Komórkowe strategie reakcji pająków na stres środowiskowy

The aim of the work was to compare cellular effects of natural (heat shock and starvation) and anthropogenic (organophosphorous pesticide) stressors in two, behaviorally different spider species: web building Agelena labyrinthica and wandering Xerolycosa nemoralis, from areas variously polluted by heavy metals. It was crucial to check whether the response of female and male spiders, chronically exposed to high metal concentrations in their habitats to additional stressors, is similar as in spiders from the slightly polluted site and decide which of the stressogenic factors exerts the strongest additional costs for an organism. Since they are the midgut glands that play a strategic role in digestion and detoxification, the analyses were conducted in this organ. Using cytometric techniques and spectrophotometric methods, the following parameters were measured: — the intensity of apoptotic and necrotic changes as well as the quantity of cells with depolarized mitochondria and the caspase-like protease activity level; — the quantitative changes in the cells that positively react with metallothionein (Mt) and heat shock protein (Hsp70) antibodies; — the level of selected antioxidative parameters (total glutathione concentration, activity of selene- -dependent glutathione peroxidase; GPOX, selene- independent glutathione peroxidase; GSTPx, glutathione S-transferase; GST, superoxide dismutase; SOD, catalase; CAT). Obtained results demonstrated that the individuals from heavily polluted areas were more sensitive to applied stressing factors than the animals that have not been exposed to environmental pollutants. The diversity in the cellular response of female and male spiders from variously polluted habitats confirms the necessity of considering the aspect of gender in this kind of comparisons. Mixing males and females together may conceal quantitative correlations between analysed parameters, when coping with long lasting pre-exposure. From among all stressing factors applied, the most pronounced changes, measured as the percentage of apoptotic cells in spider midgut glands, were caused by hunger. Pro-apoptotic effect of the exposure to the applied factors diminished according to the following order: hunger > heat shock + dimethoate > dimethoate > heat shock. Female X. nemoralis seemed more resistant to starvation, since the stressing factor did not enhance necrotic changes in the cells of their midgut glands. In heat shock and/or dimethoate exposed groups the degree of necrotic changes in the organ was higher than in respective A. labyrinthica groups. This may indicate higher sensitivity of the wandering spider species to the remaining stressing factors. Enzymatic neutralization of reactive oxygen species in female X. nemoralis was revealed mainly by the increased activity of SOD and CAT. In spiders also glutathione concentration and activity of glutathione- dependent enzymes were elevated in response to stressing factors. Antioxidative responses registered in A. labyrinthica midgut glands were poorly differentiated and appeared mainly as increased glutathione concentration and gender-related GPOX (females and males) or GSTPx (males) activity. The exposure to stressing factors resulted also in the induction of stress proteins in the spiders. Female X. nemoralis had an increased Mt synthesis while males, under the same conditions, had higher Hsp70 production. In case of female A. labyrinthica stressing factors stimulated the Hsp70 synthesis, while in males both Hsp70 and Mt production was enhanced. In case of wolf spiders (X. nemoralis) the cytoprotective role of SOD and CAT may be verified by positive correlations found for their activity and necrosis and/or apoptosis intensity in the organ. In case of tunnel spiders (A. labyrinthica) both parameters were either negatively correlated or there were no correlations at all. Cytoprotective role in A. labyrinthica is likely played by Hsp70, since the percentage of cells dying according to necrotic pathway was lower in those groups where higher percentage of Hsp70 positive cells was found. Analysed indices of cell death in case of both spider species are good biomarkers of general stress, caused by the exposure to both natural and anthropogenic factors. However, the lack of unequivocal correlations between the level of measured parameters and the kind of stressor is the reason why it is impossible to point out precisely the cause of the changes, but only to confirm the stress itself that, dependently on its intensity, increased apoptotic and/or necrotic changes. The analyses of quantitative ratios may be used in the comparisons of sensitivity of the species to applied stressing factors. Obtained results confirmed inter-species differences in antioxidative strategies found in previous investigations on spiders. Irrespectively of the type of stressor, in case of X. nemoralis an important defensive role in midgut glands is played by SOD and CAT while in case of A. labyrinthica — glutathione and Hsp70. Quantitative changes in the Mt-positive cells in particular experimental groups may indicate that in case of spiders the proteins contribute rather to antioxidative defense than in direct metal binding

Cell death in the epithelia of the intestine and hepatopancreas in Neocaridina heteropoda (Crustacea, Malacostraca)

The endodermal region of the digestive system in the freshwater shrimp Neocaridina heteropoda (Crustacea, Malacostraca) consists of a tube-shaped intestine and large hepatopancreas, which is formed by numerous blind-ended tubules. The precise structure and ultrastructure of these regions were presented in our previous studies, while here we focused on the cell death processes and their effect on the functioning of the midgut. We used transmission electron microscopy, light and confocal microscopes to describe and detect cell death, while a quantitative assessment of cells with depolarized mitochondria helped us to establish whether there is the relationship between cell death and the inactivation of mitochondria. Three types of the cell death were observed in the intestine and hepatopancreas- apoptosis, necrosis and autophagy. No differences were observed in the course of these processes in males and females and or in the intestine and hepatopancreas of the shrimp that were examined. Our studies revealed that apoptosis, necrosis and autophagy only involves the fully developed cells of the midgut epithelium that have contact with the midgut lumen-D-cells in the intestine and B- and F-cells in hepatopancreas, while E-cells (midgut stem cells) did not die. A distinct correlation between the accumulation of Ecells and the activation of apoptosis was detected in the anterior region of the intestine, while necrosis was an accidental process. Degenerating organelles, mainly mitochondria were neutralized and eventually, the activation of cell death was prevented in the entire epithelium due to autophagy. Therefore, we state that autophagy plays a role of the survival factor

Effective activation of antioxidant system by immune-relevant factors reversely correlates with apoptosis of Eisenia andrei coelomocytes

Oxidative stress is harmful to the microbes but also to the host, and may result in bystander damage or death. Because of this, respiratory burst triggered in phagocytes by pathogens is counteracted by production of antioxidative factors. The aim of this work was to examine effectiveness of the latter system in earthworms Eisenia andrei by induction of reactive oxygen species, lipofuscin and phenoloxidase by natural (LPS, zymosan, Micrococus luteus) and synthetic (phorbol ester, PMA) stimulants. The compounds impaired numbers, viability (increased apoptosis) and composition of coelomocytes, and triggered the antioxidant activity of catalase and selenium-dependent glutathione peroxidase. The natural pathogenic compounds, unlike PMA, strongly activated antioxidative responses that diminished cell apoptosis. Moreover, repeated exposure to the same or different pathogenic compounds did not induce respiratory burst exhausted phenotype showing that coelomocytes are constantly at bay to withstand numerous infections. The current study reveals importance and efficiency of the oxidative–antioxidative systems in annelids but also confirms its evolutionary conservatism and complexity even in lower taxa of the animal kingdom

Relationship between ROS production, MnSOD activation and periods of fasting and re-feeding in freshwater shrimp Neocaridina davidi (Crustacea, Malacostraca)

The middle region of the digestive system, the midgut of freshwater shrimp Neocaridina davidi is 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 shrimps Neocaridina 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

Biomechanical and morphological stability of acellular scaffolds for tissue-engineered heart valves depends on different storage conditions

Currently available bioprosthetic heart valves have been successfully used clinically; however, they have several limitations. Alternatively, tissue-engineering techniques can be used. However, there are limited data concerning the impact of storage conditions of scaffolds on their biomechanics and morphology. The aim of this study was to determine the effect of different storage conditions on the biomechanics and morphology of pulmonary valve dedicated for the acellular scaffold preparation to achieve optimal conditions to obtain stable heart valve prostheses. Scaffold can then be used for the construction of tissue-engineered heart valve, for this reason evaluation of these parameters can determine the success of the clinical application this type of bioprosthesis. Pulmonary heart valves were collected from adult porcines. Materials were divided into five groups depending on the storage conditions. Biomechanical tests were performed, both the static tensile test, and examination of viscoelastic properties. Extracellular matrix morphology was evaluated using transmission electron microscopy and immunohistochemistry. Tissue stored at 4 °C exhibited a higher modulus of elasticity than the control (native) and fresh acellular, which indicated the stiffening of the tissue and changes of the viscoelastic properties. Such changes were not observed in the radial direction. Percent strain was not significantly different in the study groups. The storage conditions affected the acellularization efficiency and tissue morphology. To the best of our knowledge, this study is the first that attributes the mechanical properties of pulmonary valve tissue to the biomechanical changes in the collagen network due to different storage conditions. Storage conditions of scaffolds for tissue-engineered heart valves may have a significant impact on the haemodynamic and clinical effects of the used bioprostheses

Influence of soil contaminated with cadmium on cell death in the digestive epithelium of soil centipede Lithobius forficatus (Myriapoda, Chilopoda)

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 deathmechanisms that take part in homeostasismaintenance 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

Psychometric properties assessment of the Meister questionnaire (Polish version) used in evaluating mental load among nurses

Background: The objective of this study was to assess psychometric properties of the Polish version of the Meister questionnaire for subjective assessment of work-related mental load among nurses. Material and Methods: A group of 211 nurses (mean age, 43.1±7.26) participated in our study. In the process of adaptation stability (test-retest) reliability and factorial validity were assessed. The Maslach Burnout Inventory (MBI) was used as a tool for assessing construct validity). Results: Factor analysis of the questionnaire confirmed a very good fit between input data and factor model (Kaiser-Mayer-Olkin measure of 0.83). Selected variables, including overload, monotony, and non-specific load, explained 57%, 41%, and 66% of the variance, respectively. The analysis proved high validity of the tool, Cronbach alpha was 0.83 for the total score. The correlation factor for questions and scores in subscales was high. Repeated measurements resulted in similar outcomes, which proved the stability of this measure. The analysis of construct validity showed that overload, monotony, and non-specific load presented positive correlation with emotional exhaustion and depersonalisation. Correlation between variables was moderate (rHO = 0.39–0.58; p < 0.001). The total score indicated the following correlations: strong positive with emotional exhaustion (rHO = 0.6, p < 0.001), negative with personal achievements, and positive and moderate with depersonalisation (rHO = –0.14; p < 0.05, rHO = 0.48; p < 0.001). Conclusion: The Polish version of the Meister questionnaire meets all psychometric criteria and may be used for evaluating mental load among nurses. Med Pr 2013;64(3):349–35

Can insecticide-free clean water regenerate the midgut epithelium of the freshwater shrimp after dimethoate treatment?

Insecticides such as dimethoate persist for a long time in freshwater environments, influencing the physiology of the animals inhabiting such environments. In aquatic organisms, toxic substances can enter the body through the epidermis and the digestive system. The midgut is part of this system in which intense processes constitute a barrier against the effects of toxic substances on the body. The aim of this study was to evaluate the toxic potential of dimethoate in the midgut epithelium of the freshwater shrimp Neocaridina davidi, emphasizing ultrastructural alterations. However, the additional and main purpose was to determine whether the midgut epithelium can regenerate after placing animals in insecticide-free clean water after various periods of exposure to dimethoate. N. davidi originates from Asia, but it has also been described in European rivers. This species is of particular interest among breeders worldwide due to its ease of breeding and reproduction. The animals were treated with dimethoate for 1, 2, and 3 weeks and then placed in clean water for 1, 2, and 3 weeks. The qualitative and quantitative analysis revealed different sensitivity of organs forming the midgut in freshwater crustaceans and the possibility for midgut regeneration after insecticide exposure. We concluded that different processes were triggered in the intestine and hepatopancreas to regenerate cells after damage, and mitochondria were the first organelles to respond to the appearance of a stressor in the living environment

Ocena sterowania potencjałami przedsionkowymi podczas stałej stymulacji serca w trybie VDD z zastosowaniem pojedynczej elektrody umieszczonej w drodze odpływu prawej komory

Stymulacja VDD pojedynczą elektrodą Wstęp: Stymulację VDD z pojedynczą elektrodą uznano za alternatywny do DDD tryb stymulacji u chorych z blokiem przewodzenia przedsionkowo-komorowego (p-k) i zachowaną prawidłową funkcją węzła zatokowego. Położenie końcówki elektrody w drodze odpływu prawej komory (RVOT, right ventricular outflow tract) powoduje w naturalny sposób łukowate ułożenie elektrody i może korzystnie wpływać na stabilizację jej pozycji względem bocznej ściany prawego przedsionka. Cel pracy: Ocena sterowania potencjałami przedsionka podczas stałej stymulacji serca typu VDD z pojedynczą elektrodą umieszczoną w RVOT u chorych z blokiem p-k w porównaniu ze stymulacją typu VDD z pojedynczą elektrodą umieszczoną w wierzchołku prawej komory (RVA, right ventricular apex) oraz stymulacją typu DDD. Materiał i metody: Do implantacji układu VDD lub DDD zakwalifikowano 60 chorych z zaawansowanym blokiem p-k, u których nie stwierdzano cech choroby węzła zatokowego, w tym niewydolności chronotropowej. Chorzy byli kwalifikowani losowo do grupy VDD lub DDD w stosunku odpowiednio 2:1, a następnie chorzy z grupy VDD byli losowo włączani do grupy RVOT lub RVA. W pomiarach telemetrycznych wykonywanych w spoczynku wartości amplitudy fali P w grupie chorych ze stymulacją typu VDD-RVOT były niższe niż w grupie chorych ze stymulacją typu DDD w pozycji leżącej, natomiast nie obserwowano takich różnic w pozycji stojącej. Podobne wyniki uzyskano podczas badania kontrolnego po 3 miesiącach od implantacji. Analiza danych wykazała, że potencjały przedsionkowe w grupie chorych ze stymulacją typu VDD-RVOT (a nie w pozostałych grupach) są uzależnione od pozycji ciała jedynie w pomiarach wykonywanych przed wypisem ze szpitala, a nie po 3 miesiącach. Wyniki: W 24-godzinnym zapisie EKG u kilku chorych w każdej z grup obserwowano undersensing. Nie stwierdzono znamiennych różnic w częstości undersensingu pomiędzy poszczególnymi grupami chorych. Wnioski: Obserwacje kliniczne potwierdziły, że stymulacja w drodze odpływu z zastosowaniem elektrod z pasywną fiksacją jest przydatnym i bezpiecznym sposobem stymulacji. Zaburzenia sterowania występują z podobną częstością w stymulacji typu VDD z końcówką elektrody umiejscowionej w drodze odpływu, jak i w stymulacji typu VDD z końcówką elektrody w wierzchołku prawej komory serca oraz w stymulacji przedsionkowo-komorowej typu DDD, a programowanie maksymalnej czułości w kanale przedsionkowym pozwoliło na optymalizację sterowania potencjałami przedsionka, bez zwiększania ryzyka zaburzeń sterowania w postaci oversensingu

Ocena sterowania potencjałami przedsionkowymi podczas stałej stymulacji serca w trybie VDD z zastosowaniem pojedynczej elektrody umieszczonej w drodze odpływu prawej komory

Stymulacja VDD pojedynczą elektrodą Wstęp: Stymulację VDD z pojedynczą elektrodą uznano za alternatywny do DDD tryb stymulacji u chorych z blokiem przewodzenia przedsionkowo-komorowego (p-k) i zachowaną prawidłową funkcją węzła zatokowego. Położenie końcówki elektrody w drodze odpływu prawej komory (RVOT, right ventricular outflow tract) powoduje w naturalny sposób łukowate ułożenie elektrody i może korzystnie wpływać na stabilizację jej pozycji względem bocznej ściany prawego przedsionka. Cel pracy: Ocena sterowania potencjałami przedsionka podczas stałej stymulacji serca typu VDD z pojedynczą elektrodą umieszczoną w RVOT u chorych z blokiem p-k w porównaniu ze stymulacją typu VDD z pojedynczą elektrodą umieszczoną w wierzchołku prawej komory (RVA, right ventricular apex) oraz stymulacją typu DDD. Materiał i metody: Do implantacji układu VDD lub DDD zakwalifikowano 60 chorych z zaawansowanym blokiem p-k, u których nie stwierdzano cech choroby węzła zatokowego, w tym niewydolności chronotropowej. Chorzy byli kwalifikowani losowo do grupy VDD lub DDD w stosunku odpowiednio 2:1, a następnie chorzy z grupy VDD byli losowo włączani do grupy RVOT lub RVA. W pomiarach telemetrycznych wykonywanych w spoczynku wartości amplitudy fali P w grupie chorych ze stymulacją typu VDD-RVOT były niższe niż w grupie chorych ze stymulacją typu DDD w pozycji leżącej, natomiast nie obserwowano takich różnic w pozycji stojącej. Podobne wyniki uzyskano podczas badania kontrolnego po 3 miesiącach od implantacji. Analiza danych wykazała, że potencjały przedsionkowe w grupie chorych ze stymulacją typu VDD-RVOT (a nie w pozostałych grupach) są uzależnione od pozycji ciała jedynie w pomiarach wykonywanych przed wypisem ze szpitala, a nie po 3 miesiącach. Wyniki: W 24-godzinnym zapisie EKG u kilku chorych w każdej z grup obserwowano undersensing. Nie stwierdzono znamiennych różnic w częstości undersensingu pomiędzy poszczególnymi grupami chorych. Wnioski: Obserwacje kliniczne potwierdziły, że stymulacja w drodze odpływu z zastosowaniem elektrod z pasywną fiksacją jest przydatnym i bezpiecznym sposobem stymulacji. Zaburzenia sterowania występują z podobną częstością w stymulacji typu VDD z końcówką elektrody umiejscowionej w drodze odpływu, jak i w stymulacji typu VDD z końcówką elektrody w wierzchołku prawej komory serca oraz w stymulacji przedsionkowo-komorowej typu DDD, a programowanie maksymalnej czułości w kanale przedsionkowym pozwoliło na optymalizację sterowania potencjałami przedsionka, bez zwiększania ryzyka zaburzeń sterowania w postaci oversensingu