potential. However, here we present how the concentration of ROS changes according to different periods of fasting and whether re-feeding causes their decrease. In addition, the activation/deactivation of mitochondrial superoxide dismutase (MnSOD) was analyzed. The freshwater shrimpsNeocaridina davidi(Crustacea, Malacostraca, Decapoda) were divided into experimental groups: animals starved for 14 days, animals re-fed for 4, 7, and 14 days. The material was examined using the confocal microscope and the flow cytometry. Our studies have shown that long-term starvation increases the concentration of free radicals and MnSOD concentration in the intestine and hepatopancreas, while return to feeding causes their decrease in both organs examined. Therefore, we concluded that a distinct relationship between MnSOD concentration, ROS activation, cell death activation and changes in the mitochondrial membrane potential occurred.

Modern multi-omics studies introduce a major challenge for biomedical data storage, processing and integration. The ability to utilize multiple different measurement techniques in order to provide a comprehensive view on the studied processes is becoming a standard in molecular biology and medicine, increasing the need for the development of new strategies used for handling various types of data in a single environment. To address this need we developed a remotely accessible BioTest platform which allows to process multi-omics data, obtained using various measurement techniques, including next-generation sequencing (NGS), oligonucleotide microarrays, and mass spectrometry. This allows to process various biomedical data from genomic level, trough transcriptomics, up to the proteomic level on a scalable environment that can handle even the largest datasets. Integrated version control for library files and intermediate results as well as the possibility of designing workflows comprised of various algorithms, ensures that all of the results are highly reproducible. Incorporated support for MapReduce jobs and Big Data processing on a high performance computer cluster allows to test multiple various hypotheses at the same time.