Predicting the Combined Effects of Multiple Stressors and Stress Adaptation in Gammarus pulex.

Global change confronts organisms with multiple stressors causing nonadditive effects. Persistent stress, however, leads to adaptation and related trade-offs. The question arises: How can the resulting effects of these contradictory processes be predicted? Here we show that Gammarus pulex from agricultural streams were more tolerant to clothianidin (mean EC50 148 µg/L) than populations from reference streams (mean EC50 67 µg/L). We assume that this increased tolerance results from a combination of physiological acclimation, epigenetic effects, and genetic evolution, termed as adaptation. Further, joint exposure to pesticide mixture and temperature stress led to synergistic interactions of all three stressors. However, these combined effects were significantly stronger in adapted populations as shown by the model deviation ratio (MDR) of 4, compared to reference populations (MDR = 2.7). The pesticide adaptation reduced the General-Stress capacity of adapted individuals, and the related trade-off process increased vulnerability to combined stress. Overall, synergistic interactions were stronger with increasing total stress and could be well predicted by the stress addition model (SAM). In contrast, traditional models such as concentration addition (CA) and effect addition (EA) substantially underestimated the combined effects. We conclude that several, even very disparate stress factors, including population adaptations to stress, can act synergistically. The strong synergistic potential underscores the critical importance of correctly predicting multiple stresses for risk assessment.

Association of common BRCA1 variants with predisposition to breast tumors in Pakistan.

BRCA1 variants are extensively associated with increased risk of breast cancer. Early detection and screening of variants is still rare in developing countries. Here, we investigated six BRCA1 variants in 300 subjects from Pakistani population using tetra amplification-refractory mutation system (T-ARMS) PCR. Our results indicate significant association of BRCA1 variants rs8176237 (AA; OR 8.2, 95% CI 3.02-22.64, p < 0.0001), rs1060915 (CC; OR 4.29, 95% CI 1.94-9.48, p = 0.0003), and rs799912 (TT; OR 3.16, 95% CI 1.44-6.94, p = 0.004) with up to 8-fold increased odds of breast cancer under recessive model. Furthermore, BRCA1 haplotypes AGCACG and AGCCCT were associated with up to 18% breast cancer cases (p < 0.05). Additionally, we found association of these variants with up to 11-fold increased odds of benign breast tumors. Linkage disequilibrium (LD) block-wise analysis revealed haplotypes GCAC and ATAC were associated with significantly increased risk. To our knowledge, this is the first study that identifies the association of these BRCA1 variants with breast tumors in Pakistani population. In conclusion, BRCA1 variants investigated in the present study are associated with high odds of benign- and malignant breast tumors. Studies with bigger sample size may help early detection and screening to reduce the odds of breast cancer.

Multiple Stress Reduces the Advantage of Pesticide Adaptation.

Under global change scenarios, multistress conditions may occur regularly and require adaptation. However, the adaptation to one stressor might be associated with the increased sensitivity to another stressor. Here, we investigated the ecological consequences of such trade-off under multiple stress. We compared the pesticide tolerance of the crustacean Gammarus pulex from agricultural streams with populations from reference streams. Under optimum temperature, G. pulex from agricultural streams were considerably more tolerant to pesticides as compared to the reference populations. Here, we assume that the increased tolerance in agricultural populations is the combination of acclimation, epigenetic effect, and genetic evolution. After experimental pre-exposure to very low concentration (LC50/1000), reference populations showed increased pesticide tolerance. In contrast, pre-exposure did not further increase the tolerance of agricultural populations. Moreover, these populations were more sensitive to elevated temperature alone due to the hypothesized fitness cost of genetic adaptation to pesticides. However, both reference and agricultural populations showed a similar tolerance to the combined stress of pesticides and warming due to stronger synergistic effects in adapted populations. As a result, pesticide adaptation loses its advantage. The combined effect was predicted well using the stress addition model, developed for predicting the synergistic interaction of independent stressors. We conclude that under multistress conditions, adaptation to pesticides reduces the general stress capacity of individuals and trade-off processes increase the sensitivity to additional stressors. This causes strong synergistic effects of additional stressors on pesticide-adapted individuals.

Environmental Stress Increases Synergistic Effects of Pesticide Mixtures on Daphnia magna.

Some widely used pesticide mixtures produce more than additive effects according to conventional combined effect models. However, synergistic effects have been so far generally observed at unrealistically high pesticide concentrations. Here, we used Daphnia magna as a test organism and investigated how food limitation-a common ecological stressor-affects the mixture toxicity of a pyrethroid insecticide and azole fungicide. We also compared three models regarding the prediction of mixture effects, including concentration addition (CA), effect addition (EA), and stress addition model (SAM). We revealed that especially under low food, the strength of synergism between esfenvalerate and prochloraz increased with an increasing concentration of prochloraz independent of the null model. Under high food conditions and at concentrations of prochloraz ≥32 µg/L, we observed a marginal synergistic effect with a model deviation ratio (MDR) = 2.1 at 32 µg/L prochloraz and 2.2 at 100 µg/L prochloraz when using CA as the null model. In contrast, the combination of both pesticides and food stress caused synergistic effects shown by an MDR = 10.9 even at 1 µg/L of prochloraz that is frequently detected in the environment. The combined effects of pesticides and food stress could be predicted best with the SAM that showed the lowest mean deviation between effect observation and prediction (mean deviation SAM = 16 [SD = 28], EA = 1072 [2105], CA = 1345 [2644]). We conclude that common environmental stressors can strongly increase the synergistic effects of toxicants. This knowledge is especially relevant considering current efforts to include the additional risk of pesticide mixtures and environmental stressors into the environmental risk assessment of pesticides.

Pesticide Body Burden of the Crustacean Gammarus pulex as a Measure of Toxic Pressure in Agricultural Streams.

Risk assessments of toxicants in aquatic environments are typically based on the evaluation of concentrations in water or sediment. However, concentrations in water are highly variable, while the body burden may provide a better time-integrated measure of pesticide exposure and potential effects in aquatic organisms. Here, we quantified pesticide body burdens in a dominant invertebrate species from agricultural streams, Gammarus pulex, compared them with pesticide concentrations in water samples, and linked the pesticide contamination with observed ecological effects on macroinvertebrate communities. In total, 19 of 61 targeted analytes were found in the organisms, ranging from 0.037 to 93.94 ng g-1 (wet weight). Neonicotinoids caused the highest toxic pressure among the pesticides detected in G. pulex. Using linear solvation energy relationships (LSERs), we derived equivalent pesticide concentrations in streamwater based on the body burden. These equivalent concentrations correlated with the concentrations in water samples collected after runoff (65% of variance explained). Pesticide pressure significantly affected the aquatic macroinvertebrate community structure, expressed as SPEARpesticides, and caused, on average, 3-fold increased insecticide tolerance in G. pulex as a result of adaptation. The toxic pressure derived from body burden and from water samples similarly explained the change in community structure (68% and 64%). However, the increased tolerance of G. pulex to pesticides was better explained by the toxicity derived from body burden (70%) than by the toxicity from water samples (53%). We conclude that the internal body burden of macroinvertebrates is suitable to assess the overall pesticide exposure and effects in agricultural streams.

Engineering Highly Interconnected Neuronal Networks on Nanowire Scaffolds.

Identifying the specific role of physical guidance cues in the growth of neurons is crucial for understanding the fundamental biology of brain development and for designing scaffolds for tissue engineering. Here, we investigate the structural significance of nanoscale topographies as physical cues for neurite outgrowth and circuit formation by growing neurons on semiconductor nanowires. We monitored neurite growth using optical and scanning electron microscopy and evaluated the spontaneous neuronal network activity using functional calcium imaging. We show, for the first time, that an isotropic arrangement of indium phosphide (InP) nanowires can serve as physical cues for guiding neurite growth and aid in forming a network with neighboring neurons. Most importantly, we confirm that multiple neurons, with neurites guided by the topography of the InP nanowire scaffolds, exhibit synchronized calcium activity, implying intercellular communications via synaptic connections. Our study imparts new fundamental insights on the role of nanotopographical cues in the formation of functional neuronal circuits in the brain and will therefore advance the development of neuroprosthetic scaffolds.

Industrial wastewater treatment in internal circulation bioreactor followed by wetlands containing emergent plants and algae.

Wastewater treatment based on ecological principles is a low cost and highly desirable solution for the developing countries like Pakistan. The present study evaluated the effectiveness of biological treatment systems including Internal Circulation (IC) anaerobic bioreactor and constructed wetlands (CWs) containing macrophytes and mixed algal cultures for industrial wastewater treatment. The IC bioreactor reduced COD (52%), turbidity (89%), EC (24%) of the industrial wastewater. However, the effluents of IC bioreactor did not comply with National Environmental Quality Standards (NEQS) of Pakistan. Post-treatment of IC bioreactor effluents was accomplished in CW containing macrophytes (Arundo donax and Eichhornia crassipes) and mixed algal culture. The CWs planted with macrophytes lowered the concentrations of COD (89%) and turbidity (99%). CWs with algal biomass were not effective in further polishing the effluent. Inhibition of algal biomass growth was observed due to physicochemical characteristics of wastewater. The integrated treatment system consisting of IC bioreactor and macrophytes was found more suitable option for industrial wastewater treatment.

Enhanced luminescence from GaN nanopillar arrays fabricated using a top-down process.

We report the fabrication of GaN nanopillar arrays with good structural uniformity using the top-down approach. The photoluminescence intensity from the nanopillar arrays is enhanced compared to the epilayer. We use finite difference time domain simulations to show that the enhancement in photoluminescence intensity from the nanopillar arrays is a result of anti-reflection properties of the arrays that result in enhanced light absorption and increase light extraction efficiency compared to the epilayer. The measured quantum efficiency of the nanopillars is comparable to that of an epitaxially grown GaN epilayer.

Revealing the cascade of pesticide effects from gene to community.

Global pesticide exposure in agriculture leads to biodiversity loss, even at ultra-low concentrations below the legal limits. The mechanisms by which the effects of toxicants act at such low concentrations are still unclear, particularly in relation to their propagation across the different biological levels. In this study, we demonstrate, for the first time, a cascade of effects from the gene to the community level. At the gene level, agricultural pesticide exposure resulted in reduced genetic diversity of field-collected Gammarus pulex, a dominant freshwater crustacean in Europe. Additionally, we identified alleles associated with adaptations to pesticide contamination. At the individual level, this genetic adaptation to pesticides was linked to a lower fecundity, indicating related fitness costs. At the community level, the combined effect of pesticides and competitors caused a decline in the overall number and abundance of pesticides susceptible macroinvertebrate competing with gammarids. The resulting reduction in interspecific competition provided an advantage for pesticide-adapted G. pulex to dominate macroinvertebrate communities in contaminated areas, despite their reduced fitness due to adaptation. These processes demonstrate the complex cascade of effects, and also illustrate the resilience and adaptability of biological systems across organisational levels to meet the challenges of a changing environment.

Seasonal hot spots of pollution and risks in Western Kenya: A spatial-temporal analysis of almost 800 organic micropollutants.

The release of chemicals into the environment presents a significant threat to aquatic ecosystems dependent on the proximity to emission sources and seasonal dynamics of emission and mobilization. While spatial-temporal information on water pollution in Europe is increasing, there are substantial knowledge gaps on seasonal pollution dynamics in tropical countries. Thus, we took Lake Victoria South Basin in western Kenya as a case study to identify spatial and seasonal hot spots of contamination, quantified toxic risks to different groups of organisms, and identified seasonal risk drivers. For this purpose, we analyzed grab water samples from five rivers with agricultural and wastewater treatment plants in their catchment in four different seasons. We used liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) with a target list of 785 organic micropollutants. A total of 307 compounds were detected with concentrations ranging from 0.3 ng/L to 6.6 µg/L. Using a Toxic Unit (TU) approach based on mixture toxicity to standard test organisms, crustaceans were identified as the most affected group followed by algae and fish. For crustaceans, chronic risk thresholds were exceeded in 96 % of all the samples, while 56 % of all samples are expected to be acutely toxic, with the highest risk in February during the dry season. High toxic unit values for algae and fish were recorded in July dry season and May wet season. Diazinon, imidacloprid, clothianidin and pirimiphos-methyl were the major drivers for crustacean toxicity while triclosan and different herbicide mixtures drive risks to algae in dry and wet seasons, respectively. A total of 18 chemicals were found to exceed acute and chronic environmental risk thresholds. With this study, strong spatial-temporal patterns of pollution, risks and risk drivers could be confirmed informing prioritization of monitoring and abatement to enhance water quality and reduce toxic risks.

Impact of Cadmium Contamination on Fertilizer Value and Associated Health Risks in Different Soil Types Following Anaerobic Digestate Application.

Cadmium (Cd) contamination in the soil potentially hampers microbial biomass and adversely affects their services such as decomposition and mineralization of organic matter. It can reduce nitrogen (N) metabolism and consequently affect plant growth and physiology. Further, Cd accumulation in plants can pose health risks through vegetable consumption. Here, we investigated consequences of Cd contamination on fertilizer value and associated health risks following the application of biogas residues (BGR) to various soil types. Our results indicate that the application of BGR to all soil types significantly increased dry matter (DM) yield and N uptake. However, the Cd contamination negatively affected DM yield and N recovery from BGR in a dose-dependent manner. Organic N mineralization from BGR also decreased in Cd-contaminated soils. The highest DM yield and N recovery were recorded in sandy soil, whereas the lowest values were observed in clay soil. Cadmium was accumulated in spinach, and health risk index (HRI) associated with its dietary intake revealed that consuming spinach grown in Cd-contaminated soil, with or without BGR, is unsafe. Among the soil types, values of daily intake of metals (DIM) and HRI were lowest in clay soil and highest in sandy soil. However, the application of BGR curtailed HRI across all soil types. Notably, the application of BGR alone resulted in HRI values < 1, which are under the safe limit. We conclude that soil contamination with Cd reduces fertilizer value and entails implications for human health. However, the application of BGR to the soil can decrease Cd effects.

High optical quality InP-based nanopillars fabricated by a top-down approach.

Dense and uniform arrays of InP-based nanopillars were fabricated by dry etching using self-assembly of colloidal silica particles for masking. The pillars, both single and arrays, fabricated from epitaxially grown InP and InP/GaInAsP/InP quantum well structures show excellent photoluminescence (PL) even at room temperature. The measured PL line widths are comparable to the as-grown wafer indicating high quality fabricated pillars. A stamping technique enables transfer with arbitrary densities of the nanopillars freed from the substrate by selectively etching a sacrificial InGaAs layer.

Junction-type photonic crystal waveguides for notch- and pass-band filtering.

Evolution of the mode gap and the associated transmission mini stop-band (MSB) as a function of photonic crystal (PhC) waveguide width is theoretically and experimentally investigated. The change of line-defect width is identified to be the most appropriate way since it offers a wide MSB wavelength tuning range. A high transmission narrow-band filter is experimentally demonstrated in a junction-type waveguide composed of two PhC waveguides with slightly different widths. The full width at half maximum is 5.6 nm; the peak transmission is attenuated by only ~5 dB and is ~20 dB above the MSBs. Additionally, temperature tuning of the filter were also performed. The results show red-shift of the transmission peak and the MSB edges with a gradient of dλ/dT = 0.1 nm/°C. It is proposed that the transmission MSBs in such junction-type cascaded PhC waveguides can be used to obtain different types of filters.

Pesticide-induced metabolic changes are amplified by food stress.

In natural ecosystems, long-term detrimental effects of pesticides may occur at very low concentrations, below those considered safe by the governmental risk assessment. Mechanisms potentially responsible for this unexpected sensitivity include environmental stress-factors such as food deficiency. To understand this so called "effect-paradox", we investigated how food stress interacts with insecticide-induced biochemical fingerprints. Therefore, we measured metabolomic perturbations in Daphnia magna following a 24 h exposure to esfenvalerate under high and low food conditions. In total, 160 metabolites covering the groups of amino acids, fatty acids, lipids, and sugars were analyzed. At 0.001 µg/L esfenvalerate - a factor of >200 below the acute lethal concentration (LC50) - the endogenous metabolome was significantly affected. Further, the effect under low food conditions was considerably stronger compared to high food conditions. Individual metabolites showed up to 7-fold stronger effects under low food conditions. In general, the metabolomic changes were largely dose-specific and increased over seven days after contamination. We conclude that the metabolic profiles are altered for at least seven days after a pulse exposure, and therefore might be a key process to understanding population level changes at ultra-low pesticide concentrations in the field.

Insecticides in agricultural streams exert pressure for adaptation but impair performance in Gammarus pulex at regulatory acceptable concentrations.

Pesticide exposure in agricultural streams requires non-target species to adapt. However, pesticides may reduce performance in between exposure events due to long-term effects and physiological fitness costs of adaptation. Here, we investigated the long-term consequences of pesticide exposure to low concentrations in the widespread crustacean Gammarus pulex. We collected populations from six German streams covering no to moderate agricultural pesticide exposure. Peak concentrations ranged up to 1/400 of their acute median lethal concentration (Toxic Unit = -2.6), resulting in significant changes in the macroinvertebrate community composition (SPEARpesticides = up to 0.12). Acute toxicity tests revealed up to 2.5-fold increased tolerance towards the most frequently found insecticide clothianidin compared to populations from non-contaminated streams. However, populations showing increased insecticide tolerance were characterized by reduced survival, per capita growth and mating when cultured under pesticide-free conditions in the laboratory for three months. We conclude that pesticide pollution triggers adaptation both at the species and the community level even at concentrations considered to be safe according to the European pesticide legislation. In G. pulex, exposure and adaptation are associated with impaired performance which potentially affects ecosystem functions such as leaf litter degradation. These long-term impairments need to be considered in deriving safe concentrations.

Drivers of pesticide resistance in freshwater amphipods.

Aquatic invertebrates exposed to pesticides may develop pesticide resistance. Based on a meta-analysis we revealed environmental factors driving the magnitude of resistance in the freshwater amphipod Gammarus pulex in the field. We showed that (i) insecticide tolerance of G. pulex increased with pesticide contamination in agricultural streams generally by a factor of up to 4. Tolerance increased even at concentrations lower than what is considered safe in regulatory risk assessment (ii) The increase in insecticide tolerance was pronounced at high test concentrations; comparing the LC50 of populations therefore potentially underestimates the development of resistance. (iii) Insecticide resistance in agricultural streams diminished during the spraying season, suggesting that adverse effects of sublethal concentrations in the short term contrast long-term adaptation to insecticide exposure. (iv) We found that resistance was especially high in populations characterized not only by high pesticide exposure, but also by large distance (>3.3 km) from non-polluted stream sections and by low species diversity within the invertebrate community. We conclude that the test concentration, the timing of measurement, distance to refuge areas and species diversity mediate the observed response of aquatic communities to pesticide pollution and need to be considered for the sustainable management of agricultural practices.

Allium cepa assay based comparative study of selected vegetables and the chromosomal aberrations due to heavy metal accumulation.

Irrigation of industrial effluents may end in the bioaccumulation of various toxic metals and consequent genetic changes in contaminated food crops. To test this hypothesis and extent of genetic modifications, Allium cepa test was performed to food crops viz. tomato (Lycopersicum esculentum) and chili (Capsicum annum) as Allium cepa test is a useful tool to assess genetic variations in plants. Prior to A. cepa test, the plants were exposed to various metal concentrations 125-1000 mg/L in the synthetic wastewater. The extracts of harvested plants were used to grow the root of A. cepa following its standard method. The root tips were fixed, stained and examined under compound microscope (almost 300-400 dividing cells) to check the extent of chromosomal variations during various stages of mitosis. The results revealed various chromosomal abnormalities including laggards, stickiness, vagrant chromosomes, binucleated cells, nuclear lesions, giant cells and c-mitosis at different level of treatment. On the whole, aberrations were increasing with the increasing doses along the positive control. In comparison, chili crop had higher level of aberrations depicting the higher chromosomal changes. Lower mitotic index (MI) with increasing level of doses was also describing the hampered cell division due to increased metal stress. The study is showing that the cell division was ceased with increasing metal stress thus increasing the rate of cell aberrations.

Breast cancer risk associated with BRCA1/2 variants in the Pakistani population.

BACKGROUND: Majority of the BRCA1 and BRCA2 mutations are associated with the risk of sporadic and familial breast cancer. Since these genes are significant in DNA repair mechanisms, we focused homology-directed DNA repair (HDDR) and BRCA complex. METHODS: We selected BRCA1 variant (rs80356932, 4491C/T) and BRCA2 variant (rs80359182, 319T/C) from the interaction region of BRCA complex and studied in 100 breast cancer patients and 100 controls using tetra-ARMS-PCR. RESULTS: Here we show that BRCA1 and BRCA2 variants are significantly associated with high breast cancer risk (BRCA1 rs80356932; Genotype T/T OR 8.66, 95% CI 3.16-23.71, p < 0.0001; Allele-T, OR 2.48, 95% CI 1.62-3.81, p < 0.0001 and BRCA2 rs80359182; Genotype C/C OR 4.32, 95% CI 1.95-9.53, p = 0.0001; Allele-C, OR 2.19, 95% CI 1.43-3.34, p = 0.0002). Additionally, bioinformatics analysis showed that BRCA2-tryptophan > arginine substitutions result in altered interaction of BRCA1/PALB2/BRCA2/protein complex and impaired HDDR pathway. We also observed that breast cancer risk was significantly increased in over-weighted and obese women. CONCLUSIONS: Our results indicate that high risk of breast cancer is significantly associated with BRCA1 and BRCA2 variants, and mutations may alter the protein interactions of BRCA complex that results in tumor genesis.

Removal of pharmaceuticals and personal care products using constructed wetlands: effective plant-bacteria synergism may enhance degradation efficiency.

Post-industrial era has witnessed significant advancements at unprecedented rates in the field of medicine and cosmetics, which has led to affluent use of pharmaceuticals and personal care products (PPCPs). However, this has exacerbated the influx of various pollutants in the environment affecting living organisms through multiple routes. Thousands of PPCPs of various classes-prescription and non-prescription drugs-are discharged directly into the environment. In this review, we have surveyed literature investigating plant-based remediation practices to remove PPCPs from the environment. Our specific aim is to highlight the importance of plant-bacteria interplay for sustainable remediation of PPCPs. The green technologies not only are successfully curbing organic pollutants but also have displayed certain limitations. For example, the presence of biologically active compounds within plant rhizosphere may affect plant growth and hence compromise the phytoremediation potential of constructed wetlands. To overcome these hindrances, combined use of plants and beneficial bacteria has been employed. The microbes (both rhizo- and endophytes) in this type of system not only degrade PPCPs directly but also accelerate plant growth by producing growth-promoting enzymes and hence remediation potential of constructed wetlands.

Breast cancer risk associated with genes encoding DNA repair MRN complex: a study from Punjab, Pakistan.

BACKGROUND: Variants of DNA repair genes are extensively reported to cause genetic instability and increase the risk of breast cancer. In combination with NBS1, MRE11 and RAD50 constitute an MRN (MRE11-RAD50-NBS1) complex that repairs DNA damage. However, certain genetic alterations in MRE11 and RAD50 produce abnormal protein that affects the repairing process and may result in malignancy. We aimed to investigate the association of MRE11 and RAD50 polymorphisms with breast risk in the female population of Punjab, Pakistan. METHODS: We collected blood samples of 100 breast cancer patients and 100 tumor-free females selected as controls. Extracted DNA was genotyped by tetra ARMS-PCR followed by gel electrophoresis. Results were analyzed by SPSS and SNPstats to analyze the association of different clinical factors and SNPs (single nucleotide polymorphisms) with the risk of breast cancer. RESULTS: We found that the increased risk of breast cancer is associated with MRE11 variant rs684507 (odds ratio-OR 3.71, 95% confidence interval-CI 1.68-8.18, p value < 0.0001), whereas, RAD50 variant rs28903089 appeared to have protective effect (OR 0.55, CI 0.29-1.02, p value = 0.003). Additionally, clinical factors such as positive family history, life style, and marital status also play significant roles in breast cancer development. CONCLUSION: In the present study, strong risk of breast cancer was associated with MRE11 gene. However, RAD50 showed protective effect. Additionally, clinical factors are also pivotal in risk assessment. We anticipate that targeting specific genetic variations confined to ethnic groups would be more effective in future therapeutic approaches for prevention and treatment of breast cancer.