Additive and dose-dependent mixture effects of Flumite 200 (flufenzin, acaricide) and Quadris (azoxystrobin, fungicide) on the reproduction and survival of Folsomia candida (Collembola).

Chemical plant protection is still a dominant agricultural practice in the world, and usually fields are treated with several pesticides many times per year. This means that not only the single substances affect the environment and the non-target organisms, but their mixtures. Our model organism was Folsomia candida (Collembola). We aimed to gain information on the toxicity of Quadris (azoxystrobin) and Flumite 200 (flufenzine aka. diflovidazine) on survival and reproduction and whether the animals can mitigate the toxicity with soil and/or food avoidance behaviour. Also, we aimed to test the effect of the mixture of these two pesticides. We used the OECD 232 reproduction test, a soil avoidance test, and a food choice test for both single pesticides and their mixture. We prepared the mixtures based on the concentration addition model, so the 50% effective concentrations (EC50) of the single materials were used as one toxic unit with a constant ratio of the two materials in the mixture. In the end, the measured mixture EC and LC (lethal concentration) values were compared to the estimate of the concentration addition model. Both materials were toxic to the Collembola in much higher concentrations than the recommended field concentrations (Flumite 200 EC50: 1.096, LC50: 1.561, Quadris EC50: 65.568, LC50: 386.165 mg kg-1). The springtails did not consistently avoid the polluted soils, this only happened in higher concentrations. The mixtures seemed to have additive effects on the reproduction and we found dose-dependent interaction with the survival (EC50: 1.022 Toxic Unit, 0.560 Flumite 200 and 33.505 Quadris; LC50: 1.509 Toxic Unit, 0.827 Flumite 200 and 49.471 mg kg-1 Quadris). The deviation from the concentration addition model suggests that the curve starts with a synergy. but above EC50 it becomes antagonistic. We conclude that both Quadris and Flumite 200 are safe for springtails until the recommended field concentration is respected. However, if higher concentrations are used the animals cannot avoid Flumite 200 and the toxic effects can fully manifest. Consequently, the dose-dependent deviation from the concentration addition model is a reason for caution as the low concentrations were synergistic for survival. That means the field concentrations can possibly cause synergistic effects. However, to clarify that further tests are necessary.

Urbanization decreases species richness, and increases abundance in dry climates whereas decreases in wet climates: A global meta-analysis.

Soil invertebrates have an essential role in decomposition, nutrient turnover and soil structure formation, all of which are strongly threatened by urbanization. Sealing, compaction by trampling and pollution destroy and degrade city soils and potentially damage soil-living invertebrates. The existing literature on how urbanization affects soil invertebrates is inconsistent, presenting both negative and positive effects. Therefore, here we aimed to synthesize the effects of urbanization on soil invertebrates considering their taxonomic (Acari, Annelida, Carabidae, Collembola, Gastropoda, Isopoda, Myriapoda, Nematoda) and functional (soil living vs. soil-related; mobility) identities, as well as to examine how the overall effect is modulated by climatic conditions (total annual precipitation, annual mean ambient temperature), urban heat island effect (based on ambient temperature differences between urban and rural areas) and city population. In a systematic review using hierarchical and categorical meta-analyses, we extracted 158 effect sizes from 75 studies on abundance and 125 effect sizes from 84 studies on species richness. Invertebrate abundance showed an increase (r = 0.085), whereas species richness significantly decreased with increasing urbanization (r = -0.168). The reason behind this could be that a few generalist species can adapt well to the urban environment and achieve strongly elevated densities. The species richness of annelids (r = -0.301), springtails (r = -0.579), and snails (r = -0.233) decreased with advancing urbanization, most probably because these animals are sensitive to soil compaction and pollution, both of which are common consequences of urbanization. The temperature did not modify the effects of urbanization, but precipitation modified the effects on abundance (r = -0.457). Abundance increased with advancing urbanization in drier climates, probably because irrigation increased soil moisture, whereas it decreased in wet climates, as urban areas were drier than their surroundings. Making future cities more climate-neutral could better sustain soil biodiversity.

Aspirin Mediates Its Antitumoral Effect Through Inhibiting PTTG1 in Pituitary Adenoma.

CONTEXT: DNA demethylation and inhibitory effects of aspirin on pituitary cell proliferation have been demonstrated. OBJECTIVE: Our aim was to clarify the molecular mechanisms behind the aspirin-related effects in pituitary cells. METHODS: DNA methylome and whole transcriptome profile were investigated in RC-4B/C and GH3 pituitary cell lines upon aspirin treatment. Effects of aspirin and a demethylation agent, decitabine, were further tested in vitro. PTTG1 expression in 41 human PitNET samples and whole genome gene and protein expression data of 76 PitNET and 34 control samples (available in Gene Expression Omnibus) were evaluated. RESULTS: Aspirin induced global DNA demethylation and consequential transcriptome changes. Overexpression of Tet enzymes and their cofactor Uhrf2 were identified behind the increase of 5-hydroxymethylcytosine (5hmC). Besides cell cycle, proliferation, and migration effects that were validated by functional experiments, aspirin increased Tp53 activity through p53 acetylation and decreased E2f1 activity. Among the p53 controlled genes, Pttg1 and its interacting partners were downregulated upon aspirin treatment by inhibiting Pttg1 promoter activity. 5hmC positively correlated with Tet1-3 and Tp53 expression, and negatively correlated with Pttg1 expression, which was reinforced by the effect of decitabine. Additionally, high overlap (20.15%) was found between aspirin-regulated genes and dysregulated genes in PitNET tissue samples. CONCLUSION: A novel regulatory network has been revealed, in which aspirin regulated global demethylation, Tp53 activity, and Pttg1 expression along with decreased cell proliferation and migration. 5hmC, a novel tissue biomarker in PitNET, indicated aspirin antitumoral effect in vitro as well. Our findings suggest the potential beneficial effect of aspirin in PitNET.

Urbanization hampers biological control of insect pests: A global meta-analysis.

Biological control is a major ecosystem service provided by pest natural enemies, even in densely populated areas where the use of pesticides poses severe risks to human and environmental health. However, the impact of urbanization on this service and the abundance patterns of relevant functional groups of arthropods (herbivores, predators, and parasitoids) remain contested. Here, we synthesize current evidence through three hierarchical meta-analyses and show that advancing urbanization leads to outbreaks of sap-feeding insects, declining numbers of predators with low dispersal abilities, and weakened overall biological pest control delivered by arthropods. Our results suggest that sedentary predators may have the potential to effectively regulate sap-feeders, that are one of the most important pests in urban environments. A well-connected network of structurally diverse and rich green spaces with less intensive management practices is needed to promote natural plant protection in urban landscapes and sustainable cities.

Pollution impacts on water bugs (Nepomorpha, Gerromorpha): state of the art and their biomonitoring potential.

As water pollution poses an increasing risk worldwide, it is timely to assess the achievements of the aquatic macroinvertebrate ecotoxicology to provide a sound basis for the discipline's future and support the development of biomonitoring. Aquatic and semi-aquatic bugs (Hemiptera: Nepomorpha, Gerromorpha) are ubiquitous in almost all water types, sometimes in high densities, and play a significant role in organic material turnover and energy flow. Nevertheless, they are ignored in the water pollution biomonitoring schemes. Here, based on 300 papers, we review and evaluate the effects of chemical pesticides, microorganism-derived pesticides, insecticides of plant origin, heavy metals, eutrophication, salinisation and light pollution which are summarised for the first time. Our review encompasses the results of 100 laboratory and 39 semi-field/field experiments with 47 pesticides and 70 active ingredients. Pyrethroids were found to be more toxic than organochlorine, organophosphate and neonicotinoid insecticides to water bugs, like other macroinvertebrate groups. Additionally, in 10 out of 17 cases, the recommended field concentration of the pesticide was higher than the LC50 values, indicating potential hazards to water bugs. The recommended field concentrations of pesticides used in mosquito larvae control were found non-toxic to water bugs. As very few replicated studies are available, other findings on the effects of pesticides cannot be generalised. The microorganism-derived pesticide Bti appears to be safe when used at the recommended field concentration. Data indicates that plant-derived pesticides are safe with a high degree of certainty. We have identified three research areas where water bugs could be better involved in water biomonitoring. First, some Halobates spp. are excellent, and Gerris spp. are promising sentinels for Cd contamination. Second, Micronecta and, to a certain extent, Corixidae species composition is connected to and the indicator of eutrophication. Third, the species composition of the Corixidae is related to salinisation, and a preliminary method to quantify the relationship is already available. Our review highlights the potential of water bugs in water pollution monitoring.

A relative quantitation method for measuring DNA methylation and hydroxymethylation using guanine as an internal standard.

Global DNA methylation and hydroxymethylation play an important role in gene expression. They can be connected with several diseases. The modification status could be a biomarker to determine the status of disease. A fast, easy and accurate liquid chromatography - tandem mass spectrometry method has been developed for the precise quantitation of 5-methylcytosine and 5-hydroxymethylcytosine. Formic acid was used for the hydrolysis of the DNA strand resulting in nucleobases. These polar hydrolysis products were separated on a normal phase column using reversed phase eluents in inverse gradient mode. Multiple reaction monitoring was applied to achieve high selectivity and sensitivity for the quantitation. A new relative quantitation model was developed by using guanine, as an internal standard, present in samples. The new method was successfully validated with excellent accuracy and precision values in the range of 0.005-0.5% for 5hmC and 1-15% for 5mC. The main advantages of this quantitation method are that, due to relative quantitation, calibration curves can be used without reacquiring the calibration points and no additional isotope labeled internal standards are required. The method was tested to identify the concentrations of 5mC and 5hmC in various sample types. The lowest level of DNA sample required in the case of 0.005% 5hmC is 0.5 µg.

Three Dimensional Cell Culturing for Modeling Adrenal and Pituitary Tumors.

In vitro monolayer conditions are not able to reproduce the complexity of solid tumors, still, there is scarce information about the 3D cell culture models of endocrine tumor types. Therefore, our aim was to develop in vitro 3D tumor models by different methodologies for adrenocortical carcinoma (H295R), pituitary neuroendocrine tumor (RC-4B/C and GH3) and pheochromocytoma (PC-12). Various methodologies were tested. Cell biological assays (cell viability, proliferation and live cell ratio) and steroid hormone production by HPLC-MS/MS method were applied to monitor cellular well-being. Cells in hanging drops and embedded in matrigel formed multicellular aggregates but they were difficult to handle and propagate for further experiments. The most widely used methods: ultra-low attachment plate (ULA) and spheroid inducing media (SFDM) were not the most viable 3D model of RC-4B/C and GH3 cells that would be suitable for further experiments. Combining spheroid generation with matrigel scaffold H295R 3D models were viable for 7 days, RC-4B/C and GH3 3D models for 7-10 days. ULA and SFDM 3D models of PC-12 cells could be used for further experiments up to 4 days. Higher steroid production in 3D models compared to conventional monolayer culture was detected. Endocrine tumor cells require extracellular matrix as scaffold for viable 3D models that can be one reason behind the lack of the usage of endocrine 3D cultures. Our models help understanding the pathogenesis of endocrine tumors and revealing potential biomarkers and therapeutic targets. They could also serve as an excellent platform for preclinical drug test screening.

The inter-individual variance can provide additional information for the ecotoxicologists beside the mean.

The hypothesis that the inter-individual parameter variability is an unexploited area of ecotoxicology was proposed several decades ago. Although some illustrative examples were presented to support this hypothesis in the last decades, it has never been tested on an extensive, coherent database. In this study, variance changes of 105 dose-response curves were analysed. All data originated from the same experiment, where the effects of the insecticide Trebon EC were investigated in a dose-response manner on 15 traits of the collembolan Folsomia candida in four subsequent generations and two types of insecticide treatments. A consistent relationship between inter-individual variance and insecticide application was found in 2 (first clutch size and growth-reproduction trade-off) out of the 15 of the parameters. Contrary to the mean, the variance of the first clutch size showed consistent differences compared to the control. Furthermore, the variance of the growth-reproduction trade-off was consistently different from the control except in one case (F3 generation of the transgenerational treatment). Higher first clutch size variances were found in F1 and a lower one in the F2 and F3 generations than in that of the control. This overall pattern of the variance changes of the first clutch size and the trade-off seems to be a quick response to the insecticide application. In the short term, we have found that variance increased with insecticide treatment (P and F1 generation), because phenotypic variance generally increases due to environmental stress. Disruptive selection could be another mechanism between the more detoxification less reproduction strategy and the more reproduction less detoxification strategy. However, in the later generations (F2-F3) the variance decreases compared to the control, which could be because on short term selection stronger on the viability parameters and in long-term selection on reproduction becomes stronger. According to our results, analysis of the variance changes of some parameters may give information about the effects of the pesticide even when the mean does not predict any impact. Testing variance changes are important in ecotoxicology because variance change can signalise toxicant impact even when the mean does not change in certain cases.

Demethylation Status of Somatic DNA Extracted From Pituitary Neuroendocrine Tumors Indicates Proliferative Behavior.

BACKGROUND: Cytosine intermediaries 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), epigenetic hallmarks, have never been investigated in pituitary neuroendocrine tumors (PitNET). OBJECTIVE: To examine methylation-demethylation status of global deoxyribonucleic acid (DNA) in PitNET tissues and to assess its correlation with clinical and biological parameters. MATERIALS AND METHODS: Altogether, 57 PitNET and 25 corresponding plasma samples were collected. 5mC and 5hmC were investigated using liquid chromatography-tandem mass spectrometry. Expression of DNA methyltransferase 1 (DNMT1); tet methylcytosine dioxygenase 1 through 3 (TET1-3); and ubiquitin-like, containing PHD and RING finger domains 1 and 2 (UHRF1-2) were measured by reverse transcription-polymerase chain reaction. Levels of 5hmC and UHRF1-2 were explored by immunohistochemistry. Effect of demethylating agent decitabine was tested on pituitary cell lines. RESULTS: 5hmC/5mC ratio was higher in less differentiated PitNET samples. A negative correlation between Ki-67 proliferation index and 5hmC, 5hmC to 5mC ratio were revealed. Higher 5mC was observed in SF-1 + gonadotroph adenomas with a higher Ki-67 index. Expressions of TET2 and TET3 were significantly higher in adenomas with higher proliferation rate. UHRF1 showed gradually increased expression in higher proliferative adenoma samples, and a significant positive correlation was detected between UHRF2 expression and 5hmC level. Decitabine treatment significantly decreased 5mC and increased 5hmC levels in both cell lines, accompanied with decreased cell viability and proliferation. CONCLUSION: The demethylation process negatively correlated with proliferation rate and the ratio of 5hmC to 5mC was higher in less differentiated adenomas. Therefore, epigenetic markers can be potential biomarkers for PitNET behavior. Altering the epigenome in adenoma cells by decitabine decreased proliferation, suggesting that this treatment might be a novel medical treatment for PitNET.

Comprehensive Analysis of Circulating miRNAs in the Plasma of Patients With Pituitary Adenomas.

BACKGROUND: Circulating miRNAs in pituitary adenomas would improve patient care, especially as minimally invasive biomarkers of tumor recurrence and progression in nonfunctioning adenoma cases. AIM: Our aim was to investigate plasma miRNA profiles in patients with pituitary adenomas. MATERIALS AND METHODS: A total of 149 plasma and extracellular vesicle (preoperative, early postoperative, and late postoperative) samples were collected from 45 patients with pituitary adenomas. Adenomas were characterized on the basis of anterior pituitary hormones and transcription factors by immunostaining. miRNA next-generation sequencing was performed on 36 samples (discovery set). Individual TaqMan assays were used for validation on an extended sample set. Pituitary adenoma tissue miRNAs were evaluated by TaqMan array and data in the literature. RESULTS: Global downregulation of miRNA expression was observed in plasma samples of pituitary adenomas compared with normal samples. Expression of 29 miRNAs and isomiR variants were able to distinguish preoperative plasma samples from normal controls. miRNAs with altered expression in both plasma and different adenoma tissues were identified. Three, seven, and 66 miRNAs expressed differentially between preoperative and postoperative plasma samples in GH-secreting, FSH/LH+, and hormone-immunonegative groups, respectively. miR‒143-3p was downregulated in late postoperative but not in early postoperative plasma samples compared with preoperative ones exclusively in FSH/LH+ adenomas. The plasma level of miR‒143-3p discriminated these samples with 81.8% sensitivity and 72.3% specificity (area under the curve = 0.79; P = 0.02). CONCLUSIONS: Differentially expressed miRNAs in pituitary adenoma tissues have low abundance in plasma, minimizing their role as biomarkers. Plasma miR‒143-3p level decreased in patients with FSH/LH+ adenomas, indicating successful surgery, but its application for evaluating tumor recurrence needs further investigation.

Transgenerational and multigenerational stress gene responses to the insecticide etofenprox in Folsomia candida (Collembola).

Insecticide exposure may cause both transgenerational and multigenerational effects on populations, but the molecular mechanisms of these changes remain largely unclear. Many studies have focused on either transgenerational or multigenerational mechanisms but did neglect the comparative aspects. This study assessed whether the pyrethroid insecticide etofenprox (formulation Trebon® 30 EC) shows transgenerational and/or multigenerational effects on the survival and reproduction of Folsomia candida (Collembola). The activation of stress-related genes was studied to detect whether etofenprox modifies the expression of reproduction-associated genes in trans- and multigenerational treatments. A laboratory study was carried out for three generations with five insecticide concentrations in LUFA 2.2 soil. In the transgenerational treatment, only the parent generation (P) was exposed, but the subsequent generations were not. In the multigenerational treatment, all three generations were exposed to the insecticide in the same manner. Multigenerational exposure resulted in reduced reproduction effects over generations, suggesting that F. candida is capable of acclimating to enhanced concentration levels of etofenprox during prolonged exposure over multiple generations. In the transgenerational treatment, the heat shock protein 70 was up-regulated and cytochrome oxidase 6N4v1 expression down-regulated in a dose-dependent manner in the F2 generation. This finding raises the possibility of the epigenetic inheritance of insecticide impacts on parents. Furthermore, CYP6N4v1 expression was oppositely regulated in the trans- and multigenerational treatments. Our results draw attention to the differences in molecular level responses of F. candida to trans- and multigenerational etofenprox exposure.

Survivin as a potential therapeutic target of acetylsalicylic acid in pituitary adenomas.

Acetylsalicylic acid (ASA) is known as a cancer preventing agent, but there is no data available regarding the effect of ASA on pituitary cells. We investigated 66 nonfunctioning (NFPA) and growth hormone (GH)-producing adenomas and 15 normal pituitary samples. Functional assays (cell viability, proliferation, flow cytometry cell cycle analysis, caspase-3 activation and DNA degradation) were applied to explore the effect of ASA, YM155 (survivin inhibitor), survivin-targeting siRNA and TNF-related apoptosis-inducing ligand (TRAIL) in RC-4B/C and GH3 cells. Pituitary adenoma xenografts were generated in immunocompromised mice. We found that survivin was overexpressed and TRAIL was downregulated in NFPAs compared to normal pituitary tissue. ASA decreased proliferation but did not induce apoptosis in pituitary cells. Additionally, ASA treatment decreased cells in S phase and increased cells in G2/M phase of the cell cycle. Inhibition of survivin using an inhibitor or siRNA-mediated silencing reversed the ASA-induced growth inhibition partially. In addition, we also found survivin-independent effects of ASA on the cell cycle that were mediated through inhibition of cyclin A, cyclin dependent kinase 2 (CDK2) and phospho-CDK2. We also aimed to test the effect of acetylsalicylic acid in an animal model using RC-4 B/C cells, but in contrast to GH3 cells, RC-4 B/C cells failed to adhere and grow a xenograft. We concluded that ASA inhibited the growth of pituitary adenoma cells. Survivin inhibition is a key mechanism explaining its antineoplastic effects. Our results suggest that inhibition of survivin with small molecules or ASA could serve as potential therapeutic agents in NFPA.

New method for automatic body length measurement of the collembolan, Folsomia candida Willem 1902 (Insecta: Collembola).

The collembolan, Folsomia candida, is widely used in soil ecotoxicology. In recent years, growth rate of collembolans has become as frequently used endpoint as reproduction rate in ecotoxicological studies. However, measuring collembolan body sizes to estimate growth rate is a complicated and time-consuming task. Here we present a new image analysis method, which facilitates and accelerates the body length measurement of the collembolan Folsomia candida. The new software package, called CollScope, consists of three elements: 1) an imaging device; 2) photographing software; 3) an ImageJ macro for image processing, measurement and data analysis. We give a complete description of the operation of the software, the image analyzing process and describe its accuracy and reliability. The software with a detailed usage manual is attached as Supplementary Material. We report a case study to demonstrate that the automated measurement of collembolan body sizes is highly correlated with the traditional manual measurements (estimated measuring accuracy 0.05 mm). Furthermore, we performed a dose-response ecotoxicity test using cadmium-sulfate by using CollScope as well as classical methods for size measurement. Size data measured by CollScope or manually did not differ significantly. Furthermore the new software package decreased time consumption of the measurements to 42% when tested on 35 animals. Consequently, methodological investigations performed in this study should be regarded as a recommendation for any other routine dose-response study where body growth is an endpoint.