KRas localizes to the plasma membrane by spatial cycles of solubilization, trapping and vesicular transport.

KRas is a major proto-oncogene product whose signaling activity depends on its level of enrichment on the plasma membrane (PM). This PM localization relies on posttranslational prenylation for membrane affinity, while PM specificity has been attributed to electrostatic interactions between negatively charged phospholipids in the PM and basic amino-acids in the C terminus of KRas. By measuring kinetic parameters of KRas dynamics in living cells with a cellular-automata-based data-fitting approach in realistic cell-geometries, we show that charge-based specificity is not sufficient to generate PM enrichment in light of the total surface area of endomembranes. Instead, mislocalized KRas is continuously sequestered from endomembranes by cytosolic PDEδ to be unloaded in an Arl2-dependent manner to perinuclear membranes. Electrostatic interactions then trap KRas at the recycling endosome (RE), from where vesicular transport restores enrichment on the PM. This energy driven reaction-diffusion cycle explains how small molecule targeting of PDEδ affects the spatial organization of KRas.

Comprehensive study of the effects of strobilurin-based fungicide formulations on Enchytraeus albidus.

Excessive application of fungicides in crop fields can cause adverse effects on soil organisms and consequently affect soil properties. Existing knowledge on the effects of strobilurin fungicides has been primarily based on toxicity tests with active ingredients, while the effects of fungicide formulations remain unclear. Therefore, this work aims to provide new data on the effects of three commercial formulations of strobilurin fungicides on the soil organism Enchytraeus albidus. The tested fungicide formulations were Retengo® (pyraclostrobin-PYR), Zato WG 50® (trifloxystrobin-TRI) and Stroby WG® (kresoxim-methyl-KM). In laboratory experiments, multiple endpoints were considered at different time points. The results showed that PYR had the greatest impact on survival and reproduction (LC50 = 7.57 mga.i.kgsoil-1, EC50 = 0.98 mga.i.kgsoil-1), followed by TRI (LC50 = 72.98 mga.i.kgsoil-1, EC50 = 16.93 mga.i.kgsoil-1) and KM (LC50 = 73.12 mga.i.kgsoil-1, EC50 ≥ 30 mga.i.kgsoil-1). After 7 days of exposure, MXR activity was inhibited at the highest concentration of all fungicides tested (6 mgPYRkgsoil-1, 15 mgTRIkgsoil-1 and 30 mgKMkgsoil-1). Furthermore, oxidative stress (induction of SOD, CAT and GST) and lipid peroxidation (increase in MDA) were also observed. In addition, there was a decrease in total available energy after exposure to PYR and KM. Exposure to fungicides resulted in a shift in the proportions of carbohydrates, lipids, and proteins affecting the amount of available energy. In addition to the initial findings on the effects of strobilurin formulations on enchytraeids, the observed results suggest that multiple and long-term exposure to strobilurin formulations in the field could have negative consequences on enchytraeid populations.

Toxicity of fungicide azoxystrobin to Enchytraeus albidus: Differences between the active ingredient and formulated product.

Due to the often-excessive usage of fungicides, increasing attention is being paid to their impact on soil and non-target organisms. Risk assessments are usually based on the pure active ingredient and not on the formulated products applied in the environment. The aim of this study was therefore to investigate how azoxystrobin, the best-selling strobilurin fungicide, affects non-target soil organisms Enchytraeus albidus. To investigate the effects of the different types of azoxystrobin, E. albidus was exposed to the pure active ingredient, AZO_AI, and the formulated product, AZO_FP. Survival, reproduction, and molecular biomarkers of E. albidus were determined for different exposure durations (seven and 21 days). AZO_FP (LC50 = 15.3 mga.i./kg) showed a slightly stronger effect on survival than AZO_AI (LC50 = 16.8 mga.i./kg), yet the impact on reproduction was much stronger. Namely, while the tested concentrations of AZO_AI (EC50≥ 8 mga.i./kg) had almost no effect on reproduction, AZO_FP (EC50 = 2.9 mga.i./kg) significantly inhibited reproduction in a dose-dependent manner. Changes in enzyme activities (superoxide dismutase, catalase, glutathione-s-transferase) and malondialdehyde levels in both treatments indicated oxidative stress. Although AZO_FP had a stronger negative effect, the impact depended on the exposure time and the tested concentration. The higher toxicity of AZO_FP was a consequence of increased bioavailability and activity of the active ingredient due to the presence of adjuvants. Overall stronger adverse effects of AZO_FP suggest that the toxicity of azoxystrobin in the agricultural environment on the enchytraeid population may be underestimated. Furthermore, the results of this study highlighted the importance of comparing the toxicity of the active ingredient and the formulated product.

Flaxseed and evening primrose oil slightly affect systolic and diastolic function of isolated heart in male but not in female rats.

Considering that sex related differences in cardiac response to flaxseed (FSO) and evening primrose oil (EPO) are insufficiently known present investigation assessed the effect of these two oils, on the cardiac function of isolated rat hearts and the possible role of sex in this. The present study was carried out on 60 adult male Wistar albino rats randomly divided into 6 groups: male rats treated with EPO, dose of 10 mg/kg/day; female rats treated with EPO, dose of 10 mg/kg/day; male rats treated with FSO, dose of 300 mg/kg/day; female rats treated with FSO, dose of 300 mg/kg/day; control group of female rats treated with regular laboratory diet for animals; control group of male rats treated with regular laboratory diet for animals. Using the Langendorff technique, markers of the heart function were evaluated: the maximum and minimum rates of pressure development in the left ventricle (LV; dP/dtmax, dP/dtmin), systolic and diastolic left ventricular pressure (SLVP, DLVP, respectively), heart rate (HR) and coronary flow (CF). Male rats treated with EPO had significantly higher (p = 0.016) mean values of dP/dtmax, dP/dtmin, SLVP and DLVP (average increase for all CPPs 20%, 25%, 30% and 110%, respectively), compared to the group of male rats treated with FSO (p = 0.914). Our study results indicate that both types of PUFA oils only slightly changed the function of the isolated rat heart in male but not in female rats. Nevertheless, the difference between oil treatments was found in male rats who had stronger cardiac response after supplementation with EPO.

Multigenerational and transgenerational effects of azoxystrobin on Folsomia candida.

Soil organisms are exposed to various pollutants during several generations. However standard toxicity tests are often based on exposure in only one generation. Research of multigenerational (MG) and transgenerational (TG) effects are still quite scarce, however evidence accumulates that effects observed in one generation can be significantly different in some of the following generations, with different effects observed. Some studies suggest adaptation to pollutants, while others report severe effects in following generations. Azoxystrobin is commonly used in the prevention and treatment of fungal diseases in a wide range of economically important crops. The main aim of this study was to assess the toxic effects of azoxystrobin (AZO) on F. candida over 3 generations through the application of biochemical and population level biomarkers. Results of reproduction tests showed a significant decrease in estimated EC50 values, with EC50 for F0 being estimated at 104.44 mga.i./kgD.W.soil and only 15.4 mga.i./kgD.W.soil for F1. In F1 a significant reduction in the number of juveniles was observed, and at AZO concentration of 50 mga.i./kgD.W and higher, F1 did not reproduce. Significant oxidative stress was observed in all generations, with increased SOD and lipid damage that slowly decreased in subsequent generations. Transgenerational effects were also observed, with a significantly reduced number of juveniles in F1 and significant oxidative stress and lipid damage in all generations. IBRv2 showed that F1 was most affected, followed by F0, and least affected was F3. When considering the whole body energy budget, F1 to F3 had significantly higher WBEB compared to F0, and a shift in proportion of energy reserves occurred in F1, where the proportion of lipids increased while protein decreased. Results of this research show that considering standard toxicity tests, risks for populations of soil organisms are possibly severely underestimated. Therefore, standard toxicity guidelines should be supplemented by multigenerational tests, when possible.

The Influence of Sociodemographic Factors on the Level of Hope in Women with Breast Cancer.

Background: We aimed to determine the level of hope in breast cancer patients along with the influence of sociodemographic characteristics on the expressed level of hope. Methods: A descriptive cross-sectional study was conducted on the sample size of 72 female patients with breast cancer diagnosis. The study was conducted at the University Clinical Center of Kragujevac, Serbia from November 2020 until March 2022. Data were collected from the patients in a form of the Sociodemographic Variables Questionnaire (Q-SV). The Herth Hope Index (HHI) was used in assessing the level of hope. Results: The value of the total HHI score was ranging from 14 to 48, whereas the mean value of the HHI score was 40.00±5.92. The significantly higher level of hope was reported in female patients who: searched the Internet for gathering information on their disease (39.24±6.23; P=0.047), were religious (38.77±6.13; P=0.003), believed in God (38.45±5.51; P=0.004), had no formal medical education (38.44±5.75; P=0.036) and after therapy (38.76±6.49; P=0.022). Conclusion: Hope is an existential internal resource possibly affecting the manner in which female patients perceive their own health state and future as well. A better understanding of the meaning of hope during treatment can be of great value in supporting cancer patients.

The first case of combined oxidative phosphorylation deficiency-1 due to a GFM1 mutation in the Serbian population: a case report and literature review.

BACKGROUND: Combined oxidative phosphorylation deficiency-1 (COXPD1) resulting from a mutation in the G elongation factor mitochondrial 1 (GFM1) gene is an autosomal recessive multisystem disorder arising from a defect in the mitochondrial oxidative phosphorylation system. Death usually appears in the first weeks or years of lifespan. CASE: We report a male patient with ventriculomegaly diagnosed in the 8th month of pregnancy. The delivery was done by caesarean section and respiratory failure occurred immediately after birth. Hypoglycemia, lactic acidosis, elevated gamma-glutamyl transferase and hepatomegaly were confirmed. The brain MRI detected hypoplasia of the cerebellar hemispheres, dilated lateral ventricles, and markedly immature brain parenchyma. Epilepsy had been present since the third month. At 5 months of age, neurological follow-up showed his head circumference to be 37 cm, with plagiocephaly, a low hairline, a short neck, axial hypotonia and he did not adopt any developmental milestones. A genetic mutation, a missense variant in the GFM1 gene, was confirmed: c.748C > T (p.Arg250Trp) was homozygous in the GFM1 gene. CONCLUSIONS: To the best of our knowledge, 28 cases of COXPD1 disease caused by mutations in the GFM1 gene have been described in the literature. COXPD1 should be considered due to symptoms and signs which begin during intrauterine life or at birth. Signs of impaired energy metabolism should indicate that the disease is in the group of metabolic encephalopathies.

In Silico Guided Design of Metal/Semiconductor Photocatalysts: A Case of Cu-Modified TiO2 for Ciprofloxacin Degradation.

(1) Background: An increasing use of pharmaceutics imposes a need for the permanent development of efficient strategies, including the tailoring of highly specific new materials for their removal from the environment. Photocatalytic degradation has been the subject of increasing interest of the researchers in the field. (2) Methods: This paper is focused on the investigation of the possibility to deposit a thin metal layer on a TiO2 surface and study its photocatalytic performance for the degradation of ciprofloxacin using a combination of theoretical and experimental methods. (3) Results: Based on the extensive DFT screening of 24 d-metals' adhesion on TiO2, Cu was selected for further work, due to the satisfactory expected stability and good availability. The (Cu)TiO2 was successfully synthesized and characterized with XRD, SEM+EDS and UV-Vis spectrophotometry. The uniformly distributed copper on the TiO2 surface corresponds to the binding on high-affinity oxygen-rich sites, as proposed with DFT calculations. The photocatalytic degradation rate of ciprofloxacin was improved by about a factor of 1.5 compared to the bare non-modified TiO2. (4) Conclusions: The observed result was ascribed to the ability of adsorbed Cu to impede the agglomeration of TiO2 and increase the active catalytic area, and bandgap narrowing predicted with DFT calculations.

Mitigation of OMW toxicity toward Enchytraeus albidus with application of additives.

The olive oil industry generates considerable amounts of olive mill wastewater (OMW) which is treated and used in agriculture, energy production, or discharged into evaporating ponds where OMW contaminated soil (OMWS) is formed. Due to the extremely high phenol content, untreated OMWS is not suitable for plants and soil organisms. This study aimed to determine the adverse effects of OMWS on Enchytraeus albidus and whether the addition of clay and diatomaceous earth can reduce these effects. Without additives, reproduction was reduced up to 35%, with high number of unhatched cocoons, indicated hatching impairment. Furthermore, acetylcholinesterase (AChE) activity decreased significantly at the highest OMWS ratio (40%), as did glutathione-S-transferase (GST) activity at two ratios (40%), indicating neurotoxic effects and oxidative stress. The application of additives (clay and diatomaceous earth) decreased phenol concentration up to 35 and 45%, respectively. Consequently, the number of juveniles increased significantly compared to the control and AChE and GST activities were not affected. However, an increased number of unhatched cocoons and lipid peroxidation were observed after diatomaceous earth treatment, indicating the occurrence of stress. Although additives can bind phenols, neutralize harmful effects and allow the use of OMWS as a fertilizer in agriculture, they should be carefully selected taking into account soil organisms as well. The use of additives to reduce the adverse effects of OMWS can be a turning point in the remediation of olive contaminated soil.

Avoidance Behaviour of Six Collembolan Species Shows Species-Specific Sensitivity-Impact of Ag NM300K.

Although standard testing guidelines use a species as a representative surrogate, species-specific sensitivity is well-known. The aim of this study was to investigate the species-specific difference in avoidance behaviour among Collembola species exposed to silver (Ag) nanomaterials (NM) (Ag NM300K). The avoidance test was performed with Folsomia candida, an international standard species in laboratory tests, and five widely distributed species with different life history traits, commonly used in small multispecies systems (Folsomia fimetaria, Proisotoma minuta, Mesaphorura macrochaeta, Protaphorura fimata and Ceratophysella denticulata). There was higher avoidance in euedaphic species, such as F. candida and F. fimetaria, compared to the epiedaphic species C. denticulata, which showed the least avoidance behaviour. An explanation may be that euedaphic species (living in deeper soil layers) are more directly exposed within the soil pores and have developed a pronounced avoidance behaviour. In contrast, species living on the surface are likely less directly exposed and hence only avoid at higher total concentrations. Additionally, difference in cuticula between the groups, providing different degrees of protection against exposure, can explain the different behaviours. The present results highlight the importance of biodiversity for the ecosystem and raise awareness on species sensitivity.

Effects of olive mill waste (OMW) contaminated soil on biochemical biomarkers and reproduction of Dendrobaena veneta.

Olive oil industry is economically important in Mediterranean countries. Disposal of olive mill waste (OMW) presents an environmental concern in those countries due to its high salinity and its high level of polyphenols. In order to reuse OMW, those properties have to change either through the filtration process and addition of adsorbents or by composting. One of the most important organisms in composting of organic wastes is earthworms. However, data on the effects of OMW on earthworms are scarce. The main aim of our study was to investigate whether OMW contaminated soil (OMW CS) causes adverse effects on molecular and organism level in epigeic earthworm Dendrobaena veneta and on microbiological activity. Changes of measured biochemical biomarkers (AChE, CAT, GST, lipids, MDA) varied depending on the quantity of added OMW CS and the exposure duration. Oxidative stress occurred after 7 days of exposure, while in most cases enzyme activity recovered after 28 days. At the highest ratio of contaminated soil (50%), reproduction was completely inhibited. The second aim was to investigate the impact of earthworms on phenol degradation and microbial activity, indicating an important role in the bioremediation of contaminated soils. Our results show that above a certain quantity an OMW CS has an adverse effect on earthworms, while the impact of earthworms on soil microbial activity was positive but transient. Yet, as the results also imply that earthworms have an impact on phenol degradation, they can be used to help remediation of OMW CS and its subsequent usage in agriculture. However, the quantity of OMW CS that can be safely added should be determined first.

Assessment of adverse effects of olive mill waste water and olive mill waste contaminated soil on springtail Folsomia candida.

In recent years significant attention has been given to the problem of olive mill waste towards the environment. Still, there is a considerable gap in the knowledge of the impact of the olive mill wastewater (OMWW) and the olive mill waste contaminated soil (OMW CS) on non-target soil organisms. Springtails, as an important group of non-target soil organisms, are frequently used in ecotoxicological research. However, information on olive mill waste impact on the model species Folsomia candida is scarce. Therefore, in this study, we determined the effects of OMWW and OMW CS on survival, reproduction, neurotoxicity, oxidative stress, and available energy in springtail F. candida. The exposure to different ratios of OMWW and OMW CS showed higher toxicity of OMW CS in terms of survival (LC50 = 32.34% of OMWW; LC50 = 45.36% of OMW CS) and reproduction (EC50 = 10.10% of OMWW; EC50 = 19.44% of OMW CS). Furthermore, neurotoxicity (AChE induction), oxidative stress (SOD, GST, and MDA induction), and changes in available energy (decrease in lipid and carbohydrate content) have been observed. Those negative effects are likely consequences of the high phenol content specific to OMWW and OMW CS. Obtained results indicate that for the ecotoxicological assessment of various wastes it is essential to consider different tier level biomarkers to have a clear insight into the mechanism of action.

Effects of strobilurin fungicides (azoxystrobin, pyraclostrobin, and trifloxystrobin) on survival, reproduction and hatching success of Enchytraeus crypticus.

Large quantities of strobilurin fungicides (SFs) are used worldwide, resulting in adverse effects on non-target organisms. SFs affect the reproduction and embryonic development of aquatic organisms, while the impact on soil organisms has been insufficiently researched. Therefore, we investigated the effects of three SFs (azoxystrobin (AZO), pyraclostrobin (PYR), and trifloxystrobin (TRI)) on the survival, reproduction, and hatching success of the non-target soil oligochaete Enchytraeus crypticus. The standard enchytraeid reproduction test (ERT) showed that, regarding survival, TRI (LC50 = 2.34 mg/kg) was the most toxic, followed by PYR (LC50 = 4.26 mg/kg) and AZO (LC50 ≥150 mg/kg). Reproduction was affected in the same order (TRI EC50 = 0.045 mg/kg, PYR EC50 = 1.85 mg/kg, and AZO EC50 = 93.10 mg/kg). Exposure to AZO and PYR showed a negative impact on hatching success with a significant increase in the number of unhatched cocoons. Prolonged hatching test was consequently carried out. As a result, a hatching delay was observed at lower AZO and PYR concentrations, while at higher concentrations hatching was completely stopped as the cocoons were no longer viable. Hence, hatching test enabled a discrimination between hatching delay and hatching impairment. Besides demonstrating the adverse effects of AZO, PYR, and TRI on the survival, reproduction, and hatching success of E. crypticus, the obtained results indicate the convenience of using several endpoints in reproduction tests. The usage of prolonged hatching tests and monitoring of hatching dynamics could fill the gap between standard reproduction tests and multigeneration tests and allow a better understanding of the adverse effects on reproduction.

Measurement of multixenobiotic resistance activity in enchytraeids as a tool in soil ecotoxicology.

The multixenobiotic resistance (MXR) mechanism is the first defense line against xenobiotics. Enchytraeids, a model organism in soil ecotoxicology, are often exposed to various xenobiotics, some of which may influence MXR activity. Since MXR activity has not been studied in these organisms, the aim of this paper was to establish a methodology for the implementation of the dye assay in enchytraeids. Enchytraeus albidus and Enchytraeus crypticus were exposed to model chemosensitizers: cyclosporine A (CA), dexamethasone (DEX), ivermectin (IVM), rifampicin (RIF), verapamil (VER), and fungicide propiconazole (PCZ). Thereafter, a dye assay with specific fluorescent dyes rhodamine B and rhodamine 123 was performed. Changes in MXR activity caused by variations in dye accumulation were measured fluorometrically. CA, IVM, and VER were found to inhibit the MXR system and increase the fluorescence 2.2-fold, while DEX and RIF induced the MXR system and decreased the fluorescence. CA was the strongest inhibitor in both E. albidus (IC50 5.48 ± 1.25 µM) and E. crypticus (IC50 5.20 ± 3.10 µM). In the validation experiment, PCZ was found to inhibit the MXR system. The IC50 varied between species and exposure substrates: water (E. albidus - IC50 0.74 ± 0.24 mg/L; E. crypticus - 1.31 ± 0.24 mg/L) or soil (E. albidus - 1.79 ± 0.42 mg/kg; E. crypticus - 1.79 ± 0.17 mg/kg). In conclusion, the tested compounds changed the MXR activity, which confirms the applicability of this method as a valuable complementary biomarker in soil ecotoxicology.

Identification of pyrazolopyridazinones as PDEδ inhibitors.

The prenyl-binding protein PDEδ is crucial for the plasma membrane localization of prenylated Ras. Recently, we have reported that the small-molecule Deltarasin binds to the prenyl-binding pocket of PDEδ, and impairs Ras enrichment at the plasma membrane, thereby affecting the proliferation of KRas-dependent human pancreatic ductal adenocarcinoma cell lines. Here, using structure-based compound design, we have now identified pyrazolopyridazinones as a novel, unrelated chemotype that binds to the prenyl-binding pocket of PDEδ with high affinity, thereby displacing prenylated Ras proteins in cells. Our results show that the new PDEδ inhibitor, named Deltazinone 1, is highly selective, exhibits less unspecific cytotoxicity than the previously reported Deltarasin and demonstrates a high correlation with the phenotypic effect of PDEδ knockdown in a set of human pancreatic cancer cell lines.