Apis mellifera) death rates globally. Clothianidin neonicotinoid appears to impair the microbe–immunity axis. We conducted cage experiments on newly emerged bees that were 4–6 days old and used a 16S rRNA metataxonomic approach to measure the impact of three sublethal clothianidin concentrations  on survival, sucrose syrup consumption and gut microbiota community structure. Exposure to clothianidin significantly increased mortality in the three concentrations compared to controls. Interestingly, the lowest clothianidin concentration was associated with the highest mortality, and the medium concentration with the highest food intake. Exposure to clothianidin induced significant variation in the taxonomic distribution of gut microbiota activity. Co-abundance network analysis revealed local dysbiosis signatures specific to each gut section  were driven by specific taxa. Our findings confirm that exposure to clothianidin triggers a reshuffling of beneficial strains and/or potentially pathogenic taxa within the gut, suggesting a honeybee’s symbiotic defense systems’ disruption, such as resistance to microbial colonization. This study highlights the role of weak transcriptional activity taxa in maintaining a stable honeybee gut microbiota. Finally, the early detection of gut dysbiosis in honeybees is a promising biomarker in hive management for assessing the impact exposure to sublethal xenobiotics.Pesticides are increasing honeybee ( NF-κB pathway  , which were tailed on the 5′ end with part of the Illumina TruSeq adapters. The first PCR was conducted in a total volume of 50 µL: reaction buffer (Q5) (5×) 10 µL; dNTPs (10 mM) 1 µL; 347-F 2.5 µL; 803-R 2.5µL; high GC enhancer (Q5) (5×) (NEB) 10 µL; Q5 high-fidelity DNA polymerase (NEB) 1 µL; H20 20 µL; and DNA template 3µL. After initial denaturation at 98 °C for 2 min, amplification was performed using 35 cycles of 10 s at 98 °C, 30 s at 60 °C and 30 s at 72 °C followed by a final extension at 72 °C for 2 min. PCR reactions were purified using the sparQ PureMag Beads PCR cleanup kit . Amplification products were run on 2.0% agarose gels to detect fragments of expected size (~450 bp), then quantified using a Nanodrop 1000 spectrophotometer .Then, a second PCR was performed to attach the remaining adapter sequences . The second PCR was conducted in a total volume of 50 µL: reaction buffer (Q5) (5×) 10 µL; dNTPs (10 mM) 1 µL; generic forward primer (5′-AATGATACGGCGACCACCGAGATCTACAC-[index1]-ACACTCTTTCCCTACACGAC-3′) (Sigma-Aldrich) (10 µM) 1 µL; reverse primer (5′-CAAGCAGAAGACGGCATACGAGAT-[index2]-GTGACTGGAGTTCAGACGTGT-3′) (Sigma-Aldrich) (10 µM) 1 µL; high GC enhancer (Q5) (5×) (NEB) 10 µL; Q5 high-fidelity DNA polymerase (NEB) 1 µL; H20 24 µL; and DNA template (10–15 ng/µL) 3µL. After initial denaturation at 98 °C for 2 min, amplification was performed using 12 cycles of 10 s at 98 °C, 30 s at 60 °C and 30 s at 72 °C followed by a final extension at 72 °C for 2 min. The quality of purified PCR products was verified on a DNA 7500 BioAnalyzer chip (Agilent) in order to detect an insert fragment size of 550 bp, then quantified using a Nanodrop 1000 dpectrophotometer.Barcoded amplicons were pooled in equimolar concentrations and sequenced at the “Plate-forme d’Analyses Génomiques” of Laval University  using Illumina MiSeq paired end technology (2 × 300 bases). We used 15–20% of the PhiX control v3 Library  in the sequencing runs as a calibration control.Please note that primers used in this work contain Illumina-specific sequences protected by intellectual property . https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on 28 March 2018)). Prior to analysis, we obtained a total of 6,283,142 sequences from which we kept a total of 3,141,571 reads after filtration. Reads were processed through the dada2 pipeline (version 1.12) [In total, 120 samples (2 replicates of 5 bees × 3 gut sections × 5 cages per group × 4 treatments) were sequenced individually. Raw sequences from all 120 samples were checked for accuracy using FastQC (on 1.12) ; the quaTaxonomic classification was carried out using blast matches from the NCBI 16S Microbial database . As the p < 0.05) between the unexposed group (control) and the group exposed  to clothianidin in each honeybee gut section. The assumption of normality was measured using a Shapiro–Wilk test . BecauseHmisc R (version 4.2-0) [p-values corrected with the false discovery rate (FDR) method [Hmisc package) was used to compute a matrix of Spearman’s rho rank correlations coefficients. It provides both the correlation coefficients and the p-value of the correlation for all possible pairs of columns in the ASV table. Afterwards, a false discovery rate test “Bonferroni” was applied to confirm the significance of correlation pairs. Pairs of ASVs with R Spearman coefficients ≥ 0.4 and ≤−0.4 and Corr p-value < 0.05 and Bonferroni p-value < 0.05 were considered as significant. The Spearman coefficient method is solid and comparable to the new methods of mutual information in inferring correlation [i) edge with significant positive or negative correlations according to Spearman’s correlation coefficient such that −1 ≤ r ≤ −0.4  and 0.4 ≤ r ≤ 1 ; p-value < 0.05 with FDR correction; and (ii), we only accounted for bee gut taxa that occurred in most replicated samples (n > 7 on 10 samples per condition). We defined low activity taxa as genera with a very low activity (<0.01%) of the total sample activity that occurred in very few samples (n < 3 on 10 per condition). Co-abundance networks were built using Rstudio (Version 1.3.1093) to identify significant taxon–taxon correlations in each gut section per experimental condition. Data were divided into subsets by condition (gut section + concentration). Correlation matrices were constructed using the n 4.2-0)  package ) method . The funrelation . Twelve relation . Each noTo further assess significant gut bacterial disturbances induced by clothianidin, we measured the distribution of the following network topological parameters: Degree (DG), Neighborhood Connectivity (NC) and Closeness Centrality (CC) obtained with the Network Analyser function built into Cytoscape. DG is a local quantification representing the communication activity inside a network. The more the DG of a node increases, the more a node is connected locally inside the network ,81. NC iOur results indicated a significant difference between the survival rate of the group exposed to 0.1 ppb clothianidin and the control group at the beginning of the experiment, with a trend that was shown to decrease over time . The othp < 0.001), suggesting a treatment effect on feeding rates (p < 0.05) (p < 0.05)  . For bee < 0.05) . However < 0.05) . Mean clothianidin concentration accumulated (per honeybee) varied differently across all experimental groups . At T 7,Bartonella, Bifidobacterium, Bombella, Frishella, Gilliamella, Lactobacillus, Parasaccharibacter, Pediococcus, Snodgrassella and unassigned ASVs) in all treatment groups versus the unexposed group (control) at the genus  had the greatest impact on all three honeybee gut sections . Frischella perrara ASVs, two Lactobacillus spp. and an unassigned ASV , with a significant increase and/or decrease in activity, were the two gned ASV . The mosactivity . Gilliamella apicola ASVs, two Frischella perrara ASVs and an unassigned ASV , with a significant increase and/or decrease in activity were two gned ASV . The mosgned ASV . Regardigned ASV . All the results presented below are summarized in Lactobacillus, Snodgrassella and Gilliamella activity was variable across concentrations. For all clothianidin treatment concentrations, Bifidobacterium (core members) activity increased, Frischella (core member) activity decreased and Flavobacterium (low activity taxa) decreased. Then, there was a gain in significant correlations with the following low activity ASV Ralstonia (0.1 and 10 ppb). Finally, there was a loss of correlations with Devosia (0.1 and 1 ppb) and Leifsonia .Midgut: At the genus taxonomic rank, the number of interacting ASVs varied from 45 (control midgut) to 35, 36 and 33, respectively, after 0.1, 1 and 10 ppb of exposure to clothianidin . Signifip = 0.045; 1 ppb: p ≤ 0.001; 10 ppb: p = 0.002) (p = 0.03 at 0.1 ppb) and significantly lower values for NC  relative to the control midgut network   and a significantly higher NC  relative to 1 ppb  relative to the control midgut network (0.1 ppb: = 0.002) . Regardi network . Our res0.1 ppb) . Additioto 1 ppb . Lactobacillus and Gilliamella activity was variable across all treatment concentrations. For all concentrations, Bifidobacterium (core members) activity increased and Snodgrassella and Frischella (core members) activity decreased. We observed a gain in significant correlations with low activity ASVs: Moraxella and Prevotella (1 ppb); Lawsonella (0.1 and 1 ppb) and Ralstonia . At 0.1 ppb, we observed a slight increase in Pseudomonas and Flavobacterium taxa activity.Ileum: At the genus taxonomic rank, the number of interacting ASVs varied from 35 (control ileum) to 42, 40 and 30, respectively, after 0.1, 1 and 10 ppb of exposure to clothianidin . Signifip = 0.018) at 1 ppb and significantly less connected (p = 0.002) at 10 ppb compared to the control ileum network, respectively (p = 0.023) and lower NC (p ≤ 0.001) at 1 ppb relative to the control ileum network, respectively (p = 0.001) and higher NC (p ≤ 0.001) at 10 ppb, respectively, relative to the control ileum network (p = 0.001) at 1 ppb relative to 0.1 ppb (p ≤ 0.001) at 10 ppb relative to 1 ppb (p ≤ 0.001) at 1 ppb relative to 0.1 ppb (p ≤ 0.001) at 10 ppb relative to 0.1 ppb (p ≤ 0.001) at 10 ppb relative to 1 ppb   activity was variable across treatment concentrations. For all concentrations, Bifidobacterium (core member) activity increased, while Frischella (core member) activity decreased.Rectum: At the genus taxonomic rank, the number of interacting ASVs varied from 34 (control rectum) to 25, 23 and 22, respectively, after 0.1, 1 and 10 ppb of exposure to clothianidin . We obsep ≤ 0.001, p = 0.004 and p = 0.001, respectively, at 0.1, 1 and 10 ppb)  and a significantly lower NC  relative to the control rectum network   compared to the control rectum network ( 10 ppb) . Then, t network . Our res 10 ppb) .First, exposure to a gradient of three doses of clothianidin left different signatures of microbiota dysbiosis in the three gut sections of honeybees. Changes in correlations in each gut section reveal a rise in beneficial ASVs with probiotic properties, which offsets the activity increase of spikes of potential opportunistic strains. Such dysbiosis patterns were expected, given as gut sections are colonized by specific microbial communities . In the Lactobacillus Firm-5 and L. Firm-4 clades [Lactobacillus Firm-5 and L. Firm-4 clades [Lactobacillus activities at 0.1  did not affect Bifidobacterium sp. abundance, but induced a significant increase in bee mortality [Drosophila that the pesticide chlorpyrifos was more toxic than its metabolite (chlorpyrifos oxon). In our case, the final toxicity of metabolites based on the initial clothianidin concentration could differentially impact honeybee physiology, and in turn, survival.Second, the clothianidin–microbiota interaction induced a toxicity tolerance scenario. The microbial strain that first metabolizes the molecule into an intermediate molecule, or a derivate influences the syntrophic exchange network. For instance, honeybee gut microbiota exposed to two fipronil concentrations did not respond similarly: the lowest concentration (0.25 μg/kg) affected ortality . Daisleyortality  showed iFlavobacterium and Pseudomonas sp. [Leifsonia sp. [Flavobacterium and Pseudomonas increased in the ileum of 0.1 ppb group  and non-core (Parasaccharibacter at 10 ppb) gut microbes in the ileum   B–D with  10 ppb)  where th 10 ppb) .Collectively, all these changes in the microbiota structure support a dysbiosis signature in the ileum gut microbiota in our study and may suggest a clothianidin’s possible relation to the honeybee microbiota–immunity axis. In , the autTo summarize, clothianidin differentially impacted the activity of certain ASVs at specific concentrations, disturbed ASV–ASV interactions in the honeybee gut microbiota and properties of clothianidin degradation are determined by specific ASV. Likewise, different members may have handled the molecule first, determining excreted metabolites and thus toxicity, which may not be proportional to the initial pesticide concentration.Leifsonia (0.1 and 1 ppb) and Devosia  relative to the control midgut  treatment group. Loss of positive correlations with increasing neonicotinoid concentrations along intestinal sections indicates dysbiosis . Bifidobacterium, Pediococcus and Commensalibacter are known either as core microbiota members or as probiotics [Lawsonella, a low activity taxa, thus supporting its role as a keystone species inside the ileum  [Pulmonaria officinalis floral nectar [Apis mellifera, where they exhibited an antimicrobial resistance to the bacterial pathogen Paenibacillus larvae [As stated above, the most extensive adverse impact, in terms of correlational network structure, was recorded at 0.1 ppb . For insobiotics ,109,110.he ileum . In thisn insect , suggestlla spp. , which se (0.5%) , the herl nectar  and, fins larvae . The othBifidobacterium, Frischella, Gilliamella, Lactobacillus, Parasaccharibacter and Snodgrassella. Each of these bee symbionts are known to be involved in either host immunity or maintenance of a homeostatic microbiota [Gilliamella apicola [Lactobacillus [Snodgrassella alvi and G. apicola ensures homeostatic microbiota in the intestinal ecosystem [Frischella perrara [Parasaccharibacter spp. [Fifth, our study highlights a local honeybee gut microbiota reaction to exposure to clothianidin. Within all three gut sections, we observed a gain in significant correlations (positive and/or negative) for the genera crobiota ,113,114. apicola  and Lactbacillus  are respbacillus . Moreove perrara  and Parater spp.  were docter spp. . TherefoLactobacillus Firm-5 and L. Firm-4 clades were restricted, evidenced by the significant decrease in the respective activities  B was mortivities  and losstivities . In contG and CC , establiathogens ,118,119 athogens . This fuBifidobacterium and Pediococcus (diminishing CC)  following exposure to clothianidin. This competition shift suggests a dysbiosis rewiring pattern [Complementarily, loss of connectivity (diminishing DG) for hing CC)  supportsactivity  at 0.1 p pattern .Giamella apicola abundance. Different experimental approaches may induce microbial composition variability [Giamella apicola, Snodgrassella alvi and Frischella perrara. We observed a decrease and/or an increase in Frischella perrara activity, while exposure to other pesticides was variable, as it either induced (nitenpyram) [F. perrara abundance [F. perrara is known to play a key role in honeybee immunity by limiting microbial resistance [F. perrara disbalance may affect honeybee gut immunity, leading to microbiota dysbiosis. Finally, the increasing activity of Bifidobacterium activity confirms results of previous studies that tested honeybee exposure to nitenpyram and thiacloprid [Previous studies highlighted fluctuations of honeybee microbiota facing stress. Applications of coumaphos, tau-fluvalinate  and tetriability , as obseenpyram)  or failebundance . F. perrsistance ; therefoacloprid ,20,21.Snodgrassella alvi is involved for the upregulation of the gene expression related to antimicrobial peptides [S. alvi, may create an overall imbalance in gut microbiota. A decrease in S. alvi activity in the ileum following exposure to pesticide is consistent with findings of two previous studies [S. alvi activity played a role in adversely impacting the immune response of Apis mellifera when facing an increase in potential opportunistic strains such as Roseburia and Pelomonas. Our results provide additional evidence on the level of bacterial activity, showing that honeybees exposed to neonicotinoids are more sensitive to microbial gut pathogens [Additionally, peptides . Destabi studies ,126. In athogens ,127,128.Ralstonia genus had a greater impact on the ileum  networks  and non-core members within each gut section. Given that these core symbionts are either involved in host immunity and/or microbiota equilibrium [More specifically, the increase of potential opportunistic ASVs activity was variable along the gut section and gradient of exposure. networks B–D. For  network , suggestilibrium ,113,114,ilibrium ,22,23. Sixth, this study highlights how environmental factors adversely shape individual honeybee health. The link established here between low exposure to clothianidin, high mortality and overall honeybee health differs somewhat from previous studies. Alkassab and colleagues  reportedFinally, this study showed how honeybees are significantly more attracted by syrup supplied with the medium sublethal clothianidin concentration. In a previous study, honeybees were found to be more attracted to contaminated than non-contaminated syrup . A dose-This work highlights the interplay between gut microbiota activity, food intake and exposure to pesticides. Our work provides unprecedented insights regarding the impact of clothianidin gradient on the activity interactions between core members  and non-core members, including potential opportunistic strains and the potential link of clothianidin pesticide with the microbiota–immunity axis. Overall, our results suggest that extent of gut microbiota dysbiosis depends on both xenobiotic exposure level and gut section. Finally, activity interaction networks appear to be a valuable tool to measure the impact of exposure to pesticide on microbiome community structure.


We provide dosimetric data for the following procedures: coiling, flow diverter (FD), Woven EndoBridge (WEB), combined techniques.Modern endovascular treatment of unruptured intracranial aneurysms (UIAs) demands for observance of diagnostic reference levels (DRLs). The national DRL  and fluoroscopy time were investigated for the following variables: endovascular technique, aneurysm location, DSA protocol, aneurysm size, and patient age.2) were 119 ± 73 (89–149) and 94  for coiling, 128 ± 53 (106–151) and 134  for FD, 128 ± 56 (102–153) and 118  for WEB, and 165 ± 102 (110–219) and 131  for combined techniques (p > .05). Regarding the aneurysm location, neither DAP nor fluoroscopy time was significantly different (p > .05). The lowest and highest fluoroscopy times were recorded for WEB and combined techniques, respectively . A low-dose protocol yielded a 43% reduction of DAP (p < .001). Significantly positive correlations were found between DAP and both aneurysm size  and patient age .Eighty-seven patients  were included. Total mean and median DAP . As tneurysms ; alternaneurysms –7 or intneurysms –10, thatAs a consequence, published data on radiation dose often only take into account coil embolisation –15. FurtRegarding endovascular treatment, a risk-benefit assessment is particularly essential in patients with an incidental, unruptured intracranial aneurysm (UIA). Recommendations on elective endovascular treatment of UIAs are (i) a high technical success and low peri-procedural complication rate, (ii) a reasonable radiation dose particularly in young patients according to the ALARA (as low as reasonably achievable) principle, and (iii) a limited intervention duration, as a prolonged fluoroscopy time is associated with an increased peri-procedural complication rate .The aim of this retrospective single-centre study is the evaluation of radiation dose and fluoroscopy time in patients with an incidental saccular UIA, who underwent an elective aneurysm treatment using the following endovascular procedures: Coiling, FD, WEB, combined techniques. The provided data may be useful for the establishment of novel DRLs in the field of modern endovascular treatment of intracranial aneurysms.This retrospective single-centre study was approved by the responsible Institutional Review Board (project number 19-813) of the Ludwig-Maximilians-University Munich, Germany. The study was performed in accordance with the Declaration of Helsinki.Age ≥ 18 yearsLocation: anterior communicating artery (ACOM); intradural segments of the internal carotid artery (ICA), including posterior communicating artery and carotid T; tip of the basilar artery (BA)Endovascular treatment: coiling, FD, WEB, combined techniquesInclusion criteria:Ruptured and/or symptomatic aneurysmsNon-saccular  aneurysmsMultiple aneurysmsOther intracranial aneurysm locationsAdditional diagnostic pan- (four-vessel) angiography during the same interventionNo 3D angiographyExclusively monoplanar digital subtraction angiography (DSA) acquisitions due to difficult working projectionExclusion criteria:We analysed patients with a saccular UIA who were endovascularly treated between January 2015 and May 2019. To increase the dosimetric homogeneity of the data pool and to reduce treatment bias, the following inclusion and exclusion criteria were defined : 2 or 4 f/s , 1 f/s (venous phase), kV 73, pulse width 50 ms, dose 1820 μGy/pNormal dose (ND): 2 or 4 f/s , 1 f/s (venous phase), kV 73, pulse width 100 ms, dose 3000 μGy/pEndovascular procedures were performed by five consultant neuroradiologists with six to more than 20 years of experience in interventional neuroradiology. The applied angiographic system was a biplane angiographic unit . A transfemoral approach was used in each patient. Regarding the vessel visualisation, a non-ionic iodinated contrast agent was applied . The angiographic workflow routinely comprised initial and final DSA acquisitions including arterial and venous phases on standard anteroposterior and lateral projections with a preferred field of view (FOV) of 32 cm, a 3D DSA with a FOV of 48 cm (or minimum of 42 cm) preset by the manufacturer, peri-procedural DSA acquisitions in arterial phase on working projections using a targeted FOV of 11 cm or 16 cm, and a pulsed fluoroscopy with a frame rate of 7.5 f/s. Regarding the DSA acquisition type, two protocols were preset by the manufacturer:In each patient, all imaging data and dose reports retrieved from a dedicated picture archiving and communication system  were reviewed by two experienced neuroradiologists with 9 (R.F.) and 10 (C.G.T.) years of experience in diagnostic and interventional neuroradiology. In detail, the following parameters were documented: aneurysm size, aneurysm location, endovascular technique, DSA acquisition count, DSA protocol, fluoroscopy time and DAP (representing a surrogate measure of energy delivered to patients ), and DSFurthermore, the impact of different DSA protocols on DAP was investigated. In detail, (1) the total DAP was compared between the LD, ND, and mixed-dose  groups, and (2) the individual dose index was calculated for each patient in the three groups, by using the following formula:t test if data were normally distributed. The Mann-Whitney U test was used if data were not normally distributed. When statistically significant differences occurred, single posttest comparisons were performed by using the Mann-Whitney U test and t test with Bonferroni’s correction for multiple comparisons. To note, though DAP values were normally distributed, we also calculated and compared the respective median DAP for different endovascular techniques and aneurysm locations, enabling adjustment to the local DRL defined by the 75% percentile [α = 0.05 was used throughout the study.Continuous data are provided as mean ± standard deviation  and/or median , and categorical data as counts and percent. Regarding the two outcome parameters DAP and fluoroscopy time, data were initially assessed for normality applying the Kolmogorov-Smirnov test considering the endovascular technique, aneurysm location, and DSA protocol. Variables were then compared according to the rcentile . The PeaPatient characteristics are summarised in Table n = 3) and a maximum diameter of 30 mm (n = 1). To note, the three patients with the smallest UIA diameter were treated, as they had a history of subarachnoid haemorrhage due to rupture of another intracranial aneurysm, consequently harbouring a statistically increased risk of re-bleeding. Thirty out of 87 (34.5%) aneurysms were located at the ACOM, 40/87 (46%) at the intradural ICA, and 17/87 (19.5%) at the BA tip. Regarding the endovascular technique, 26/87 (29.9%) patients were treated by coiling , 24/87 (27.6%) by FD , 21/87 (24.1%) by WEB (1 device in 21/21 patients), and 16/87 (18.4%) by a combined technique . In detail, 15/30 (50%) ACOM aneurysms were treated by coiling, 1/30 (3.3%) by FD, 11/30 (36/7%) by WEB, and 3/30 (10%) by a combined technique . Intradural ICA aneurysms were treated by coiling in 8/40 (20%), using a FD in 23/40 (57.5%), and a combined technique in 9/40 patients . BA tip aneurysms were treated by coiling in 3/17 (17.7%), using a WEB in 10/17 (58.8%), and a combined technique in 4/17 patients .The median aneurysm size was 6.7 mm, with a minimum diameter of 2 mm (Results of radiation dose and fluoroscopy time are illustrated in Table 2) of 87 patients were 130 ± 65 (116–144) and 116 , respectively. In detail, total mean and median DAP (Gy cm2) were 119 ± 73 (89–149) and 94  for coiling, 128 ± 53 (106–151) and 134  for FD, 128 ± 56 (102–153) and 118  for WEB, and 165 ± 102 (110–219) and 131  for combined techniques. We calculated the lowest mean and median DAP for the coiling group, and the highest mean DAP for the combined-technique group; however, pairwise comparison of total mean and median DAP between groups did not reach statistical significance  were 134 ± 68 (109–159) and 116  for ACOM aneurysms, 130 ± 69 (108–152) and 120  for intradural ICA aneurysms, and 133 ± 87 (89–178) and 110  for BA tip aneurysms . In contrast, pairwise comparison of median fluoroscopy time between the coiling, FD, and WEB groups was not significantly different  for coiling, 34 min  for FD, 26 min  for WEB, and 94 min  for combined techniques. Median fluoroscopy time of the latter group was significantly higher when compared with the three other groups  was 102 ± 45 (83–121) for the LD group, 144 ± 78 (118–170) for the ND group, and 144 ± 77 (113–176) for the MD group. The mean dose index  was 4.49 ± 1.76 (3.77–5.22) for the LD, 7.89 ± 2.97 (6.90–8.88) for the ND, and 6.78 ± 3.06 (5.52–8.04) for the MD groups. Values were significantly lower in the LD group when compared with those in the ND and MD groups , whereas difference of values between the ND and MD groups did not reach statistical significance (p = .159). According to the mean dose index, a LD protocol yielded a 43% reduction of DAP per DSA acquisition when compared with a ND protocol. A LD protocol was most commonly applied in patients undergoing coil embolisation , whereas a ND protocol was preferentially chosen in FD  and WEB cases .A LD protocol was applied in 25/87 (28.7%), a ND protocol in 37/87 (42.5%), and a MD protocol in 25/87 patients (28.7%). Mean DSA acquisition count (biplane) did not significantly differ between groups (LD 27 ± 13 (19–35), ND 28 ± 17 (19–37), MD 25 ± 13 (18–32); n = 87), we found a significantly positive correlation between the aneurysm size and both total DAP  and fluoroscopy time . Moreover, a significantly positive correlation was found between patient age and total DAP , whereas correlation between patient age and fluoroscopy time did not reach statistical significance .Considering the entire study population (2) [2 for a cerebral four-vessel angiogram. According to the Federal Office of Radiation Protection [In the present study, we provide detailed data of radiation dose and fluoroscopy time for modern endovascular treatment techniques in patients with saccular UIAs. With regard to the Euratom Basic Safety Standards (BSS) directive , we beli2) . Moreove2) –14, 19 a2 and 116  Gy cm2, respectively. In detail, the calculated 75th percentile was 130 Gy cm2 for coiling, 176 Gy cm2 for each FD and WEB, and 209 Gy cm2 for combined techniques. The measured difference in radiation dose between the treatment groups clearly reflects the grade of aneurysm complexity, with a comparably lower DAP in simple coiling and a higher DAP in aneurysms treated by combined techniques; however, this difference did not reach statistical significance. To note, as the data pool was homogenised in order to reduce inter-individual dosimetric variations as described above, we indeed noted normally distributed DAP values, additionally enabling reliable report of the statistical mean considering the different endovascular techniques, aneurysm locations, and applied DSA protocols.According to the ICRP 135 publication , applica2 [2 [With regard to the literature, the median DAP for coiling was within the range of previously published data by other authors, e.g. Hassan et al 78.7 59.5; 111.9) Gy cm1.9 Gy cm2 [2 . Further2 [2 , 23, 24.2 [2 , 20, 22,r = .320, p = .003) and patient age .Considering the aneurysm location, neither the DAP nor fluoroscopy time was significantly different when comparing the three most common anatomic sites treated in our institution . We therefore assume that neither radiation dose nor fluoroscopy time is necessarily dependent on the aneurysm location as reported by Acton and colleagues , but ratp < .001).We observed a median fluoroscopy time of 49 min for coiling, 34 min for FD stenting, and 26 min for implantation of a WEB. These values are clearly in the range of published data on endovascular aneurysm embolisation –13. In c2, p = .018). The impact of a LD protocol on radiation dose reduction was more objectively illustrated by calculating the dose index, which reflects the DAP per single DSA acquisition . A LD protocol was most commonly applied in aneurysms treated by coiling. Contrarily, a ND protocol was preferentially chosen in FD and WEB cases. This distribution in turn explains the slightly increased radiation dose in the FD and WEB groups when compared with the coiling group as illustrated above. However, the provided DRLs are still clearly below the official local DRL for coil embolisation [The standard initial and final DSA runs of the relevant vascular territory (FOV 32 cm) should preferentially be conducted in LD mode, as this protocol is both appropriate for aneurysm visualisation and robust enough to detect/rule out catheter-associated complications such as thromboembolism, vasospasm, and arterial dissection.Regarding peri-procedural targeted DSA runs in working projections (FOV 11–16 cm), application of a LD protocol is particularly useful in endovascular aneurysm treatment using materials with a high X-ray opacity (e.g. platinum coils). Contrarily, a ND protocol is reasonable when applying materials with a comparably lower fluoroscopic visibility (e.g. nitinol FD or WEB), allowing for a more detailed visualisation of the implanted device with respect to the aneurysmal sac and parent vessel.With regard to the DSA acquisition mode, application of a LD protocol yielded a significantly lower DAP when compared with a ND protocol . Second, the following parameters were not documented: peri-procedural change of strategy, size of the aneurysm neck, type of aortic arch. Third, several aneurysm locations were excluded due to procedural in-house management in our neurovascular centre  and/or rare occurrence ; thus, dosimetric data observed in the present study cannot be generalised for all endovascular procedures. However, we believe that our selected study population may serve as a representative cohort of patients harbouring saccular UIAs at common anatomic sites accessible for endovascular treatment, as comparable data—particularly with regard to FD and WEB—are missing.In conclusion, the present study introduces novel DRLs in the field of modern endovascular treatment of UIAs, including FD and WEB. Radiation dose was not significantly different between the endovascular procedures. However, radiation dose was comparably low in simple coiling and higher when using combined techniques, which are particularly applied in patients characterised by complex aneurysms. Aneurysm location did significantly alter neither radiation dose nor fluoroscopy time, whereas both aneurysm size and patient age were associated with radiation dose. Fluoroscopy time was the lowest for WEB and highest for combined techniques. A low-dose DSA protocol yielded a significant reduction of radiation dose and is particularly useful when applying high-opacity materials (e.g. platinum coils). With regard to the next Euratom version, we recommend a prospective collection of dosimetric data derived from multiple centres for definition of DRLs, considering different manufacturers and dose reduction techniques.


Higher growth was obtained on liquid 1/2 MS medium supplemented with 60 g/L sucrose for HRs grown over 20 days. In this medium, 200 µM H2O2 applied on day 12 induced anthocyanin accumulation by 20%. The activity of superoxide dismutase (SOD)—which generates H2O2 from O2•−—increased by over 50%, whereas the activity of H2O2-scavenging enzymes was not enhanced. Elicitation in the HRs can result in a controlled oxidative burst, in which SOD activity increased H2O2 levels, whereas anthocyanins, as effective reactive oxygen species scavengers, could be induced to modulate the oxidative burst generated. Moreover, given the proven stability of the HR lines used and their remarkable productivity, this system appears as suitable for elucidating the interplay between antioxidant and secondary metabolism.Hairy roots (HRs) grown in vitro are a powerful platform for plant biotechnological advances and for the bio-based production of metabolites of interest. In this work, black carrot HRs able to accumulate anthocyanin as major secondary metabolite were used. Biomass and anthocyanin accumulation were improved by modulating growth medium composition—different Murashige & Skoog (MS)-based media—and H Rhizobium rhizogenes (formerly Agrobacterium rhizogenes) causes the proliferation of “hairy roots” (HRs) on a range of dicotyledonous plants  containing 7.5 g/L agar, on 9-cm diameter Petri dishes, as reported by  and incubated at 16 h light .First, an H−2 s−1)/8 h darkness (25 °C constant) on an oscillatory shaker  at 85 rpm.All flasks were incubated at 16 h light  was collected, washed, gently dried, and weighed. Then, HRs were ground in the presence of liquid nitrogen using a mortar. The resulting powder was stored at −80 °C for further analysis.w/w). The generated homogenate was vigorously mixed with distilled water  and incubated at room temperature for 1 h. Then, the mix was centrifuged for 20 min at 4000 g. The supernatant (extract) was used to measure monomeric anthocyanin content according to the pH differential method with minor modifications [v/v), and the absorption of the mix was registered between 350 nm and 700 nm using a UV/Vis V-630 Bio spectrophotometer . Based on a cyanidin-3-glucoside standard, data are expressed as concentration (µg anthocyanin g−1 FW) and yield ).A 2-g aliquot of the generated powder was blended in a 3% sulfuric acid solution  into 50 mM Tris–acetate buffer containing 0.1 mM EDTA, 2 mM cysteine, and 0.2% (v/v) Triton X-100 (pH 6.0). The resulting extract was centrifuged for 15 min at 10,000× g . Finally, the supernatant was passed through Sephadex G-25 NAP columns , and the filtrate used for enzymatic determinations using a UV/Vis V-630 Bio spectrophotometer (Jasco). CAT, POX, SOD, APX, MDHAR, GR and DHAR activities were assayed according to [2O2 decomposition to water and oxygen. POX activity was measured by following the oxidation of 4-metoxy-naphtol at 595 nm. SOD activity was based on the enzymatic system xanthine–xanthine oxidase, measuring the reduction of cytochrome C at 550 nm. APX activity was calculated following the oxidation of ascorbate at 290 nm. MDHAR activity was based on the reduction of ascorbate coupled to the oxidation of NADH followed at 340 nm. GR activity was measured by monitoring the oxidation of NADPH, reflected as a decrease in absorbance at 340 nm. Finally, DHAR activity was assayed by measuring the increase in absorbance at 265 nm due to the reduced ascorbate formation.Enzyme extraction was done as described ,57,58. Brding to ,60: CAT p ≤ 0.05), whereas data from the accumulation curves were analysed by Welch t-test. The R Program for Statistical Computing  was used.Analyses were performed on five to six biological replicates. All experiments were conducted independently twice. Data were expressed as the mean ± SE. Normality and homoscedasticity of variances for all data were checked by a Shapiro and Bartlett tests, respectively. Data from single time point-experiments were compared using a one-way analysis of variance (ANOVA) followed by a Tukey HSD post hoc test (


Thirty non-specifically resistance-trained university standard male rugby union players (age (years): 20 ± 2; height (cm): 181 ± 7; body mass (kg): 92 ± 17) completed the study), who were moderate habitual caffeine consumers (118 ± 110 mg), completed the study. Using a within-subject double-blind, placebo-controlled experimental design, the acute effects of caffeine intake on upper and lower limb maximal voluntary concentric and eccentric torque were measured using isokinetic dynamometry (IKD) prior to and immediately following a resistance training intervention. Participants were split into strength-matched groups and completed a resistance-training program for seven weeks, consuming either caffeine or a placebo before each session. Irrespective of group, acute caffeine consumption improved peak eccentric torque of the elbow extensors (p < 0.013), peak concentric torque of the elbow flexors (p < 0.005), total eccentric work of the elbow flexors (p < 0.003), total concentric work of the knee extensors (p < 0.001), and total concentric and eccentric work of the knee flexors (p < 0.046) following repeated maximal voluntary contractions. Many of these acute caffeine effects were still prevalent following chronic exposure to caffeine throughout the intervention. The training intervention resulted in significant improvements in upper and lower body one-repetition maximum strength (p < 0.001). For the most part, the effect of the training intervention was equivalent in both the caffeine and placebo groups, despite a small but significant increase (p < 0.037) in the total work performed in the participants that consumed caffeine across the course of the intervention. These results infer that caffeine may be beneficial to evoke acute improvements in muscular strength, with acute effects prevalent following chronic exposure to the experimental dose. However, individuals that consumed caffeine during the intervention did not elicit superior post-intervention training- induced adaptations in muscular strength.The present study uniquely examined the effect of 3 mg·kg A wealth of scientific evidence, summarised in a recent umbrella review , documen−1 of caffeine in low habitual caffeine users (<75 mg/day−1) results in intolerance after 4 weeks [Several meta-analyses have reported an ergogenic effect of acute caffeine ingestion for muscular strength ,3,5. Due 4 weeks . Further 4 weeks . Direct  4 weeks . However−1 of caffeine consumed before every exercise session resulted in superior improvements in the bench press and squat one repetition maximum (1RM) compared to the placebo group following 6 weeks of resistance training. Using the same dose and a similar population, recent work by Giráldez-Costas et al. [Only two studies examined if chronic caffeine ingestion can augment resistance training adaptations. In resistance-trained males, Kemp et al.  demonstrs et al.  indicateThe disparity in caffeine-induced improvements in maximal strength may be related to the difference in the training regimes and duration of the program. Furthermore, previous studies did not account for baseline strength when assigning participants to training groups, which may have influenced the results. Given the limited research in the area and the inconclusive findings, further investigation is required. Whilst these initial studies offer important insight, work is now needed to understand if chronic caffeine consumption during resistance training induces regional and contractile mode-specific changes given that caffeine’s effect on strength may differ between concentric and eccentric models of activity, contractile velocity, and specific muscle groups ,18,19,20−1 of caffeine consumption on the maximal voluntary concentric and eccentric strength of the knee and elbow flexors and extensors in a population of male rugby union players; (2) using a between-subjects experimental design to assess the effects of 3 mg·kg−1 of caffeine consumption on adaptations to a 7-week resistance training program in participants matched for baseline strength; (3) using a within-subjects experimental design to reassess the acute effect of 3 mg·kg−1 of caffeine consumption on the maximal voluntary concentric and eccentric strength of the knee and elbow flexors and extensors post completion of the training regime. As such, the present work provides the most detailed examination of the effects of chronic caffeine consumption on regional and contractile mode-specific adaptations to resistance training and uniquely considers the impact of caffeine habituation in inhibiting augmentation of the training response. It was hypothesised that (1) acute caffeine consumption would evoke improved muscular strength, which would manifest in a small but significant superior training adaptations; (2) that the group that consumed caffeine during the training intervention would demonstrate a reduced acute effect of caffeine on measures of muscular strength following the intervention.Given the outlined important gaps in the literature, the aims of the present study were threefold: (1) using a within-subjects experimental design the present work sought to assess the acute effect of 3 mg·kgn = 3) , illness (n = 2), or for reasons not stated (n = 5) leaving a total sample of 30 (mean ± standard deviation (SD); Age (years): 20 ± 2; height (cm): 181 ± 7; body mass (kg): 92 ± 17).Following ethics approval from Coventry University  and informed consent, 40 apparently healthy participants from the Coventry University Men’s rugby union team agreed to participate in the study. The current study was also in compliance with the declaration of Helsinki . ParticiFollowing baseline assessments of maximal strength and a familiarisation trial, the acute effects of caffeine on maximal strength were assessed, which was immediately followed by chronic caffeine consumption through a resistance training program . Finally−1; Myprotein, UK) or a placebo  issued in a transparent capsule . The ergogenic effect of caffeine supplementation at this dose is well established [Prior to the assessment of the acute effects of caffeine, participants were asked to abstain from caffeine at least 12 h before testing and intense physical activity at least 48 h prior. As outlined below, when prescribed participants consumed caffeine .The first visit was to familiarise participants with the experimental procedures to be used for the assessment of the acute caffeine effect. Initially, assessments were made barefoot in shorts and a t-shirt, and measures of height (cm) and body mass (kg) were taken using a SECA 213 portable stadiometer  and electronic weighing scales , respectively. Participants were then asked to complete a caffeine consumption questionnaire  to deter−1, followed by static and dynamic stretching of the elbow flexors . Then, the participants completed a lower-body warm-up that consisted of 5 min of cycling on an exercise ergometer (Monark 824E Ergomedic) with an unloaded cradle fixed at 70 revs·min−1 immediately followed by static and dynamic stretching, focusing on the knee extensors .Prior to completion of the physical assessments, participants were asked to complete a standardised warm-up of the upper and lower body. The upper body warm-up consisted of 5 min of arm crank ergometry  with an unloaded cradle fixed at 70 revs·minAfter completing the warm-up procedure, participants completed the strength assessment. Skeletal muscle contractile performance was assessed using isokinetic dynamometry in accordance with previously published protocols ,20,22. IMaximal voluntary isokinetic torque (Nm) of the elbow flexors and extensors for the dominant side was measured using an isokinetic dynamometer  set up following the manufacturer’s instructions. The rotational axis of the dynamometer head was aligned with the lateral epicondyle of the humerus on the dominant side, with an elbow rest positioned relative to this. A handgrip bar at the opposing end of the lever arm was adjusted relative to the length of the hand and forearm to allow the participant a comfortable grip. During concentric measures, participants were instructed to pull upwards on the bar as hard a possible through a fixed range of motion (80°–120° relative to anatomic zero). During eccentric measures, participants were asked to resist the movement of the lever arm moving through the same range of motion. Average and maximal concentric and eccentric forces were measured at fixed angular velocities of 60, 120, and 180 deg/s. Participants warmed up with 3 submaximal attempts at each speed to become familiarised with the movements and test speeds. During the assessment of maximal voluntary torque, participants performed 3 attempts separated by 60-s rest. The best attempt of the three was used for the analysis. Each speed was separated by a two-minute rest period. Following the measure of maximal concentric and eccentric force, the participants performed 3 sets of 10 maximal repetitions at 60 deg/s with individual repetitions being summed across the 30 repetitions. Each set was separated by 10 s. All torque values collected were corrected for gravity effects by estimation of limb weight prior to the assessment of maximal voluntary torque. Following each set, Rate of Perceived Exertion (RPE) was measured [Participants then completed an assessment of the maximal voluntary isokinetic torque (Nm) of the knee flexors and extensors. Each participant was strapped to the dynamometer chair in a seated position, and the lever arm axis of rotation was aligned with the lateral femoral epicondyle of the dominant limb. The distal end of the lever arm was fitted with a shin pad which was aligned with the lateral malleolus. A strap was placed across the midpoint of the upper limb of the participant’s dominant leg. Throughout the testing, participants were instructed to keep their arms fixed across the chest. The range of motion was fixed at 10°–80° relative to anatomic zero. The testing protocol was then carried out in the way that described for the assessment of maximal voluntary torque of the elbow flexors and extensors.−1 caffeine or a placebo 45 min prior to completion of the warm-up. Treatments were issued 45 min prior to the warm-up so that the experimental trial commenced 60 min post-ingestion. Previous work indicates that maximal blood plasma concentration of caffeine occurs 60 min post-consumption [The experimental protocol followed the procedure outlined above but was proceeded by a treatment ingestion period . Particisumption . Readinesumption  and Feltsumption  were mean = 15) or caffeine (n = 15) group. Groups were matched based on habitual caffeine use, using the data obtained from the caffeine consumption questionnaire, and for baseline strength, using the procedures outlined in the statistical method. Following the assessment of 1RM, all participants then completed the same training regime as indicated below , deadlifts (DL), chest press (CP), seated shoulder press (SSP), power clean (PC), and hang clean (HC). All exercises were completed using a 20 kg Eleiko barbell and in accordance with published protocols ,34,35. P−1 caffeine and those in the placebo group an equivalent dose of maltodextrin in the same manner as previously outlined. The intention of the resistance training intervention was to develop upper and lower body maximal strength and strength endurance. Following a standardised warm-up consisting of static and dynamic stretches focusing on the upper and lower body muscles participant completed a circuit training program consisting of 8 exercises. The exercises were CP, SSP, SQ, DL, PC, HC, sit up, and press-ups. The load for CP, SSP, SQ, DL, PC, HC was set at 70% of 1RM; sit-ups and press-ups were performed without any external load. Throughout the course of the intervention, all exercises were performed with “repetitions until failure” (RTF), meaning that the participants lifted 70% of 1RM until exhaustion. Previous resistance training interventions commonly used loads of 60–80% of 1RM [The effect of caffeine on resistance training was assessed using a double-blind, between-subject experimental design. Participants were asked to abstain from caffeine 12 h prior to the commencement of each training session. Forty-five minutes before the commencement of each training session participants in the caffeine group consumed 3 mg·kg% of 1RM ,40,41,42% of 1RM ,43. Prev% of 1RM ,45,46. R% of 1RM ,41,47. Up = 0.048) and DL and HC , were significantly correlated . Given that 28 of 30 1RM comparisons demonstrated a significant correlation, all the data were transformed to a Z-score using the formula (x-μ)/σ, summed and compared using an independent sample t-test. The acute effects of caffeine on measures of peak torque (PT), total work (TW), and RPE, were assessed using a 4-factor mixed-model ANOVA. The between factor being Group (caffeine and placebo) and within factors being Time (pre-and post-intervention), Treatment (caffeine or placebo), and Speed/Set . Furthermore, RTE and FAS were assessed using a 4-factor mixed-model ANOVA. The between factor being Group (caffeine and placebo) and within factors being Time (pre-and post-intervention), Treatment (caffeine or placebo), and Minutes . Maximal strength  measures were assessed using a single factor ANOVA with the fixed factor of Time (pre-and post-intervention). All intervention data (RTF and RPE) were analysed using an imputation method [2) was reported with significant ANOVA main effects as a measure of effect size [d) from the pairwise comparison (bias-corrected (Hedges) was calculated using the difference in means divided by the SD of the compared variables. Effect size was reported using the following categories: trivial < 0.20, small 0.20–0.49, medium 0.50–0.79, and large > 0.80 [p < 0.05.Data analysis was performed using Statistical Package for the Social Sciences (IBM SPSS Statistics Version 25) and Excel (Microsoft Windows Version 16.41 2020). Initially, appropriate tests of normality and homogeneity were performed. To match groups for baseline strength prior to completion of the resistance training program, a Pearson’s or Spearman’s correlation was performed between all measures of 1RM. All measures, other than DL and PC , height , or body mass  did not differ between individuals assigned to the caffeine or placebo groups. Average caffeine consumption was 133 ± 123 mg per day and 121 ± 95 mg per day for the caffeine and placebo groups, respectively, with four participants reporting no caffeine use . Average caffeine consumption did not differ between groups . The perceived effect of caffeine in energy/work enhancement and sleep disturbance were significantly higher in the placebo group ; however, there were no other significant differences for any other caffeine expectancy subscale . Baseline strength was comparable between the groups prior to completing the resistance training program .Age . Pairwise comparison indicated that eccentric PT at 120 and 180°/s was higher following caffeine supplementation compared to placebo . Furthermore, eccentric PT was greater at 180°/s compared to that at 60 and 120°/s, but only in the caffeine trial . For both concentric and eccentric PT there were no other significant interactions . Concentric PT was significantly affected by Speed  with PT at 180°/s being lower than that at 60 and 120°/s (p < 0.001 in both cases). There were no other significant main effects .For eccentric PT of the elbow extensors, there was a significant Treatment*Speed interaction (p = 0.024 ηp2 = 0.168) and for both concentric and eccentric PT a significant Time*Treatment*Speed interactions (p < 0.034 ηp2 > 0.112). Pairwise comparisons indicated that for the caffeine group, concentric PT was higher post intervention and increased in the placebo group but only when the pre to post caffeine trail was compared . Eccentric PT was not significantly increased post-intervention  and was not affected by acute caffeine treatment either pre- or post-the intervention . However, acute caffeine treatment increased concentric PT measured at 60 and 120°/s both pre and post the training intervention . In some cases, PT was affected by speed. Following the intervention concentric PT at 180°/s was higher than that at 60 and 120°/s (p < 0.003 in both cases) and following the placebo treatment PT at 60°/s was higher than 120 and 180°/s .For concentric PT of the elbow flexors, there was a significant Group*Time*Treatment interaction (p = 0.016 ηp2 = 0.138). Pairwise comparison indicated that the caffeine group had an increase in PT at 60°/s post the exercise intervention at a level that was approaching significant , whereas the placebo group had no significant training effect . There was no effect of acute caffeine supplementation or speed (p > 0.166 ηp2 < 0.001). For concentric PT, there was a significant Group*Time interaction (p = 0.010 ηp2 = 0.212). Pairwise comparison indicated PT was improved following the intervention in the caffeine group  but unchanged in the placebo group . For concentric PT, there were no other significant interactions (p > 0.097 ηp2 < 0.114) or a main effect of acute caffeine treatment (p = 0.344 ηp2 = 0.114). However, PT was significantly affected by Speed (p = 0.001 ηp2 = 0.665) with PT at 180°/s being lower than that at 60 and 120°/s (p < 0.001 in both cases d > 0.27).For eccentric PT of the knee extensors, there was a significant Group*Time*Treatment*Speed interaction (p > 0.094 ηp2 > 0.002) and no main effect of Group (p > 0.417 ηp2 > 0.004). For concentric PT, there was no main effect of Treatment (p = 0.075 ηp2 = 0.109), but there was a main effect of Time (p = 0.007 ηp2 = 0.230) with PT post the exercise intervention being significantly higher than pre-intervention exercise. There was also a main effect of Speed (p = 0.001 ηp2 = 0.418) with performance at 180°/s being lower than that at 60 and 120°/s (p < 0.003 in both cases d > 0.16). For eccentric PT, there was no main effect of Speed (p = 0.316 ηp2 = 0.040). However, there was a main effect with treatment (p = 0.007 ηp2 = 0.223) where acute caffeine increased eccentric PT compared to the placebo trial.For concentric and eccentric PT of the knee flexors there were no significant interactions (p > 0.065 ηp2 > 0.003), no main effects of Group (p > 0.762 ηp2 > 0.003), Treatment (p > 0.151 ηp2 > 0.072), or Time (p = 0.578 ηp2 > 0.006). However, there were significant main effects of Set (p = 0.001 ηp2 > 0.527) with performance at Set1 being higher than that at Set2 and Set3 (J)  and performance at Set2 being higher than that at Set3 .For concentric and eccentric TW of the elbow extensors there was no significant interactions (p < 0.047 ηp2 > 0.113). Pairwise comparison for concentric TW indicated that pre-exercise intervention, Set1 TW was higher than post-intervention . Furthermore, both pre-and post-the intervention performance in Set2 and Set3 was significantly lower than at Set1 (p < 0.001) for both concentric and eccentric TW. No other significant interactions were identified (p > 0.096 ηp2 < 0.931) and there was no main effect of Group (p > 0.271 ηp2 < 0.035). Concentric TW was not affected by Treatment (p = 0.231 ηp2 = 0.051); however, there was a main effect of Treatment for eccentric TW (p = 0.003 ηp2 = 0.272), where TW following acute caffeine treatment was greater than the placebo treatment.For concentric and eccentric TW of the elbow flexors, there was a significant interaction between Time*Set (p > 0.130 ηp2 > 0.032). For concentric TW there was a significant Time*Treatment interaction (p = 0.001 ηp2 = 0.350). Pairwise comparison indicated an increase in TW post-intervention . Furthermore, TW was significantly increased in the caffeine trial prior to the exercise intervention , however, post-intervention there was no significant difference between the caffeine and placebo trial . For concentric TW there were no other significant interactions (p > 0.184 ηp2 < 0.933). For both concentric and eccentric TW there was no effect of Group (p > 0.494 ηp2 < 0.018). Eccentric TW reported no other main effects (p > 0.140 ηp2 < 0.077) but for concentric TW there was a main effect of Set (p = 0.001 ηp2 = 0.400). TW at Set1 was higher than Set2 and Set3  and performance at Set2 was higher than that at Set3 .For eccentric TW of the knee extensors there were no significant interactions (p > 0.113 ηp2 < 0.075), but for eccentric TW there was a Group*Treatment*Set interaction (p = 0.029 ηp2 = 0.119). Pairwise comparison indicated that there was no effect of Group . However, eccentric TW was greater in the placebo group during caffeine trials . In the caffeine group Set1 TW was significantly higher during the caffeine trials . For eccentric TW there was no main effect of Time (p = 0.007 ηp2 = 0.235) and for concentric TW there was no main effect of Group (p = 0.256 ηp2 = 0.046) or Time (p = 0.148 ηp2 = 0.073). However, concentric TW was significantly increased following caffeine treatment (p = 0.014 ηp2 = 0.198). Concentric and eccentric TW was effected by Set (p = 0.001 ηp2 = 0.629) with performance at Set1 being higher than that at Set2 and Set3  and performance at Set2 being higher than that at Set3 .For concentric TW of the knee flexors there were no significant interactions (p = 0.025 ηp2 = 0.167), Time*Treatment (p = 0.003 ηp2 = 0.267), Time*Set (p = 0.011 p = 0.025 ηp2 = 0.186), and Treatment*Set (p = 0.050 p = 0.025 ηp2 = 0.115) interactions. Pairwise comparison indicated no group difference in RPE following MVC . The caffeine group had increased RPE following MVC post-training intervention (p = 0.001 d > 0.097). Furthermore, the placebo group had a treatment effect post-intervention, with RPE during the caffeine trial being higher than the placebo trial (p < 0.006) at Set2 (d = 0.87) and Set3 (d = 0.76). Irrespective of treatment, RPE following Set3 was higher than that following completion of Set1 and Set2 (p = 0.001 d > 0.97.) There were no other significant interactions (p > 0.085 ηp2 < 0.936).RPE following repeated MVC of the elbow extensors there were significant Group*Time (p = 0.001 ηp2 = 0.222). Pairwise comparisons indicated an increase in RPE following MVC post-exercise intervention (p = 0.001 d > 0.48). Both pre-and post-exercise intervention, RPE following Set 3 was higher than that following Set 1 and Set 2 (p < 0.001 d > 0.62). There were no other significant interactions (p > 0.072 ηp2 < 0.064) and no main effect of Group (p = 0.771 ηp2 < 0.014) or Treatment (p = 0.961 ηp2 = 0.001).For RPE following repeated MVC of the elbow flexors, there was a significant Time*Set interaction (p = 0.017 ηp2 = 0.135). Pairwise comparison indicated no group effect pre-or post-exercise intervention (p > 0.130 d < 2.18). However, both treatment groups indicated increased RPE post the exercise intervention across (p < 0.001 d > 0.40). RPE following Set3 was significantly higher than Set1 and Set2 (p < 0.001 d< 2.18.). There was also a Time*Treatment interaction (p = 0.001 ηp2 = 0.396), where RPE was higher post-intervention in both treatments (p = 0.001 d > 0.97). Furthermore, prior to the exercise intervention, RPE was significantly higher following caffeine treatment (p = 0.001 d > 0.76).For RPE following repeated MVC of the knee extensors, there was a significant Group*Time*Set interaction (p > 0.061 ηp2 < 0.975). However, there was a main effect with Time (p = 0.001 ηp2 = 0.748) with post-training intervention RPE being increased. There was also a main effect with Set (p = 0.001 ηp2 = 0.898) with RPE following Set3 being higher than Set1 and Set2. There were no main effects of Group (p = 0.760 ηp2 = 0.002) or Treatment (p = 0.666 ηp2 = 0.006).For RPE following repeated MVC of the knee flexors, there was no significant interaction (p > 0.060 ηp2 > 0.001). RTE Physical, RTE Mental, and FAS were significantly affected by time post-ingestion (p < 0.001 ηp2 > 0.245). Both RTE Physical and FAS reported 45 min post-treatment were significantly higher than pre, 15- and 30-min post-treatment . However, RTE Mental 30 min post-treatment was higher than pre, 15-, and 45-min post-treatment . There were no significant main effects of Group (p > 0.150 ηp2 > 0.007), Time (p > 0.057 ηp2 > 0.124) or Treatment (p > 0.415 ηp2 > 0.003).For RTE Physical, RTE Mental, and FAS there were no significant interactions . Following the resistance training intervention, 1RM improved in all lifts (p < 0.001 ηp2 > 0.633). There was no significant effect of Group in all exercises (p > 0.850 ηp2 < 0.071) apart from DL (p = 0.001 ηp2 = 0.493) with the placebo group having a greater performance during 1RM assessment.For 1RM performance, there were no significant Group*Time interaction (p = 0.998). However, RTF across the training intervention was higher in the caffeine group for SSP, SQ, DL, and HC (p < 0.037 d < 0.13). The placebo group outperformed the caffeine group in PC (p = 0.005 d < 0.18), and there were no between-group differences for CP (p = 0.618). For all exercises, there was also a main effect of Session (p < 0.005). Whilst there were some differences between specific sessions, there were clearer trends for an increase in CP, SSP, DL, and PC RTF as the duration of the intervention increased. Furthermore, for all exercises, there was a main effect of Set with RTF being higher in Set1 compared to Set2 (p < 0.001 d < 0.14).For all exercises, there were no significant interactions (p < 0.035 d < 0.62). For CP, SQ, and DL there was a main effect of Session (p < 0.034). Pairwise comparisons show some session-specific differences for SQ and DL, but no clear trends. Furthermore, for all exercises, there was a main effect of Set, with RPE being higher in Set1 compared to Set2 (p < 0.044 d > 0.49). There were no significant interactions (p > 0.072).For both SQ and HC, sessional RPE of the caffeine group was lower than that of the placebo group  3 mg·kg−1 may be useful to evoke acute enhancements in muscular strength; (2) relatively short-term chronic ingestion of 3 mg·kg−1 of caffeine may not lead to habituation to its effects; (3) chronic caffeine supplementation for 7-week may not enhance resistance-training induced gains in strength.Data from the present study indicate that prior to the training intervention the acute ingestion of 3 mg·kgRecent meta-analyses indicate that acute caffeine consumption may enhance muscular strength, including strength assessed using isokinetic dynamometry ,3,5. In +2 [Mechanistically, the ability of caffeine to evoke improved exercise performance is primarily attributed to its effects as a central adenosine receptor antagonist ,54,55,56+2 ,65,66, r+2 .Although it was not the purpose of the present study to determine the mechanisms underpinning the acute effects of caffeine on muscular strength, the influences of some of these mechanisms can be determined. When measured in the arm of the experiment examining the acute effects of caffeine, post-exercise RPE was in the most part unaffected by caffeine, and on some occasions was increased following acute caffeine ingestion. This coincides with previous work where caffeine ingestion had a limited effect on perceived effort or pain in scenarios where exercise performance was unchanged ,67,68 orInterestingly, caffeine did not have an effect on RTE physical and mental effort when measured in the arm of the experiment examining the acute effects of caffeine. This coincides with previous research ,72. WhilThere is a debate in the literature as to whether the acute benefits of caffeine on physical performance are reduced following chronic exposure, with some evidence indicating habituation to caffeine’s effect ,13 and o−1 of caffeine, twice per week for 7-weeks, in a population of 30 moderate habitual caffeine consumers, did not reduce the caffeine’s ergogenicity. Although there are varying methodological approaches to assess caffeine habituation, the findings of the present work agree with previous evidence that indicates that caffeine evokes equivalent performance-enhancing effects in low and high habitual caffeine users [−1 of caffeine improved cycling time trial performance in low (58 mg/day), moderate (143 mg/day), and high (351 mg/day) habitual caffeine users. Still, although models stratifying participants by typical caffeine consumption habits for examining the potential for habituation have value, the results from these studies may be limited by the reliance on subjective caffeine recall used to quantify caffeine use.These results suggested that consuming 3 mg·kgne users ,11,74,76−1) for 28 days. Results indicated that the acute effect of caffeine on cycling performance was evident pre-supplementation and was no longer apparent after 4 weeks of caffeine supplementation. Lara et al. [−1 supplementation for 20 consecutive days, during which performance in a Wingate test was evaluated. While caffeine increased cycling power, the ergogenic effects attenuated over the course of the study, suggesting progressive tolerance. Indirect assessments of the impact of habituation have also be made by studies that stratify their participant groups by habitual caffeine use. Several studies demonstrate an ergogenic effect in high caffeine users across anaerobic and aerobic modes of exercise [The disparity between the current data and studies that have directly measured and have indicated habituation to caffeine effects is likely due to differences in the population characteristics, the dose, and duration of caffeine administration, and the outcome variables assessed. For example, Beaumont et al.  includeda et al.  used a cexercise ,73,74,76−1 caffeine before each session of a 6-week resistance training intervention had superior improvements in the bench press and squat 1RM compared to those that consumed a placebo for the duration of the intervention. However, our data support work by Giraldez-Costas et al. and Pakulak et al. [Irrespective of the supplement provided, the training intervention resulted in a significant increase in all assessments of 1RM and specific measures of PT and TW. The more pronounced effects of the training intervention of 1RM were expected due to training specificity . Post-ink et al. ,78 who iWe progress previous findings by demonstrating this relationship following a longer intervention period and across a larger range of strength assessments considering not only 1RM in different exercises but also maximal voluntary concentric and eccentric muscle functions across a range of joint angular velocities. The disparity between the present findings and those of Kemp et al.  may be rThis study is the first to monitor the acute effect of caffeine during each session of a training intervention. Interestingly, across the training intervention, RTF was higher in SSP, SQ, DL, and HC for the caffeine group. While this may add further weight to the acute benefits of caffeine, it demonstrates the small but significant increase in the relative total weight lifted was not substantial enough in magnitude to evoke a superior training response. Furthermore, sessional RPE was either unchanged or in some cases decreased in the caffeine group, this again indicates that the ability of caffeine to suppress perception of pain/ effort may be more prevalent during sustained contractions ,67,68.The present study offers valuable insight with respect to the effects of chronic caffeine supplementation as a nutrition strategy to augment resistance training, though it is not without limitation. Firstly, a resistance-training program with a weekly training frequency greater than that adopted in the present study would likely evoke greater adaptations in the measured outcomes . HoweverOur data indicate a small but significant increase in training volume across the course of the intervention, whilst this did not manifest in an enhanced training response in the measured outcomes there is the possibility that a sustained increase in training volume over a longer period may evoke enhanced adaptations. This is an avenue for future investigation.We did not measure genetic influencers that potentially may influence the caffeine’s performance-enhancing effect ,85,86. A−1 for the present study as it typically considered the minimum dose needed to evoke an improved exercise performance [Furthermore, this study used individuals that were physically conditioned but not specifically resistance trained. Although the demonstrated effects may be relevant to a broad population that is relatively naive to resistance training, they may not be generalisable to individuals that are specifically resistance trained. Future work should look to directly compare trained against untrained participants using a similar approach to that adopted in the present study. Future work should also look to repeat this study using different doses of caffeine. We selected 3 mg·kgformance ,89,90 anformance ,3,4,5,7 formance . Whilst formance , future −1 caffeine consumption on muscular strength and explored if chronic use of caffeine could be used as an effective nutrition strategy to augment the response to resistance training. The data presented herein indicate a muscle and contractile mode-specific acute benefit of caffeine consumption, effects that were not dampened following twice-weekly consumption over the time course of a seven-week resistance training intervention. Whilst the training intervention resulted in significant improvements in 1RM performance and specific measures of isokinetic PT and TW of the upper and lower limb musculature, for the most part, the effects were equivalent in both the group ingesting caffeine and in the group ingesting a placebo. These effects were prevalent despite a small but significant increase in RTF across the training intervention in the caffeine group. In summary, our results infer that caffeine may be beneficial to evoke acute improvements in muscular strength but has limited benefits across the course of a resistance training intervention.This study examined the acute effects of 3 mg·kg


Chemotherapeutic drug-induced p53-dependent crosstalk among tumor cells affects the sensitivity of tumor cells to chemotherapeutic drugs, contributing to chemoresistance. Therefore, pharmacological targeting of p53 may contribute to overcoming drug resistance. The localization of p53 is closely related to its function. Thus, we assessed the effect of p62 on the coordination of p53 mitochondrial localization under chemotherapeutic drug treatment in ovarian cancer cells. We found that the combined use of the proteasome inhibitor epoxomicin and cisplatin led to the accumulation of p53 and sequestosome1(p62) in the mitochondria, downregulated mitochondrial DNA (mtDNA) transcription, inhibited mitochondrial functions, and ultimately promoted apoptosis by enhancing cisplatin sensitivity in ovarian cancer cells. Moreover, the ubiquitin-associated (UBA) domain of p62 was involved in regulating the mitochondrial localization of p53. Our findings suggest that the interaction between p62 and p53 may be a mechanism that determines the fate of tumor cells. In conclusion, p62 coordinated the mitochondrial localization of p53 through its UBA domain, inhibited mtDNA transcription, downregulated mitochondrial function, and promoted ovarian cancer cell death. Our study demonstrates the important role of p53 localization in tumor cell survival and apoptosis, and provides new insights into understanding the anti-tumor mechanism of targeting the ubiquitin–proteasome system in tumor cells. Ovarian cancer is the most lethal gynecological malignancy . SurgeryPuma, Noxa, and Bax, indirectly promoting apoptosis. Furthermore, Chipuk et al. found that p53 localized in mitochondria directly activated Bax to mediate mitochondrial membrane permeabilization and apoptosis [p53 performs its tumor suppressor function by inducing tumor cell apoptosis and cell cycle arrest . In addipoptosis ,17. Thesmt) is a retrograde signaling pathway from the mitochondria to the nucleus, which attenuates mitochondrial stress, adapts tumor cells to various forms of mitochondrial dysfunction, maintains mitochondrial protein stability, and prevents mitochondria-induced cell death in vivo [mt can be activated upon loss of oxidative phosphorylation (OXPHOS) function or unfolded protein accumulation in the mitochondria. A study in Caenorhabditis elegans by Nargund et al. showed that the UPRmt is a transcriptional response mediated by the transcription factor activating transcription factor associated with stress-1(ATFS-1) [mammals [mt activates the transcription of genes that assemble OXPHOS complexes to promote cell survival [PGC-1α and PGC-1β, thereby inhibiting the transcription of PGC-1α transcriptional targets including transcription factor A, mitochondrial (TFAM) in various mouse tissues [mt following denervation-induced muscle disuse of skeletal muscle, suggested that p53 affected mitochondrial protein homeostasis by mediating a series of mitochondrial quality control processes including the UPRmt [mt in tumors may provide evidence supporting the inhibition of the proteasome pathway to increase chemotherapeutic drug sensitivity [Recent evidence suggests that the mitochondrial unfolded protein response  , activat[mammals ,which pr[mammals . Moreovesurvival . Studies tissues . The finhe UPRmt . Therefositivity ,27.p62 is well known as the most typical autophagy receptor participating in the degradation of cellular proteins . Numeroumt, and mediating cell death. These findings suggest that targeting the proteasome pathway may inhibit tumor cell proliferation.In this study, we hypothesized that p62 played a role in the subcellular localization of p53 in ovarian cancer cells. We found that the combined use of epoxomicin (epox) with cisplatin increased mitochondrial localization of p53 through the UBA domain of p62, thereby downregulating mitochondrial functions, affecting the transcription of important subunits of the mitochondrial OXPHOS system and the UPRA2780 and SKOV3 cells were treated with cisplatin(CDDP) for 24 h and cell viability was evaluated using MTT assays. We found that A2780 cells were more sensitive to cisplatin than SKOV3 cells a,b. Undemt protects cells from a wide range of mitochondrial stresses that include oxidative phosphorylation (OXPHOS) dysfunction, disturbance of protein import due to mitochondrial protein misfolding, ATP depletion, and dissipation of the mitochondrial inner membrane potential [LONP1, mtHSP70, HSP60, and HSP10, which play a protective role as the downstream of ATF5, were increased, but their levels in the combined CDDP and epox group were lower compared with those in the epox group  or cisplatin (2 μg/mL) or both for 0, 6, 12, and 24 h to assess mitochondrial functions. The mitochondrial membrane potential at 24 h and ATP production at 12 h decreased in the epox, CDDP, and combined CDDP and epox groups. These changes were the most obvious in the combined CDDP and epox group a,b. Prodotential . Mitochoox group g–i. ThesMT-ND2 and MT-CO1  or cisplatin (2 μg/mL) or both for 0,6,12 h and the mitochondria and nucleus were isolated to determine the subcellular localization of p53. We found that the levels of p53 and p62 increased in a time-dependent manner in the epox and the combined CDDP and epox groups, while only the levels of p53 showed a time-dependent increase in the CDDP group in the mitochondria a. ComparMT-CO1 and MT-CO3), but were not significantly altered in the ΔLIR group . Both cells lines were cultured in RPMI 1640 ,supplemented with 10% fetal bovine serum, 100 IU/mL streptomycin and 100 IU/mL penicillin, at 37 °C under humidified air containing 5% COCisplatin and epoxomicin were purchased from MedChemExpress . 3--2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich . The following antibodies were used: anti-p62, anti-Mcl-1 ; anti-p53,anti-β-actin, anti-VDAC1, anti-LaminA/C, anti-βtublin, anti-p53, antiBax ; anti-PORLMT ; anti-Bcl-2 ; anti-OXPHOS ; All antibodies were diluted at a ratio of 1:1000.Cells were seeded in 96-well plates at a density of 8 × 103 cells per well (A2780) or 10 × 103 cells per well (SKOV3) overnight. The cells were first treated with different reagents for the indicated times and then treated with the MTT reagents. After 4–6 h, we measured the absorbance at 490 nm using a Multiskan Spectrum .2 for 20 min in the dark. Then, the cells were washed with staining buffer and centrifuged at 600× g for 4 min at 4 °C thrice. Finally, cells were suspended in staining buffer and analyzed using a BD Accuri C6 flow cytometer .A2780 cells were seeded at a density of 4 × 105 cells per well in a 6-well plate, treated with cisplatin or epox or both for 12 h, and then stained using a Mitochondrial Membrane Potential Assay Kit with JC-1 , following the manufacturer’s instructions. Briefly, cells were incubated with JC-1 dye at 37 °C under humidified air containing 5% COg, 5 min). The ATP level was determined by mixing 10 μL of the supernatant with 10 μL of luciferase reagent using a luminometer  RLU value or CPM.ATP measurement was conducted using an Enhanced ATP Assay Kit . Briefly, cells were lysed with a lysis buffer and centrifuged at 4 °C  following the manufacturer’s instructions. Briefly, cells were stained with mitosox dye, incubated at 37 °C for 10 min, and washed thrice with cold PBS. The samples were analyzed using Accuri C6 Flow Cytometry .Total DNA was extracted using a TIANamp Genomic DNA kit . qPCR was used to measure the mitochondrial-encoded nicotinamide adenine dinucleotide dehydrogenase 1 (ND1) level relative to that of nuclear-encoded gene 18S rRNA (18S).The following primer sequences were used:18srRNA:5′-TAGAGGGACAAGTGGCGTTC-3′ (forward) and5′-CGCTGAGCCAGTCAGTGT-3′ (reverse).ND1:5′-CACCCAAGAACAGGGTTTGT-3′ (forward) and5′-TGGCCATGGATTGTTGTTAA-3′ (reverse).Cells were lysed using RIPA lysis buffer containing proteasome inhibitors. Lysates were centrifuged at 4 °C and 4500 rpm for 10 min, boiled in loading buffer and resolved using SDS-PAGE. Proteins were transferred to PVDF membranes, which were later blocked with 5% skim milk and then incubated with primary antibodies overnight at 4 °C. The next day, the membranes were incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies  according to the manufacturer’s instructions. The ECL reagent  was used for immune-detection and band visualization using Syngene Bio Imaging . β-actin was used as an endogenous control, VDAC as a mitochondrial control, and LaminA/C as a nuclear control.The pcDNA3.1 vector (NC), pcDNA3.1-p62(wt-p62), pcDNA3.1-ΔLIR-p62, and pcDNA3.1-ΔUBA-p62 were purchased from Sangon Biotech . Cells were seeded in 6-well plates overnight and then transiently transfected with pcDNA-plasmid prepared in the laboratory using the Lipofectamine Transfection Reagent  according to the manufacturer’s protocols.β-actin . Primers sequences are presented in Total RNA extraction and RT-qPCR was performed as previously described before . The prig at 4 °C for collection. The supernatant was discarded and cells were resuspended in PMSF-supplemented mitochondrial separation reagent and placed in an ice bath for 10–15 min. The cell suspension was transferred to a glass homogenizer of appropriate size, and homogenized approximately 10–30 times. The cell homogenate was centrifuged at 600× g and 4 °C for 10 min, and the supernatant was transferred to another centrifuge tube and centrifuged at 11,000× g and 4 °C for 10 min. The supernatant was subsequently discarded. The pellet contained the isolated cell mitochondria. The supernatant, which contained the cytoplasmic protein, was centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was obtained to isolate mitochondrial cytoplasmic proteins. Cells were harvested after treatment with CDDP or epox or both for 0, 6, and 12 h. Mitochondria extraction was conducted using a cell mitochondria isolation kit  following the manufacturer’s instructions. Briefly, cells were washed with cold PBS, digested with trypsin-EDTA solution, and centrifuged at 600× g for 5 min at 4 °C. The supernatant contained the extracted cytoplasmic protein. PMSF-supplemented nuclear protein extraction reagent was added to the cell pellet and the sample was vortexed at maximum speed for 15–30 s, then placed back in the ice bath, and vortexed at maximum speed for 15–30 s every 1–2 min for a total of 30 min. Next, samples were centrifuged at 12,000–16,000× g for 10 min at 4 °C. The supernatant contained the extracted nuclear protein. Cells were harvested after treatment with CDDP or epox or both for 12 h. Nuclear protein extraction was conducted using a Nuclear and Cytoplasmic Protein Extraction Kit  following the manufacturer’s instructions. Briefly, cells were washed with cold PBS, scraped with cell scrapers, centrifuged, and collected. The supernatant was discarded, and PMSF-supplemented cytoplasmic protein extraction reagent A was added to the cell pellet, followed by vortexing at maximum speed for 5 s. The sample was subsequently placed in an ice bath for 10–15 min. Cytoplasmic Protein Extraction Reagent B was then added, and the sample was vortexed at top speed for 5 s, and placed in an ice bath for 1 min, followed by vortexing at top speed for 5 s, and centrifugation at 12,000–16,000× The cells were seeded in 24-well plates and treated with drugs at a cell confluence of 70–80%. Cells were stained with mitotracker red for 20 min in the dark at 37 °C, and then, washed thrice with PBS and fixed with 4% paraformaldehyde for 25 min. Next, cells were washed with PBS and permeabilized with 0.1% Triton-PBS for 7 min. After being washed with PBS and blocked with 5% horse serum for 30 min, cells were incubated with primary antibodies overnight at 4 °C. The next day, cells were washed with PBS and incubated with with FITC/Texas Red–conjugated secondary antibodies  for 30 min at room temperature in the dark. The cells were washed with PBS, stained with Hochest33342 for 5 min. The images were acquired using an Echo Lab Revolve microscope .t-test and Tukey’s tests were used to compare differences among groups.Statistical analysis was performed using GraphPad Prism 8 . All of the data are presented as means ± standard deviation (SD). All experiments were repeated three times. A one-way ANOVA followed by Student’s