Independent of temperature, Day 22 tolerance trial skin temperatures decreased from Day 1 , but training did not influence core temperature (p = 0.598). Average sweat rates were higher in the 33 °C group vs. the 20 °C group  but did not change due to training (p = 0.571). Pre-training PGC-1α mRNA increased 4h-post-exercise , was lower post-training , and was not different between temperatures (p = 0.455). While training induced some diminished transcriptional stimulus, generally the training temperature had little effect on genes related to mitochondrial biogenesis, mitophagy, and metabolic enzymes. These female participants increased aerobic fitness and maintained an exercise-induced PGC-1α mRNA response in the heat equal to that of room temperature conditions, contrasting with the blunted responses previously observed in men.Recent aerobic exercise training in the heat has reported blunted aerobic power improvements and reduced mitochondrial-related gene expression in men. It is unclear if this heat-induced blunting of the training response exists in females. The purpose of the present study was to determine the impact of 60 min of cycling in the heat over three weeks on thermoregulation, gene expression, and aerobic capacity in females. Untrained females  were assigned to three weeks of aerobic training in either 20 °C (n = 12) or 33 °C . Maximal aerobic capacity (39.5 ± 6.5 to 41.5 ± 6.2 mL·kg Current exercise recommendations apply specifics to timing, frequency, intensity, and type of exercise in order to produce optimal chronic adaptations . HoweverPGC-1α is the initial step toward multiple downstream alterations to enhance mitochondrial biogenesis and thus aerobic power [PGC-1α, NRF1, GABPA, VEGF, TFAM, and ESRRa), mitophagy , and genes related to the production of metabolic enzymes , one can gain insight into the initial adaptive improvements due to acute exercise, over training, and heat acclimation. However, an altered gene expression does not necessarily translate to an equivalent protein translation, hence there is a need for functional performance testing . Recent heat acclimation-associated diminished gene expression responses were validated by attenuated aerobic performance outcomes when compared to similar men exercising in temperate conditions [An acute bout of aerobic exercise can initiate strong mitochondrial-related gene expression associated with positive mitochondrial development ,22,23,24ic power . By linknditions . HoweverBased on known sex related thermoregulatory differences ,16,18, iTwenty-four untrained females  who did not engage in regular aerobic exercise and volunteered by completing the Institutional Review Board approved informed consent document guided by the ethical principles outlined in the Declaration of Helsinki. Subjects were considered healthy and free of any disease that may interfere with exercise training and/or thermoregulation  as assessed via an approved questionnaire. Selection bias was minimized via the application of a concealed random sequence (generated via MS Office Excel) to sort participants into one of two counterbalanced temperature conditions . All available attempts to reduce the likelihood of bias were applied; however, the assigned temperature conditions prevented potential performance bias. The design consisted of two (pre- and post-training) fitness assessment visits conducted 3 days before (Day −3) and 3 days after (Day 25) the completion of the 3-week training protocol. To examine the subject’s acclimation status, a fixed-intensity cycling temperature tolerance trial, conducted at 50%Wpeak, was conducted within the assigned temperature condition (20 °C or 33 °C) on Day 1 (pre) and Day 22 (post). Therefore, Day 1 was dual purposed to act as an acclimation status trial and Day 1 of cycle training. Training sessions were conducted during each weekday over a consecutive 3-week span (Days 2–21). This resulted in a total of 14 weekday (no training on weekends) training sessions of 60 min of cycling at a self-selected RPE  of 15 (h2peak). To determine body composition, body density was measured via hydrodensitometry in a custom-designed tank and measured via an electronic load-cell based system . Participants were asked to void prior to testing and instructed to completely submerge themselves while seated on the scale. After adjusting for estimated residual lung volume, body density was used to estimate body fat % [2peak (Wpeak) was calculated by multiplying the relative time spent in the final stage by the 35 W-stage interval and adding it to the workload of the previously completed stage. This absolute intensity (50%Wpeak) was used as the absolute fixed intensity for the pre- and post-temperature tolerance trials.Fitness assessments were conducted 3 days prior to and 3 days after the 3-week training intervention within room temperature (20 °C) conditions. The use of temperate conditions for pre- and post-testing allowed for direct fitness comparisons between groups, without the acute impact of temperature. Fitness assessments consisted of height , weight , body composition (body fat %), and maximal aerobic capacity  calculated from the pre-training fitness assessment. These fixed-intensity temperature tolerance trials were aimed at the detection of changes related to acclimation. Subjects were instructed to refrain from exercise for the previous 48 h and arrive in a fasted state. Subjects completed a self-reported 24-h dietary log prior to the Day 1 temperature tolerance trial and were instructed to follow the same diet prior to the Day 22 temperature tolerance trial. Nude body weight and the weight of any water ingested were recorded prior to and directly after the exercise bout on a digital scale  to calculate sweat rate. Core temperature was monitored continuously during exercise via rectal probe  inserted 12 cm past the anal sphincter. Skin temperature was measured continuously and averaged among 2 sites (chest and back) via skin surface probes . Rectal and skin temperatures were recorded via a 4-channel thermometer SD logger . Heart rate was monitored continuously via a watch and chest strap . Muscle biopsies were collected before and 4 h after the completion of the temperature tolerance trials on Day 1 and Day 22.Temperature tolerance trial procedures have been previously utilized and published in detail previously ,27. BrieSubjects completed a 60-min exercise bout on a cycle ergometer  within their randomly assigned intervention temperature (20 °C or 30 °C) on days 2–5, 8–12, and 15–19 (weekends off) at an intensity associated with an RPE of 15 (hard) and were encouraged to increase the intensity progressively to induce a natural overload stimulus, as previously utilized . Thus, avastus lateralis from each subject using the percutaneous needle biopsy technique with the aid of suction [Muscle biopsies were obtained before and 4 h post-exercise during the pre- and post-training temperature tolerance trials. Muscle biopsies were extracted from the belly of the  suction . Briefly suction ,23,24,30−1) was quantified using a nano-spectrophotometer  from each sample of muscle tissue (15.7 ± 3.2 mg). The RNA Integrity was measured using an Agilent RNA 6000 Kit and a 2100 Bioanalyzer  and indicated high quality intact RNA . First strand cDNA synthesis was achieved from the extracted RNA using Superscript IV LILO master mix with ezDNAase Enzyme . qRT-PCR was run in triplicate on a stratagene mx3005p PCR system  using a two-step protocol. Each 10 µL reaction volume contained 0.5 µL of primer and probe mix , 5 µL of qPCR master Mix , and 4.5 µL of sample adjusted to contain 0.5625 ng·μL−1 of cDNA. All probes and primers were designed and obtained by Integrated DNA Technologies  and presented in the RNA was extracted from skeletal muscle samples using Trizol  methodologies, as previously reported . The RNA−ΔΔCT method [beta-actin (ACTB), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), cyclophilin (CYC), beta-2-microglobulin (B2M), and ribosomal protein S18 (RPS-18). NormFinder software [Quantification of gene expression was calculated using the 2T method  relativesoftware  was usedp2, partial squared eta) are reported. SPSS Statistics for Windows  was used for all statistical analysis. A probability of type I error less than 5% was deemed significant (p < 0.05).Two-way, mixed design (time × temperature group) ANOVAs were used to determine differences between fitness assessment data , temperature tolerance trial data , and daily training data . Gene expression data was log transformed prior to statistical analyses to achieve a normal distribution. If Mauchly’s test for sphericity was violated, the Greenhouse-Geisser correction was applied. If a significant F-ratio was detected, Fisher’s protected least significant difference was used to determine differences, appropriate confidence intervals and effect sizes  and did not change due to training , and no interaction was observed . Aerobic capacity  and peak power increased with training  but were not different between the temperature groups , and no interaction was observed . See Anthropometric data were similar between the temperature groups  but were not different between the temperature groups (p = 0.310), and no interaction was observed (p = 0.448). Independent of training status, average core  and skin  temperatures were higher in the 33 °C group than the 20 °C group. Independent of temperature, Day 22 average skin temperatures decreased from Day 1 , but training did not influence core temperature (p = 0.598). Interestingly, both core temperature  and skin temperature  trended toward interaction but did not reach statistical significance. The 33 °C group had a higher sweat rate than the 20 °C group , but sweat rate was not influenced by training (p = 0.571), and no interaction was observed (p = 0.224). See The fixed intensity exercise trial also acted as Day 1 of the training intervention and allowed for pre- and post-testing (on Day 22) comparisons at the same absolute intensity within the assigned temperature condition (20 °C or 33 °C). Average heart rates during the 60 min trial were lower for the same power output during the post-intervention trial (p = 0.724) and was not different between the temperature groups (p = 0.427), and no interaction was observed (p = 0.659). The daily self-selected power produced during the cycling sessions increased overtime . More specifically, power produced during the daily workouts within weeks 2 and 3 (Days 8–19) were each individually greater than Day 2 , and the daily power outputs for each day within week 3 (Days 15–19) were greater than Day 8 . More importantly, the power output between the temperature groups were not different (p = 0.941), and no interaction was observed (p = 0.225). Post-exercise tympanic temperature did not change due to training (p = 0.798) but was greater in 33 °C than 20 °C , and no interaction was observed (p = 0.244). Sweat rate was calculated after each exercise session. Sweat rates were higher in 33 °C than in 20 °C . Besides Day 9 (p = 0.128), all of weeks 2 and 3 (Days 8–19) had higher sweat rates than Day 2 ; however, no interaction was observed (p = 0.227). See Heart rate did not change over the duration of the progressive-intensity training  and Day 22 , and there was no interaction . However, the 3-week intervention blunted Day 22 mRNA responses, when compared to Day 1 . Temperature did not influence PGC-1α (p = 0.455) and VEGF (p = 0.962) mRNA. NRF1 mRNA decreased in response to the acute exercise on Day 1  and Day 22  but was not different between days (p = 0.842) or temperatures (p = 0.517), and there was no interaction . The acute GABPA mRNA response did not change on Day 1 (p = 0.234) but decreased in response to acute exercise on Day 22 . However, the acute GABPA mRNA responses were not statistically different between days (p = 0.466), nor between temperatures (p = 0.688), and there was no interaction . TFAM and ESRRa did not change due to acute exercise, between days , or between temperatures , nor were there any interaction effects . See BNIP3-L mRNA did not change due to the acute exercise on Day 1 (p = 0.093) and Day 22 (p = 0.346); however, after the 3-week intervention, Day 22 BNIP3-L mRNA was higher than Day 1 . Temperature did not influence BNIP3-L mRNA (p = 0.486). PINK1, PARK2, and BNIP3 did not change due to the acute exercise, between days , nor between temperatures , and there was no interaction . See CS, p = 0.626; COX4, p = 0.558; BHAD, p = 0.925), and there was no interaction . CS, COX4, and BHAD mRNA were not influenced by acute exercise on Day 1 . However, after the 3-week training intervention, COX4  and BHAD  had a blunted response compared to Day 1. There were no differences for CS between days (p 0.188). See Gene expression for metabolic-related genes were not influenced by temperature , yet no change was detected within the 33 °C . Regardless of temperature, resting PGC-1α mRNA demonstrated an increased trend overtime  on Day 22. Temperature had no influence on resting PGC-1α mRNA (p = 0.087). Regardless of temperature, resting VEGF mRNA was higher on Day 22  when compared to Day 1. However, temperature had no influence on resting VEGF mRNA (p = 0.099). There were no detectable changes to resting mitochondrial biogenesis-related gene expression due to training or temperature for TFAM  and ESRRa . See The influence of this training and temperature intervention on resting mRNA was determined by comparing the resting biopsy on Day 22 relative to resting on Day 1. Resting PINK1 , PARK2 , and BNIP3  were not altered due to training or temperature, except for a main effect trend due to training for BNIP3-L, suggesting a potential reduction on Day 22 . However, temperature had no influence on resting BNIP3-L mRNA (p = 0.854). See For the most part, resting mRNA for mitophagy-related genes CS and COX4 resting mRNA were increased on Day 22  relative to Day 1, but temperature had no effect . BHAD mRNA did not change with training (p = 0.180); nor did temperature (p = 0.282). See Regardless of temperature, BNIPL-3 was influence by temperature; and (iii) this 3-week applied heat acclimation exercise routine improved aerobic fitness, peak power, and altered thermoregulation (via reduced skin temperatures). Overall, these results did not detect any temperature group acclimation differences and limited gene expression differences due to the fixed, absolute intensity tolerance trial. As such, an improved training status in both temperature conditions suggests that females may acclimate in a non-classical manner, i.e., differently from males.Historically, “classical” heat acclimation markers (decreased core temperature and increased sweat rate) are based on the physiological changes of males ,13,14,19peak) temperature (33 °C or 20 °C at 40% RH) tolerance trial induced acute increases (PGC-1α and VEGF) and decreases (NRF1) in mitochondrial biogenesis-related mRNA gene expression that were similar between temperature-groups. This suggests the skeletal muscle transcriptional responses were not impeded by the heat in these females. Although the intensity of this fixed-intensity exercise session was moderate, it was sufficient to initiate an upregulated mRNA for PGC-1α and VEGF within this group of untrained females. Increased PGC-1α and VEGF mRNA suggest an upregulated stimulus for mitochondrial and angiogenic growth potentials and have been consistently demonstrated after aerobic exercise within our lab and others [PGC-1α and VEGF) when performed within heated conditions. One likely explanation for these differences may be the underutilization of female participants within these heat-associated acute exercise projects [PGC-1α [VEGF [PGC-1α and VEGF mRNA increased similarly among the temperature groups on Day 1. Further, the acute decreases of NRF1 mRNA were similar among the female temperature groups. This was also different from previous gene expression in males [NRF1 downregulation in the heat compared to the room temperature. Together this suggests, prior to any heat acclimation training intervention, these females were more physiologically prepared to endure the heat and their acute gene expression data supports such a claim. Further, the Day 1 temperature tolerance trial was effective at separating the temperature groups via core temperature, skin temperature, and whole-body sweat rates. Therefore, females may physiologically compensate for ambient heat differently from males, independent of acclimation status.Prior to any heat acclimation training, the Day 1 fixed-intensity . An attenuated adaptive stimulus for mRNA expression suggests a reduced need to produce more protein due to training adaptations [PGC-1α, VEGF, COX4, and BHAD) represents an abundance of proteins associated with these genes, and thus an attenuated transcriptional stimulus is needed to maintain homeostasis. In applied terms, the absolute intensity on Day 22 no longer provided an overload stimulus. The fixed, absolute intensity of the Day 22 tolerance trial may have contributed to a reduced adaptive stimulus because it did not account for improvements in aerobic fitness but did allow for direct comparison. Resting mRNA gene expression comparisons (Day 22 vs. Day 1), designed to detect alterations due to training, point toward increased mRNA baseline values, as evidence by increased resting mRNA for VEGF, CS, and COX4 on Day 22. Previous post-exercise skeletal muscle gene expression data, using similar Day 22 methods, demonstrated a PGC-1α acclimation effect in men [PGC-1α mRNA responses were similar between temperate (20 °C) and heated (33 °C) men. Interestingly, non-statistical comparisons indicate that this applied heat acclimation protocol induced similar Day 22 PGC-1α relative reductions in mRNA expression, with both sexes decreasing ~2 fold change within 20 °C and ~4 fold change within 33 °C [This investigation’s Day 22 attenuated gene expression stimulus echoed fitness assessment improvements (increased VOptations . A Day 2t in men . In othein 33 °C . TherefoNRF1 and NRF2 (GABPA) expression within 20 °C but not 33 °C, which were the only temperature dependent differences. The proteins encoded from these genes are instrumental cofactors coordinating the response between the nuclear and mitochondrial genome [NRF1 and NRF2 (GABPA) mRNA of this small magnitude is unclear. Finally, mRNA gene expression for the mitophagy-associated gene BNIP3-L increased after the Day 22 tolerance trial. Intuitively, this suggests an improved sensitivity for mitophagy-associated signaling; however, no other mitophagy-related mRNA alterations were detected, including its predecessor of a similar likeness, BNIP3. However, attaching too much importance to these limited fold changes in resting mRNA comparisons, without applied functional alterations, should be cautioned against. Especially, considering there were no other mitochondrial-related resting mRNA changes , these data should not be overinterpreted.Additional Day 22 resting mRNA revealed a reduced l genome . This suAs previously demonstrated , an RPE-Here, we report that 3 weeks of exercise and heat acclimation training in untrained females did not hinder applied performance outcomes in comparison to room temperature conditions. Specifically, this applied approach to heat acclimation suggests that (i) temperature did not influence the pre-training acute exercise gene expression, (ii) there was some post-training attenuated gene expression responses attributable to the improved fitness, and (iii) this 3 week-routine improved aerobic fitness, peak power, and altered skin thermoregulation. Therefore, while this investigation did not demonstrate many classical heat acclimation temperature and exercise statistical differences, when placed in the context of previous similar work in males ,22, thesFrom a practical standpoint, caution should be taken when making exercise prescription and work physiology, as well as military training and operations recommendations that are not sex-specific. Although these females were untrained, they were better equipped to deal with the heat than untrained males. Leaders in their respective fields should tailor their heat-associated recommendations to take advantage of this unknown heat-resilient physiological mechanism within females. This can be especially useful where ambient conditions limit the ability for evaporative cooling via sweating . However, one must be cautioned, these results are based upon controlled settings and do not account for all factors that impact the heat index intensity.


This data was used to engineer spatially-controlled DNA-scaffolded bivalent ligands, which we used to compare trends in spatial-selective binding observed across HUVEC, CHO and HeLa in resting versus activated conditions in confocal microscopy images. For HUVEC and CHO, the data demonstrated an improved selectivity and localisation of binding for smaller spacings ~7 nm and ~24 nm, in good agreement with the model. A deviation from the mode predictions for HeLa was observed, indicative of a clustered, instead of homogeneous, integrin organization. Our findings demonstrate how low-technology imaging methods can guide the design of spatially controlled ligands to selectively differentiate between cell type and integrin activation state.Targeting cells specifically based on receptor expression levels remains an area of active research to date. Selective binding of receptors cannot be achieved by increasing the individual binding strength, as this does not account for differing distributions of receptor density across healthy and diseased cells. Engaging receptors above a threshold concentration would be desirable in devising selective diagnostics. Integrins are prime target candidates as they are readily available on the cell surface and have been reported to be overexpressed in diseases. Insights into their spatial organization would therefore be advantageous to design selective targeting agents. Here, we investigated the effect of activation method on integrin α A significant hurdle in the administration of therapies, and, in particular, in combatting infections, cancers and genetic diseases, is in the specific delivery of the therapeutic agent to targeted elements of an organ, tissue or cell type, i.e., active targeting . Cell re5β1 has been implicated in several diseases, ranging from cancer, inflammation [5β1 expression in epithelia is associated with inflammation [5β1 expression on the cell surface, versus relocation to the cell interior, of fibroblasts have also been implicated in resisting cancer progression [5β1 subtype could lead to applications spanning the improvement in cellular uptake of drugs [The integrin αammation , respiraammation , neuroloammation  and viruammation , besidesammation . Upregulammation  and actiammation . Changesammation . Integrigression  and has gression . Altogetof drugs  and geneof drugs , the modof drugs  and cellof drugs .5β1 on the cell surface has been reported to segregate into discreet focal adhesion clusters of active and inactive integrins, with active clusters having a higher local order [5β1 was also shown to have nanoscale ligand spatial preferences distinct from other integrin subtypes with different ligand binding behaviors in the range of spacings below 60 nm [5β1 expressed ubiquitously across cell types, are attributed to their primary function in cell adhesion [Integrins were first demonstrated to regulate their ligand-binding activity through conformational changes . The conal order . When noow 60 nm . The oveadhesion . TherefoGiven the different expression levels of subtypes of integrin receptors, based on cell type and state, engineering a targeting system capable of differentiating between these situations would have the potential to engage selective cell responses. From the pioneering works of Seeman ,30 folloIn this study, our aims are threefold. First, we focus on obtaining quantitative information of receptor spacing on surfaces of different cell types, and in different activation states, using readily available laboratory techniques and standard tools. The latter is important to generate a workflow that is generally applicable to any cell surface target, without the need for specialized equipment. Secondly, we hypothesize that receptor distributions are affected by activation state and that this metric can be a critical parameter in the design of selective targeting. We, thus, focus on the analysis of various activation methods and the quantitative effect of activation on receptor spacing. Finally, translating the spatial information of receptor distribution into DNA-scaffolded particles, we aim to demonstrate that by using spatially-controlled ligands, a selective interaction within cells, between activation states and between cell types can be obtained. This selective interaction can be observed already with bivalent ligands, if the scaffold spacing is chosen accurately.5β1 is implicated in cell proliferation and angiogenesis essential for endothelial cell function [5β1 in cervical cancer has previously been correlated with negative chemotherapeutic response and recurrence [5β1 expression is associated with inflammatory response [5β1 expression levels in HUVEC , CHO  and HeLa  cells for studies probing inter-receptor spacing and distribution.Integrin αfunction  and demofunction . An overcurrence . Additioresponse  and actiresponse . Based h2+ [5β1 as they are key modulators of endothelial cell function in regulating cell engagement with the ECM. Interestingly, the difference in the effect of the respective activation conditions on both HUVEC morphology and integrin α5β1 distribution was pronounced  were calculated assuming an even distribution between pixel intensities, factoring a jamming limit of ~0.55  a match of inter-receptor spacing to ligand presentation, (ii) number and density of integrins within a cluster [The trends in binding efficacy observed across RGD scaffolds in function of α cluster  (iii) lo cluster  (iv) pot cluster ,60. To e cluster . From th cluster  and canc cluster  the tren cluster . The 7 n cluster , and hig cluster , might f5β1 integrin targeting using bivalent, spatially controlled scaffolds designed by spatial input from a low-technology, easy-to-implement method to obtain quantitative information on global receptor spacing on the cell surface. We demonstrated the workflow using the α5β1 integrin, a critical regulator of cell-adhesion but equally important in many diseases. Indeed, a significant cell-type dependent spacing-signature profile was obtained, with clear differences when cells were activated. Translating the spatial information of receptor distribution into DNA-scaffolded multivalent particles, we demonstrated selective interaction within HUVEC and CHO cells, and between their activated and resting state. As the methodology homogenizes the receptor spacing within a pixel, deviations from reality in the special scenario when receptors form nanoclusters, can be expected. This was indeed observed when analyzing HeLa cells. Taken together, the accessibility of the presented approach, combined with the demonstration of differential targeting based on divalent ligand spacing, presents a broadly applicable strategy when selective cell targeting is envisioned.Concluding, our work presents the cell selective α® 488 (Cat.No. ab150077) 1:500, and Goat Anti-Mouse IgG H&L Alexa Fluor® 488 (Cat.No. ab150113) 1:500 were purchased from abcam, Cambridge, UK. Integrin alpha 5/CD49e Antibody (SNAKA51) (Cat.No. NBP2-50146) 1:1000 was purchased from Novus Biologicals, CO, USA. Mouse IgG2a kappa, clone eBM2a, eBioscience (Cat.No. 15287367) and 4%PFA (Cat.No. 15424389) were purchased from Fisher Scientific, NH, USA. Fmoc-Rink Amide MBHA resin , DBCO-maleimide, . All ssDNA strands listed below were ordered from Integrated DNA Technologies (IDT), Coralville, IA, USA.Unless otherwise specified, reagents were used as received without further modification. Deionized water obtained from a Milli-Q water purification system was used for all experiments. Penicillin/streptomycin (Cat.No. 15140122), DMEM + GlutaMAX (Cat.No. 31966021), Fetal Bovine Serum (Cat.No. 10500064), DPBS (Cat.No. 14040091), 0.05% Trypsin-EDTA (Cat.No. 25300054), BlockAid blocking solution (Cat.No. B10710) and BSA 7.5% solution (Cat.No. 15260037) were purchased from Thermo Fisher Scientific, Waltham, MA, USA. All buffers were filtered with 0.22 µm PES syringe filters  or PES bottle filters . HeLa Ohio cells were obtained from SuNMIL Lab, EPFL and CHO from the EPFL Protein Facility, Lausanne, Switzerland. HUVEC (Cat.No. C2517AS), Reagent Pack™ Subculture Reagents (Cat.No. CC-5034) and EGM2 endothelial cell growth medium-2 bulletKit (Cat.No. CC-3162) was purchased from Lonza, Basel, Switzerland. 96 well ibidi angiogenesis µ-plates , Poly-L-lysine solution . Rabbit anti-Integrin alpha 5 antibody EPR7854 (ab150361) 1:200, Recombinant Rabbit IgG, monoclonal (Cat.No. ab172730) 1:1500, Goat Anti-Rabbit IgG H&L Alexa Fluor2, 95% relative humidity. When relevant, cells were incubated in media supplemented with activator  for 45 min. The following steps were performed at RT. Cells were fixed in 2% PFA for 15 min, washed with DPBS, and blocked for 1 h with BlockAid blocking solution. The cells were then incubated on a shaker for 1 h with the respective primary antibodies diluted in BlockAid. Cells were washed with DPBS supplemented with 3% BSA and incubated with the respective secondary antibodies diluted in BlockAid for 1 h.HUVEC, CHO and HeLa cells were seeded in 96 well ibidi angiogenesis µ-plates, 3000 cells per well. Cells were incubated overnight at 37 °C, 5% CO® 488 conjugated secondary antibodies for calibration of antibody concentrations in experimental wells. Calibration wells were washed with DPBS prior to fluorescence measurements. Fluorescence intensities were measured on a BioTek™ Cytation 5™ Cell Imaging Multi-Mode Reader, BioTek Instruments, Inc. using the Gen5 software, Version 3.10. Monochromators were used to measure the fluorescence intensities of the Alexa Fluor® 488 secondary antibodies /519(10)). A 20X PL FL Phase  phase objective was used. Imaging settings were kept constant across cell lines when comparing different conditions. Dimensions 4X images: 1224 × 904 pixels (0.619 pixels µm−1) Dimensions 20X images: 1224 × 904 pixels (3.115 pixels µm−1).For calibration, 96 well ibidi angiogenesis µ-plates were coated with poly-L-lysine (PLL) for 1 h and washed with DPBS prior to incubation with a serial dilution of the Alexa Fluorv/v) ratio to Pierce™ Immobilized TCEP Disulfide Reducing Gel and incubated on a shaking platform for 90 min. Zeba™ Spin Desalting Columns 7 K were used to remove excess DBCO-maleimide according to the manufacturer’s instructions. The solution was incubated for 2 h, characterised and stored at −20 °C in solution.Peptide synthesis of RGD peptides with a GC linker was conducted, based on previously reported synthesis . A 65 µmBivalent, rigid DNA scaffolds were adapted from Mohri et al.  with seq2, 95% relative humidity. The following morning, cells were incubated in full media (supplemented with activator SNAKA51 primary antibody 1 µg mL−1 for activation conditions) for 1 h. Buffers used for fixation, staining and imaging were adapted from Strauss et al. Cells were fixed in 2% PFA and 1X cytoskeleton buffer  for 15 min then washed with DPBS + 0.05%Tween. When non-activated, cells were incubated with SNAKA51 primary antibody for 30 min prior to the following steps. DNA scaffolds were added to cells at 7.5 µM in Bivalent Scaffold Staining Buffer and incubated for 30 min. Cells were staining with Goat Anti-Mouse IgG H&L Alexa Fluor® 488 secondary antibody and DAPI prior to imaging. Cells were washed with Bivalent Scaffold Buffer and stored in Bivalent Scaffold Imaging Buffer for confocal imaging. Confocal imaging was conducted at the EPFL Bioimaging and Optics Platform on a Zeiss LSM980 with Colibri 5 illumination for fluorescence and ZEN Blue software ver. 3.4.91. The buffers used were the following: (i) Bivalent Scaffold Staining Buffer  (ii) Bivalent Scaffold Buffer  (iii) Bivalent Scaffold Imaging Buffer HUVEC, CHO and HeLa cells were seeded at 3000 cells per well in 96 well ibidi angiogenesis µ-plates. Cells were serum-starved incubated overnight at 37 °C, 5% COConfocal microscopy images were acquired in 3 channels, DAPI, Cy5 and Alexa488. The images were acquired as z-stacks (0.2 µm) of 15 images. Images were post-processed in FiJi software as follows in all channels for a minimum of 9 images per condition: (i) sum slices (ii) measured for signal % area and integrated signal density for plots. The heatmaps were prepared in MATLAB ver. R2018b through application of a custom colormap  after ct-tests between each spatial scaffold in resting and activated state.To quantify scaffold selectivity, we calculated the integrated density on the cell, defined with a mask based on antibody SNAKA51 presence. The quantification of the Cy5 signals was done identically for all datasets (9 per cell type and scaffold condition) using MATLAB: (i) Segmentation of cells from background using hard thresholding and morphological operators on the SNAKA51 channel, (ii) segmentation of the nuclei using the same approach in the DAPI channel, (iii) summation of the Cy5 signal in the slices when present within the defined mask of (i) but not (ii), (iv) computation of average background intensity and hard thresholding (v) computation of the average intensity of the Cy5 signal. Resulting images were exported to FiJi and identical LUT for Cy5 were applied. For statistical analysis  an ANOVAOriginal topology and configuration files prepared with vHelix were converted in oxDNA topology and configuration files using tacoxDNA. Simulations were performed using the oxDNA simulations software with the oxDNA2 forcefield . When no−4, 5 × 106, (ii) 10 °C, 5 × 10−4, 5 × 105, (iii) 20 °C, 5 × 10−4, 5 × 105, (iii) 20 °C, 5 × 10−4, 5 × 105, (iv) 25 °C, 5 × 10−4, 5 × 105. For the final production runs we used 25 °C, 1 × 10−3, 4.0 × 107 and saved configurations for analysis every 5 × 103 steps, excluding the first 0.5 × 107 steps. The topologies and trajectories were then used as input for an inhouse Python script to generate files in PDB, PSF and MDCRD format for analysis in physical units using VMD [At first, we performed a 1e7 steps long minimization in which the maximum force acting on the backbone was kept to 5. Then we performed 4 relaxation runs in the NVT ensemble in which we progressively warmed up the structure to 25 °C and lifted the limits on the maximum force. The respective combinations of temperature, time step and total number of steps for each relaxation run were: (i) 1 °C, 5 × 10sing VMD . Removal


Phytoestrogens and cold negatively influence male fertility. However, the combined effects of these two factors on male reproduction remain unknown. Herein, we studied the changes of sperm parameters and identify potential biomarkers involved in testis of rats, which were intervened by phytoestrogens combined with cold stress. Male Sprague–Dawley rats were randomly divided into control and model groups. The rats in the model group were fed an estrogen diet and placed in a climate chamber  for 12 h/daily. When compared with the control group after 24 weeks, the rats in the model group showed increased food intake, urine and stool outputs, and higher estradiol and follicle-stimulating hormone levels. However, lower sperm concentration, motility, and viability, and reduced testosterone levels were detected. The epithelial cells of the seminiferous tubules and epididymal ducts presented morphological abnormalities. Proteomic analysis showed that 24 testicular proteins were upregulated and 15 were downregulated. The identified proteins were involved in reticulophagy and stress response. Our findings suggest that the phytoestrogens combined with cold stress had negative effects on the reproductive function of male rats and provide the basis for the establishment of “course simulation” type of oligospermia animal model. SpermatExposure to environmental estrogen pollution can significantly affect the male reproductive system. Phytoestrogens are natural heterocyclic phenol compounds, which possess structural similarity with estrogen steroids and present estrogenic activity. The classical phytoestrogens, so far known, constitute a group of plant-derived compounds which include mainly isoflavones, lignans, coumestanes, stilbenes, and the flavonoids quercetin and kaempherol. Aliments, such as spinach, bean sprouts, soybeans, cabbage, and hops, contain high levels of phytoestrogens. Several preclinical experiments have demonstrated that the ingestion of high levels of phytoestrogens can negatively affect male reproduction, leading to decreased semen quality and an increase in congenital malformations, such as hypospadias and cryptorchidism. Moreover, phytoestrogens can adversely affect brain differentiation and reproductive development in rodents during embryogenesis  and can Cold is one of the strongest physical and psychological environmental stressors, leading to a significant defense response in the body, known as the cold shock response. The body responds to cold shock with long-term adaptive changes in metabolic, physiological, biochemical, and hormonal parameters ,5,6. TheXinjiang is located at the following geographical coordinates: 73°40′ E∼96°23′ E, 34°22′ N∼49°10′ N. In this location, the winter season is cold and long. Additionally, environmental pollution has become increasingly serious and estrogen pollutants  are part of the environmental contaminants.In this study, phytoestrogens combined with cold stress intervention were experimentally reproduced, and changes in sperm parameters in rats were evaluated. Alterations of the testis proteins were detected using label-free proteomic technology. This study aims to set the basis for establishing a “course simulation” type of oligospermia animal model and preliminarily identify its molecular mechanism.22.1The experimental animals were bred and provided by the Animal Experiment Center of Xinjiang Medical University. In a 24-week phytoestrogen and cold stress intervention study, 8-week-old Sprague–Dawley rats (180–220 g) were randomly assigned to two groups of 12 males each. Group N served as the control group. Rats in group M were fed phytoestrogens – diet  – and placed, every day for 24 weeks, in a climate chamber  from 9 in the morning to 21 o ‘clock in the evening  ,12. BodyEthical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals and in accordance with the Animal Care and Use Committee of Xinjiang Medical University (Res: IACUC-20210302-46).2.2At the end of the experiment, after fasting for 12 h, the rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg). The left epididymis cauda was removed, and sperm suspension was prepared by mincing the tissue with scissors in 4 mL of M199 medium (containing 2% BSA). After incubation at 37°C for 15 min, the tissue fragments were removed from the suspension. Semen analysis was performed according to World Health Organization guidelines . The sperm concentration was determined using a Neubauer counting chamber. An aliquot of the sperm suspension was charged into the counting chamber, and spermatozoa were counted. The sperm count was expressed in millions per mL. Sperm motility was determined by placing a drop of 10 µL of the sperm suspension on a 37°C prewarmed slide and covered with a cover slip. At least 200 sperm were assessed for each semen sample using a bright-field microscope, and the percentage of motile sperm was calculated. Sperm viability was assessed using the eosin–nigrosin staining technique, where the dead cells were stained. A sample of 10 µL of sperm suspension was mixed with 0.5 μL of eosin for 30 s, then added 3 μL nigrosin staining solution, and a smear was made on a prewarmed slide. The slides were examined under a microscope with oil immersion  to determine the percentage of viable sperms.2.3g for 15 min at 4°C. The collected serum samples were stored at −80°C. Serum sex hormone levels of testosterone (T), estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (PRL) were measured by using radioimmunoassay kits  according to the manufacturer’s instructions.Blood was collected from the abdominal aorta, and samples were centrifuged at 3,000 × 2.4-PCR) analysis.The right testis and epididymis were collected from both experimental groups. The tissue was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and evaluated by Periodic Acid-Schiff-hematoxylin (PAS-H) staining and immunofluorescence staining. The left testis was cryopreserved at −80°C for proteomic studies, Western blotting, and quantitative real-time polymerase chain reaction  were acquired at a mass resolution of 70 K, followed by 15 sequential high-energy collisional dissociation (HCD) MS/MS scans at a resolution of 17.5 K. In all cases, one microscope was recorded using a dynamic exclusion of 30 s. For MS/MS, precursor ions were activated using 27% normalized collision energy.Testicular tissues were lysed in the presence of protease inhibitors, and total protein was extracted. Protein concentration was determined using a bicinchoninic acid (BCA) kit , and protein quality was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). The proteins were hydrolyzed via enzymatic digestion using trypsin. After trypsin digestion, the peptides were resuspended in 25 μL solvent A (A: ACN with 0.1% formic acid), separated by nanoLC, and analyzed by online electrospray tandem mass spectrometry (MS/MS). The experiments were performed on a NanoAquity UPLC system  connected to a Q Exactive hybrid quadrupole Orbitrap-mass spectrometer  equipped with an online nano-electrospray ion source. An aliquot of the peptide sample (8 μL) was loaded onto a Thermo Scientific Ac-claim PepMap C18 column , with a flow rate of 10 μL/min for 3 min, and subsequently separated on an analytical column  with a linear gradient from 5% B to 45% B in 75 min. The column was re-equilibrated under initial conditions for 15 min. The column flow rate was maintained at 300 nL/min, and the column temperature was maintained at 40°C. An electrospray voltage of 1.9 kV versus the inlet of the mass spectrometer was used. The Q Exactive mass spectrometer was operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition. Survey full-scan MS spectra  database. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to identify the pathways that involved target proteins.2.72.7.1g for 10 min at 4°C, the supernatants were collected. The protein concentration was determined using the standard BCA kit . Then, 40 µg of each protein sample was loaded and separated by 10% sodium dodecyl SDS–PAGE and transferred to polyvinylidene fluoride (PVDF) membranes . After blocking in 5% nonfat milk for 2 h at 37°C, the membrane was incubated with the primary antibody overnight at 4°C: integrin alpha 6 (ITGA6) , alpha 1 chains of type VI collagen (Col6α1) , alpha 2 chains of type VI collagen (Col6α2) , then with a Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG(H + L) secondary antibody  for 2 h at 37°C. Finally, the membrane was washed with tris base solution-tweet (TBST) and visualized using enhanced chemiluminescence detection (ProteinSimple Flour Chem. E). Afterward, the protein bands were quantified and analyzed using ImageJ software.Testicular tissues from each rat were separately homogenized and lysed in ice-cold radio-immunoprecipitation assay lysis buffer . After centrifugation at 12,000 × 2.7.2Paraffin-embedded testicular Section (4 µm thick) were dewaxed with dimethylbenzene and gradient ethanol and then washed with phosphate-buffered saline (PBS). After incubation with 3% hydrogen peroxide, antigen retrieval was performed using 0.01 mol/L sodium citrate buffer (pH 7.0). The sections were then washed thrice with TBST and blocked with goat serum. Primary antibody was incubated overnight at 4°C: ITGA6 , Col6α1 , and Col6α2 . After washing three times, the samples were incubated with a secondary antibody to fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG for 30 min at 37°C  and washed three times with TBST; 4′,6′-diamidino-2-phenylindole (DAPI) was used to stain the nuclei of cells in the testis, and the sections were observed for epifluorescence using a Leica Microsystems CMS GmbH microscope.2.7.3n = 10) were randomly selected, and total RNA was isolated using an RNA Easy Fast Animal Tissue Total RNA Extraction Kit . Total RNA concentration was measured using a Nanodrop 2,000 spectrophotometer. RNA (1 µg) was then converted to cDNA using a FastKing RT Kit (with gDNase)  in a 20 μL reaction mixture. Aliquots of 1 μL cDNA samples were subjected to quantitative PCR (qPCR) in a total volume of 10 μL using SuperReal PreMix Plus (SYBR Green)  in a QuantStudio 6 Flex. The primers used were GAPDH F-TGAATACGGCTACAGCAACAGG, R-GTGAGGGAGATGCTCAGTGTTG, ITGA6 F-CGCCGCCGCTCAGAATATCAAG, and R-CAAGCACAGCCAGGAGGATGATC, Col6α1 F-CCTCCTGGTCCCTCCCTCTTTG, R-TTGTCTCTGCTTGCTGCCGTTAC, Col6α2 F-TCCTCCTCCTCTTCCTCCTCCTC, R-AGCAAGTTCCAGAACAGTCAAGGC. GAPDH mRNA was used as an internal control.Testicular tissues from each group (2.8t-test using SPSS version 26.0 software (IBM), and P < 0.05 was considered significant.Comparison between experimental groups was performed by the independent two-tailed 33.1P < 0.05, To determine the effect of estrogen pollution combined with cold shock, 12 rats were exposed to a diet rich in estrogen and placed for 12 h daily at a lower temperature (10 ± 2°C) for 24 weeks (group M) and were compared with a control group . Body weight, daily food and water intake, and urine and stool weights were measured. Compared with the control group, no apparent changes in the final body weight were observed. However, significant increase in daily food intake, weight of urine output, and stool output as well as reduction in water intake were detected , motility (P < 0.001), and viability (P < 0.05) in the model group (M) were significantly lower , LH, FSH, and PRL were measured. Compared with group N, T levels in group M were significantly decreased, whereas E2 and FSH levels were increased (P < 0.05). However, no significant change in LH and PRL levels was observed , estradiol , H1.2 linker histone , alpha 1 chain of type VI collagen , alpha 2 chain of type VI collagen , 40 S ribosomal protein S6-like , immunoglobulin heavy constant mu , macroH2A.1 histone , sorbin and SH3 domain-containing 2 , B-cell receptor-associated protein 31 , and transgelin . The top ten downregulated proteins included the hypothetical LOC287798 , translocase of the outer mitochondrial membrane 70,3′(2′) , 5′-bisphosphate nucleotidase 1 , structure-specific recognition protein 1 , dynein light chain LC8-type 1 , coiled-coil domain containing 58 , doublecortin domain containing 2 C , similar to 60 S ribosomal protein L38 , TOR signaling pathway regulator, and metallothionein 3 , included alpha 5 chains of type VI collagen  enrichment analysis was performed to classify the biological processes, cellular components, and molecular functions of these proteins . The difPathway analysis of the differentially expressed proteins was performed using the KEGG. The top five pathways containing the largest number of proteins were focal adhesion (FAs), human papillomavirus infection, PI3K-Akt signaling pathway, metabolic pathway, and cancer proteoglycan . Specifi4Spermatogenesis in mammals is a cyclic process of spermatogenic cell development occurring in the seminiferous tubules. The formation, maturation, and capacitation of spermatogenic cells are susceptible to dietary changes and environmental factors. Temporary sterility syndrome in livestock (ewes) is associated with high phytoestrogen intake during grazing . Humans Temperature is an important environmental factor affecting the metabolism and reproduction of small mammals . The inh2 and FSH levels increased after treatment with phytoestrogens combined with cold stress. This is related to the competitive binding of phytoestrogens to the estrogen receptor, which inhibited its negative feedback regulation, promoted the positive feedback regulation of LH secretion, and stimulated the secretion of E2. FSH is essential for the initiation of spermatogenesis, but the abnormal increase of serum FSH level often means that the seminiferous epithelium has been damaged. At the same time, when testicular cells were exposed to a high-estrogen environment, the expression of steroid synthase decreased, which could also lead to a further decrease in T level [Morphological study of the testis and epididymis showed that the epithelial cells of seminiferous tubules and ductus epididymis were obviously different between the control and model groups. Compared with group N, the number of sperm in the lumen of the epididymis decreased dramatically. The changes in hormone levels were responsible for abnormal morphology. Serum sex hormone results showed that the testosterone level in the model group significantly decreased, whereas the E T level . In addiFurther proteomic studies identified 39 testicular proteins differentially expressed between the two experimental groups. Among these proteins, integrin α6 (ITGA6) and collagen6 (chain α1 (Col6α1) and chain α2 (Col6α2)) were detected with a high fold-change. Integrins play a key role in cell adhesion to extracellular matrix (ECM) ligands and adjacent cells and serve as links between extracellular contacts and the intracellular cytoskeleton. In addition, integrins work together with receptor tyrosine kinases to communicate bidirectional signals between cells and ECM . They arGO enrichment analysis showed that the differentially expressed proteins were mainly involved in reticulophagy and response to stimulus. The endoplasmic reticulum (ER) is a biosynthetic organelle in eukaryotic cells. Its capacity to produce proteins, lipids, and oligosaccharides responds to physiological and pathologic demands . ReticulPathway analysis showed that the differentially expressed proteins were involved in 51 signaling pathways, among which the PI3K-Akt signaling pathway, FA signaling pathway, metabolic pathway, protein digestion, and absorption pathways involved more proteins. Several studies have shown that the PI3K/Akt pathway plays an essential role in the self-renewal of SSCs and the differentiation of spermatogonia . In Akt−5Rats treated with phytoestrogens combined with cold stress showed higher food intake, increased urine and stool output, and lower sperm concentration, motility, viability, and testosterone level. The epithelial cells of seminiferous tubules and epididymis presented abnormal morphology. Moreover, ITGA6 downregulation and upregulation of alpha 1 and alpha 2 chains of type VI collagen in the testis suggest that alterations of PI3K/AKT and FA pathways might represent the molecular mechanism behind the estrogen/cold-induced infertility. Therefore, this study lays the foundation for the establishment of “course simulation” type of oligospermia animal model.


Acartia tonsa. The more toxic NEOs were ACE (EC50 = 0.73 μg L−1), TMX (EC50 = 1.71 μg L−1) and CLO (EC50 = 1.90 μg L−1), while the less toxic compound was IMI (EC50 = 8.84 μg L−1). Early life-stage mortality was unaffected by NEOs at all of the tested concentrations. The calculated toxicity data indicated that significant effects due to ACE (EC20 = 0.12 μg L−1), THI (EC20 = 0.88 μg L−1) and TMX (EC20 = 0.18 μg L−1) are observed at concentrations lower than established chronic aquatic life benchmarks reported by USEPA for freshwater invertebrates. Nevertheless, since environmental concentrations of NEOs are generally lower than the threshold concentrations we calculated for A. tonsa, the effects may be currently of concern only in estuaries receiving wastewater discharges or experiencing intense runoff from agriculture.Neonicotinoids (NEOs) are neurotoxic pesticides widely used in agriculture due to their high effectiveness against pest insects. Despite their widespread use, very little is known about their toxicity towards marine organisms, including sensitive and ecologically relevant taxa such as copepods. Thus, we investigated the toxicity of five widely used NEOs, including acetamiprid (ACE), clothianidin (CLO), imidacloprid (IMI), thiacloprid (THI), and thiamethoxam (TMX), to assess their ability to inhibit the larval development of the copepod  Neonicotinoids (NEOs) are neurotoxic pesticides that disrupt synaptic transmissions by binding with the nicotinic acetylcholine receptors (nAChRs), leading to membrane depolarization, ion channels activation, and propagation of the action potential. In particular, NEOs have a high affinity for nAChRs located in the insects’ central nervous system, and once they bond with nAChRs, they cause neuronal hyper-excitation that produces different sub-lethal effects and also leads to death ,2. In adTheir high effectiveness against pest insects, combined with high water solubility and low mammalian toxicity, favored their widespread use in agriculture and made them the most popular class of pesticide in the past decades ,4. ResisConsequently, NEOs have been frequently detected in surface waters worldwide, raising concern for the risk they may pose for non-target aquatic and terrestrial communities supported by these ecosystems ,7,8. Several studies explored the toxic effects of NEOs on freshwater non-target invertebrates ,10, and Acartia tonsa, Centropages sp., Eurytemora affinis and Nitocra spinipes [Acartia tonsa are very sensitive to pesticides and other organic chemicals acting as endocrine and nervous transmission disruptors [A. tonsa a useful bioindicator organism for assessing the effects of toxic substances [Crustaceans are the marine invertebrates most possibly susceptible to NEO’s toxic action due to their nervous system’s similarity with insects ,17. Planspinipes ,20,21. Isruptors ,23,24,25bstances .A. tonsa were exposed to four concentrations of each pesticide and let to develop for five days to assess the effects of the pesticides on larval development. At the end of the five days exposure, point-estimate toxicity data  were generated for each pesticide [The present study aimed to provide a first screening of the toxicity of five synthetic commercially available NEO pesticides on the larval development of marine planktonic copepods. The selected active compounds are the first generation NEOs acetamiprid (ACE), imidacloprid (IMI) and thiacloprid (THI), and the second generation NEOs clothianidin (CLO) and thiamethoxam (TMX) . In partesticide ,29. ThesA. tonsa was performed as outlined in Picone et al. [A. tonsa were purchased from Guernsey Sea Farms Ltd., Port Vale, Guernsey, UK. In-house laboratory cultures were started by adding 800–900 freshly released eggs to 1.8-L of a 20‰ salinity culture medium prepared according to ISO 16778 [Tetraselmis suecica, Pavlova lutheri and Tisochrysis lutea), was provided four times per day through a timer-controlled peristaltic pump. All algal clones were cultured in Guillard’s F/2 medium, at 20 ± 1 °C, under continuous aeration and 16:8 light:dark photoperiod.Culturing of e et al. . BrieflySO 16778 . The culThe eggs were removed daily from cultures by siphoning off the medium from the bottom of the culture flask and then filtering it through two sieves with mesh sizes of 170-μm and 50-μm, respectively. Adult copepods, retained by the 170-μm mesh sieve, are then reintroduced in the culture, while eggs and nauplii, passing through the 170-μm sieve but retained by the 50-μm sieve, were collected and stored separately. Each culture was maintained for testing for up to 6 weeks. Acartia cultures) were used during the testing period for a total of five experiments. Eggs from culture AT14/19 were used for testing IMI (14 October 2019), while ACE was tested using eggs from culture AT17/20 (29 June 2020); culture AT18/20 was used for testing TMX (6 July 2020) and THI (13 July 2020), while eggs from culture AT19/20 were used for testing CLO (10 August 2020).Different parental groups . The physicochemical properties of the pesticides are reported in −1 were prepared for each compound in 99% ethanol and then diluted to test concentrations using the 20‰ salinity medium used for culturing the copepods. The solvent percentages in the different NEO tested concentrations were included in the range 0.0001–0.1%. Possible solvent-induced effects were tested by exposing eggs to a series of ethanol concentrations (0.0002–2%) diluted in the 20‰ salinity medium. Ethanol did not significantly affect the larval development of A. tonsa at the tested concentrations (0.0002–2%) . At 0.2% and 2% we observed a minor reduction of LDR as compared with control  but it was not statistically significant  [Stock solutions at 10 mg Lctively) .−1 for ACE; 0.02, 0.08, 1.32 and 12.4 μg L−1 for CLO; 0.02, 0.14, 1.01 and 10.1 μg L−1 for IMI; 0.03, 0.14, 1.13 and 11.0 for THI; 0.01, 0.16, 1.01 and 11.0 for TMX). These concentrations bracket the effect-concentrations reported in the literature for acute and early-life stage tests with marine and estuarine crustaceans [Four NEO concentrations were tested for each compound  mixture of T. suecica, T. lutea, and P. lutheri obtained by centrifuging cultured algae per 5 min at 4000× g.Test vessels were then maintained for five days in a thermostatic incubator  at 20 ± 1 °C, with a 16-h light 8-h dark photoperiod and under a LED illumination to minimize the ultraviolet (UV) emission and avoid photolysis. On day-2, an additional 30 mL of test solution was added to each beaker to refresh the medium. Larvae were fed on day-0 and day-2 with 100 µL of a concentrated . Lugol’s solution kills, stains, and preserves unhatched eggs, nauplii, and copepodites [Exposures ended on day-5 when approximately 40% of the larvae in negative controls reached the copepodite-I stage. The ratio of nauplii to copepodites was first determined in one control replicate after exactly 5-d by staining the beaker’s content with 0.5 mL of Lugol’s solution (100 g Lepodites . The tesDissolved oxygen (DO) and pH were measured on day-0 in one beaker per concentration, before the inoculation of the eggs, and on day-5, before staining with Lugol’s solution. ELS-m represents the ratio of hatched larvae that die within the fifth day of exposure, and it was calculated as follows:LDR is the ratio between copepodite-I larvae and the total number of early stages (nauplii plus copepodite-I larvae) recovered at the end of the test:LDR values obtained for each test concentration were then normalized to the average control LDR to compare results obtained in different testing sessions. Standard error was used as a measure of data dispersion.v/v) in water (10 mL). After extraction, the cartridges were dried for 5 min and finally NEOs were eluted with 10 mL of methanol. Eluates were reduced to 200 µL under nitrogen flow at 30 °C  and reconstituted with 800 µL of ultrapure water. Linearity ranges, limits of detection (LoDs) and limits of quantification (LoQs) are reported in NEO testing concentrations were measured using the HPLC/(-)ESI-MS/MS analytical technique, using an Agilent 1100 series HPLC system  coupled to an API 4000 triple quadrupole mass spectrometer . Briefly, 100 mL of each test solution were diluted with ultrapure water to a final volume of 1 L and spiked with 10 ng of the corresponding deuterated internal standard. The ultrapure water  was produced by a Chorus system . Samples were cleaned up and pre-concentrated using OASIS HLB cartridges  previously conditioned with 10 mL of methanol and equilibrated with formic acid (0.2% 10s), effective concentrations 20 (EC20s), and effective concentrations 50 (EC50s) were calculated using a statistical program for generating point-estimate toxicity data for variables with a continuous response, developed at the Technical University of Denmark [Effective concentrations 10, . Acceptability criteria for negative controls on day-5 include an average LDR of 0.5 ± 0.2 and an average ELS-m less than 0.3. The average LDR obtained in the five experiments for the controls was 0.45 ± 0.05 (n = 5), with a minimum of 0.36 ± 0.02  and a maximum of 0.64 ± 0.01 (culture AT14/19). ELS-m was less than 0.3 in all tests, with a minimum value of 0.13 ± 0.04 (culture AT14/19) and a maximum of 0.26 ± 0.06 . All data concerning LDR and ELS-m obtained in negative controls and NEO treatments are reported in Five experiments were performed by using eggs collected from four different 50 is 31–250 μg L−1 of 3,5-DCP [50s obtained for 3,5-DCP in the four positive control tests performed with the different parental groups  were within the control chart’s acceptability interval (31–250 μg L−1). Oxygen saturation was always >90% at the beginning and end of the test; pH variation was always < 0.5 units. Positive control tests with 3,5-dichlorophenol  as a reference toxicant were used to verify the relative sensitivity of the eggs used in toxicity tests with NEOs, as well as the precision and reliability of the data produced by the laboratory. The control chart acceptability interval for the EC 3,5-DCP . EC50s oELS-m was the less sensitive endpoint: none of the tested treatments provided significant mortality compared to negative control. A summary of ELS-m data is reported in 50s, the most toxic NEO pesticide toward A. tonsa larval development was ACE, with an EC50 of 0.73 μg L−1, while the least toxic NEO was IMI (EC50 = 8.84 μg L−1). The overall toxicity gradient based on EC50 was ACE > TMX = CLO > THI > IMI. This gradient was similar to the EC20s, with a toxicity gradient of ACE = TMX > CLO > THI > IMI.In contrast, LDR was heavily affected by all five NEOs . Accordi−1. At concentrations approximating 1.00 μg L−1, toxic effects increased markedly for all pesticides, but IMI, TMX and CLO provided an inhibition of the larval development averaging 50% at 1.01 μg L−1 and 1.32 μg L−1, respectively, while ACE increased its inhibiting effect on larval development up to 75% at 2.30 μg L−1. The toxicity of THI increased moderately, following the log-linear trend exhibited at the lowest tested concentrations (30% inhibition of LDR at 1.13 μg L−1).The effect-concentration curves differed considerably among pesticides . None ofA. tonsa (over 90%) at 11.0, 12.4 and 21.4 μg L−1, respectively. TMX and IMI were less toxic than the other pesticides at the highest tested concentrations, with inhibition reaching a maximum of 78% for TMX and 58% for IMI.THI, CLO, and ACE inhibited almost completely the larval development of 10s, EC20s and EC50s are still a good proxy for the assessment of the toxicity of the tested NEOs towards A. tonsa, these data may be not appropriate for the derivation of environmental quality criteria. However, the NEO toxicity data showed that the larval development of A. tonsa was inhibited at lower concentrations than most of the acute and chronic effect-concentrations reported in the literature for marine crustaceans, including also brackish water copepods, mysids, and prawns  was observed at concentrations ranging from 24 μg L−1 (96h-LC50 for ACE) up to 4100 μg L−1 (96h-LC50 for TMX) [50s in the range 19–4100 μg L−1 [A. bahia at concentrations significantly lower than 96h-EC/LC50s calculated for acute exposure test [−1  to 3600 μg L−1 (growth), while the 28d-EC50 calculated for ACE was 4.7 μg L−1 (growth).As concern other orders, mysids were the marine crustaceans more often used for testing NEOs ,13: signfor TMX) . Sub-let0 μg L−1 . Chronic50s ranged from 14 μg L−1 (ACE) to 940 μg L−1 (TMX) for Penaeus japonicus, and 260 μg L−1 (CLO) to 3500 μg L−1 (ACE) for Crangon uritai. All of these data are at least one order of magnitude higher than the EC50s calculated for A. tonsa LDR in the current study, ranging from 0.73 μg L−1 (ACE) to 8.84 μg L−1 (IMI). Butcherine et al. [P. monodon postlarvae, and obtained 48h-LC50s values ranging from 190 μg L−1 (CLO) up to >500 μg L−1 (ACE). However, the authors also observed an increased antioxidant activity at concentrations as low as 5 μg L−1.Hano et al.  also expe et al.  evaluate50s for juveniles (1.1 mg L−1) and megalopae (10 μg L−1) of the blue crab Callinectes sapidus showed similar or even higher sensitivity as the early-life stages of A. tonsa [Penaeus monodon (48h-LC50 = 175 μg L−1) and the brine shrimp Artemia sp. (48h-LC50 > 1.000 μg L−1), were by far more tolerant [Data on other marine crustacean species are available only for IMI; only 24h-LCA. tonsa . Other mtolerant ,36.A. tonsa are comparable or even lower than effect-concentrations calculated for the most sensitive freshwater species, including Diptera (Aedes sp. and Chironomus dilutus) and Ephemeroptera  (50s ranging from 0.8 μg L−1 (THI) to 36.8 μg L−1 (TMX) for the midge Chironomus dilutus, and from 1.6 μg L−1 (ACE) to 5.5 μg L−1 (TMX) for the mayfly Neocloen triangulifer.As compared with freshwater taxa, our data for nia sp.) . As an e50s we calculated in this study  and the fragrance materials amyl-salicylate (5d-EC50 = 0.13 μg L−1) and hexyl-salicylate (5d-EC50 = 0.06 μg L−1) provided EC50s lower than NEOs [50s similar to those calculated for the NEO pesticides [Based on the ECis study , NEOs sthan NEOs ,40. Othesticides ,25,41. 50 values ranging from 490 μg L−1  [−1 (DEHP) [50 obtained for NEOs in the present study   and othenalide™)  to 1400 1 (DEHP) , markedlnt study .A. tonsa evidenced a different sensitivity toward the different NEOs, with ACE (EC50 = 0.73 μg L−1) characterized by a higher inhibitory potential than the other NEOs. A different tolerance towards NEO compounds is a typical output for several species, sensitive freshwater insects , oligochaetes (Lumbriculus variegatus) and both freshwater  and marine crustaceans  [The effect-concentration data obtained with pinipes) ,15,21. C. riparius and C. dilutus) evidenced that factors such as nAChR density, receptor binding affinity and compound-specific binding affinity may be responsible for species-specific responses amongst different species and life-stages [A. tonsa compared with ACE and CLO could be defined by an IMI lower binding affinity to the nAChR of the naupliar stage compared with the other NEOs, similarly to that observed for chironomids [The binding properties at the nicotinic cholinergic receptors (nAChR) may explain the different species-specific sensitivities towards NEOs. Recent studies on chironomids . In particular, EC10s and EC20s calculated for A. tonsa are equal or below the CALBs for ACE, THI and TMX, as reported in 10 and EC20 are higher than the USEPA CALBs and the standards proposed for long-term exposures by European Commission (8.3 ng L−1) [The effect-concentrations calculated for  ng L−1) . These d10s we obtained for A. tonsa larval development for CLO, IMI, THI and TMX are higher than the annual average environmental quality standards (AA-EQS) recently proposed for saltwater  for saltwater [On the other hand, the ECaltwater . At the altwater . Based oaltwater  seem morA. tonsa and the benchmark proposed for preserving aquatic invertebrates. For example, in Jiaozhou Bay, China, only IMI and ACE were detected, but their concentration was <1 ng L−1 in all of the sampling stations [−1 [10s for both pesticides and EC20 for ACE  [−1) approached the EC10 calculated for A. tonsa only during intense application in agricultural land, from June to September, while in other seasons, pesticide concentrations were lower [Moreover, the available literature data attest that NEOs in brackish and coastal waters generally occur at concentrations at least one order of magnitude lower than the effect-concentrations calculated for LDR in stations ; similarions [−1 . Monitorand IMI) . Conversre lower .A. tonsa were observed at concentrations lower than established chronic aquatic life benchmarks reported by USEPA for freshwater invertebrates. However, effect concentrations calculated for A. tonsa larval development are higher than the recently proposed EQS for saltwater, and environmental concentrations in estuarine and coastal areas seldom exceed these benchmarks and effect-concentrations calculated for A. tonsa. Based on the actual contamination levels, larval development of copepods might be impaired only in estuaries receiving wastewater treatment plant’s discharges or intense runoff from agricultural land during the season of pesticide’s application.The calculated effect-concentration data indicated that NEOs are potent inhibitors of larval development. Significant effects due to ACE, THI and TMX on