Many studies have found that memory for affective material is better than memory for neutral information and memory for positive material compared to negative material is better in older adults. Behavioral, neurophysiological as well as single polymorphism differences have been advanced to account for these effects. Here, we aimed to examine whether the combination of two polymorphisms (ADRA2B and CB1) in older adults influences active maintenance and manipulation of emotional information in aging working memory. We examined genotype data from 207 older adults  who performed a verbal operation span-like task with positive, negative and neutral words. We found that subjects carrying both ADRA2B and CB1 variants generally remembered a higher number of words. In addition, double carriers showed positivity effects while single carriers showed more general emotional enhancement effects, especially as strings lengthened. These findings are amongst the first to suggest a haplotype account of positivity effects in older adults’ memory. Different explanations have been advanced to explain these declines and although working memory may be affected by more general cognitive abilities such as speed of processing2 declines in processing resources3 and/or inhibition4 other studies show how age-related changes in mechanisms responsible for maintaining to-be-remembered information may also be implicated6. Indeed, working memory impairments may also be linked to factors involved in the active maintenance of information in working memory.Older adults generally show poorer performance in working memory and especially in complex span tasks that require active maintenance of the to-be-remembered information5 posit that verbal traces are stored in the phonological loop through rehearsal, or a subvocal repetition of the to-be-remembered information. This is particularly interesting when testing older adults with span tasks where performance on short span tasks is generally better than performance on longer spans. Indeed, if information is maintained through rehearsal, short spans that require shorter articulation times, should be rehearsed more than long spans and subsequently lead to an increase in memory performance.In particular, Baddeley and Hitch6, attentive processes are involved and since limited, must be shared, in both the processing and maintenance of information in working memory. In particular, if attentive resources are employed for processing, they are not available for maintenance and vice versa. Importantly, in span tasks where attention is necessarily directed at one item at a time, memory traces for the other items decline as attention must be continuously shared between processing and maintenance.Differently, information may be maintained in working memory through an attention-based mechanism. In line with the Time-based Resource-sharing model7 and many studies have highlighted how memory for affective information is maintained with age8 and in particular, how older adults often remember positive information better than negative information (positivity effect). Carstensen and Mikels9 posited that advantage for positive information is linked to affective goals that modify attentive processes in older adults. That is, older adults who prefer affective information, direct their attentive resources to this information and subsequently show an increase in memory for affective information in general, and for positive information, in particular (but see11 for different results).Nonetheless, recent behavioral studies highlight how affective information processing in working memory is preserved in aging12, are less affected by aging than other regions involved in general cognitive processing. In addition, older adults generally show an increase in amygdala activity in response to positive information and a decrease in amygdala activity in response to negative information14.Neurophysiological explanations suggest that older adults remember affective information better than neutral information because the ventromedial prefrontal cortex (PFC), the anterior cingulate gyrus, and the temporal pole, involved in affective information processing15. In particular, ADRA2B, a functional deletion in the adrenoceptor gene 2B, and CB1, a cannabinoid receptor type 1, have been separately linked to emotional memory performance18. To our knowledge however, no study has investigated the combined action of these genotype differences on an affective working memory task in older adults.Another possible explanation for affective working memory performance in older adults may be linked to genotype differences19). Behavioral studies have that found a bias for negative information21, an association with suicidal behavior22 and a decrease in false memories23 in ADRA2B carriers. Others have detected more general emotion effects since ADRA2B carriers were sensitive to both positive and negative stimuli27. In particular, Mammarella et al.28 suggested that ADRA2B may also mediate traumatic memories in older adults since ADRA2B carriers remembered positive words better than negative and neutral words even when these were pronounce with a negative prosody . fMRI data also shows an increase in the neural activity in the amygdala while encoding affectively connoted images in older adults and especially in ADRA2B carriers20 and several studies28 have underlined how the increase in the noradrenergic system generally found in older adults may reflect a persistent focus on affective information in these individuals, especially in ADRA2B carriers. Finally, since the noradrenergic system may also be, in part, responsible for cognitive-affective flexibility and cognitive reserve29, and therefore crucial for general emotion regulation processes in aging, ADRA2B carriers may benefit even more28.Studies on the interaction between ADRA2B variant and emotion, with a specific focus on valence effects, show a very complex picture  in the third intracellular loop, reduces receptor functionality and increases central noradrenergic transmission, through the presynaptic inhibition of NA release.31. The endocannabinoid system is also associated with stress release and CB1 receptors in the prefrontal cortex may modulate anxiety, chronic stress, motivation, and, more generally, social functioning30. For example, CB1 receptors are essential for the extinction of conditioned fear associations, indicating an important role for this receptor in neuronal emotional learning and memory as shown by fMRI studies investigating the cognitive control processes regulating fear extinction and, in particular, on the role of prefrontal cortex32.Recent studies have also begun investigating the role that the endocannabinoid system may play on cognitive-affective flexibility that depends on WM functions including goal maintenance, inhibition, shifting and updating36 focused on the rs2180619 single-nucleotide cannabinoid receptor type 1 polymorphism (CB1 receptor) located in the q14-ql5 region of chromosome 6. This receptor regulates the attenuation of synaptic transmission and psycho-activity by interfering with the release of other neurotransmitters. In this manner, CB1 protects the central nervous system from overstimulation or inhibition by other neurotransmitters.Studies on the endocannabinoid systemConsidering the established role of the noradrenergic and cannabinoid systems in affective processing mentioned above, our study aimed to investigate the combined effect of ADRA2B and CB1 carriers on affective working memory performance in older adults. Indeed, the combination of multiple polymorphisms that interact in the same pathway may amplify the effect of the single variants. In general, we expect double carriers to show better memory performance than single and/or no variant carriers due to cognitive flexibility and cognitive resources. Finally, in line with the large corpus of data available in literature regarding positivity effects in older adults, double carriers may show a positivity effect and remember more positive words than negative and/or neutral ones.2 test P value > 0.05).The ADRA2b polymorphism analysis identified 80 carriers and 127 non-carriers. The HRM analysis identified 57 homozygous carriers of the CB1 variant allele , 91 heterozygous subjects , and 59 homozygote subjects for the reference allele   X 3  X 2  mixed ANOVA on accuracy scores as the dependent measure. Valence and length were within participant factors. We introduced the length of the WM task in the analysis since WM memory performance generally decreases as length increases and valence effects may emerge more clearly as available cognitive resources necessary for completing the task decrease.p2 = 0.06. A Tukey-Kramer test for unequal sample size confirmed that carriers of both variants outperformed carriers of a single variant (ADRA2B or CB1). ADRA2B or CB1 single variant carriers showed comparable performance. The main effect of Valence was also significant, F = 38,883, p < 0.001, ɳp2 = 0.19. A post-hoc Tukey-Kramer test for unequal sample size confirmed that participants, in general, remembered positive words better than negative ones and negative words better than neutral ones, indicating classical positivity and emotional enhancement effects. A significant length effect was found F = 539,80, p < 0.001, ɳp2 = 0.76 since performance was better on short strings than on longer strings. The 2-way interaction between genotype and length was significant, F = 11,590, p < 0.001, ɳp2 = 0.12. A post-hoc Tukey-Kramer confirmed that performance was in all three groups decreased as strings lengthened and that carriers of both variants remembered more words than carriers of the single CB1 variant (p < 0.001) and carriers of the single ADRA2B variant (p < 0.05).The analysis revealed a significant genotype effect, F = 5,790, p < 0.01, ɳp2 = 0.04 was found. A Tukey-Kramer test for unequal sample size confirmed that carriers  and non-carriers showed comparable performance on short strings. Instead, performance differed on longer strings. In particular, ADRA2b + CB1 carriers remembered positive words better than negative ones and negative words better than neutral ones whereas single variant carriers (ADRA2B or CB1) showed a more general preference for emotional words compared to neutral ones  = 2,506, p = 0.02, ɳ The total number of words remembered, regardless of valence, was considered as the outcome variable. The estimation procedure produced a partition of the joint distribution of affective O-span performance and related covariates into six clusters, composed of individuals with similar covariates and affective O-span performance. As a result, it was possible to evaluate the typical profile of participants for each identified group Table . ClusterTo highlight specific features of the identified clusters, the estimated probability for each covariate was summarized to be equal to the different categories in the groups associated with lower  and higher levels of performance  on the O-span test , shows a prevalence of subjects carrying both variants (ADRA2B and CB1 genes), whereas CB1 variant carriers are underrepresented. Moreover, participants belonging to this cluster are more likely to be between 71–93 years of age and have lower verbal fluency scores. Results in Table Different results characterize the first two clusters, associated with a lower WM capacity. The lowest performing cluster (group 1) is mainly composed of females, aged between 71–93 years, with lower levels of education. They also record low scores on the Forward and Backward Digit, positive PANAS and verbal fluency. Conversely, these subjects score higher on the Geriatric Depression Scale (GDS). As for group 2, the only significant variables were education and verbal fluency. No other covariates were significant.In particular, this study aimed at investigating the role of genetic variability and the neuropsychological and demographic characteristics on affective O-span test performance, differentiated according to valence. The analyses including the number of positive, negative and neutral words remembered as response variable confirmed that enhanced WM performance was associated with a prevalence of subjects carrying both variants ADRA2B and CB1). The empirical percentages of affective O-span performance across clusters, for different outcomes, are shown in the box-plots of Fig. B and CB1The present study aimed at investigating whether carriers of both ADRA2B and CB1 polymorphisms show different performance on an affective WM task in older adults compared to single variant carriers and no carriers. Moreover, for the first time, an attempt to establish a genetic and cognitive profile of older adults during affective processing was delineated. Results may be summarized as follows.Regarding the influence of the combined effect of ADRA2B and CB1 compared to ADRA2B, CB1 or no variant on memory in an affective working memory task, we found that ADRA2B + CB1 carriers outperformed single variant carriers and no variant carriers with longer strings. That is, double carriers may have more cognitive resources and are able to use them to direct attentive resources towards motivated positive information during the processing and maintenance of information in the working memory task. Indeed, we found, a positivity bias in affective WM in carriers of both ADRA2B and CB1 polymorphisms whereas single variant carriers showed more general emotional enhancement effects since they remembered both positive and negative words better than neutral ones.Working memory performance requires both efficient sub vocal rehearsal processes and the ability to share cognitive attentive resources between processing and maintenance requests. Older adults generally show good performance on simple span tasks that principally require rehearsal mechanisms for remembering. On the contrary, performance declines when cognitive resources must be shared. However, working memory performance, even on complex span tasks, increases in older adults when the task contains affective information. Moreover, in our study, older double carriers showed a preference for positive information during the affective WM task, suggesting that WM may function according to motivational goals  that direct attentive cognitive resources to the processing and maintenance of positive information and that this may be enhanced by the genetic profile.The BPR analysis identified two groups of subjects with a prevalence of carriers of both analysed variants (ADRA2B and CB1), cluster 5 and 6 respectively, associated with enhanced WM performance. Although the older adults included in those clusters did not always show a stable cognitive baseline , both groups showed higher WM performance. Interestingly, those clusters showed better memory for positive words . This emerging profile indicated that WM resources are crucial for focusing on positive information and that genotype-related differences may contribute to this valence bias.8. Moreover, this process may be modulated by ADRA2B and CB1 haplotype. Mather8 showed that NA influences stimulus processing priority and that priority becomes biased towards affective, and in particular, positive information during aging.In conclusion, our study confirmed a significant relationship between the noradrenergic system and memory for affective information in healthy older adults. Results confirm that affective information processing requires cognitive control mechanisms that selectively attend and remember positive information38. Our study also confirms recent studies38 that show that both the noradrenaline and cannabinoid systems are crucial in affective information processes, and in positivity effects in memory in particular, in the aging brain12 (Mather and Harley29 Robertson38). Moreover, this may be also linked to the haplotypes of ADRA2B and CB1. The present data represent a novel starting point for evaluating how genetic differences may modulate affective working memory performance in older adults as well as in the healthy and pathological aging brain.Our study also highlighted, for the first time, specific cognitive and genetic profiles. In particular, we found a high prevalence of both variant carriers in healthy agingThe Departmental Ethics Committee at the University of Chieti approved the study. In accordance with the Declaration of Helsinki, all participants gave written informed consent prior to inclusion in the study.2) of 0.07 of genetic correlates of memory studies13. We required 80 participants per genotype for a behavioral study. In line with the finding that 30% of the White population show the ADRA2B deletion variant, we recruited 221 right-handed native Italian speakers from a group of healthy community dwelling older adults from the Chieti area to participate in the study. Participants were between the ages of 64 and 91 . Exclusion criteria included a history of significant head injuries, stroke, epilepsy and learning disabilities. Eight participants were excluded because they could not be genotyped for ADRA2B or CB1. A further 6 participants with previous or current diagnosis or treatment for anxiety and depression, revealed by a self-report demographics questionnaire were excluded leaving a total of 207 participants.Sample size was established following the typical effect size (η39) and the Positive and Negative Affective Scale (PANAS40) to assess current mood.Neuropsychological and demographic data are reported in Table 39) was used. The O-span requires participants to solve a series of math equations while remembering series of semantically unrelated words. For each trial, an equation/word string appeared at the center of a computer monitor. Participants read the equation aloud, determined whether the given solution was true or false and then read the following word. The task was self-paced and participants pressed the space bar after reading the word in order to view the next equation–word string. The equation–word strings were presented in sets of three to six items. Three question marks in the center of the monitor cued the end of each set and to recall the words in the order of presentation  Test for emotional words  and a mean arousal level of 5.9 (2.8), negative words had a mean valence of 2.4 (1.8) and a mean arousal level of 5.9 (2.7) and neutral words had a mean valence of 5.5 (1.9) and a mean arousal level of 2.7 (2.3). Valence set order was counterbalanced across participants. For a detailed description and psychometric properties of the O-span task, see previous studies41. The proportion of correctly words recalled regardless of serial position was considered as the WM measure.We constructed three to six equation-word strings for each affective valence  with two trials for each set size. The order of set size varied randomly. An 85% accuracy criterion on the math equations was required in order to ensure that participants were not trading off between solving the equations and remembering the words. The experimenter transcribed accuracy for math equations on a dedicated protocol. Two training trials preceded the experimental task. Thirty-six math equations and 36 target wordsGenomic DNA was isolated from buccal swabs using the NucleoSpin Tissue kit  according to manufacture instructions. DNA quality and quantity were assessed by Qubit 2.0 .23. In line with previous studies26 homozygote and heterozygote ADRA2B deletion carriers were treated as a single group due to the low number of homozygotes. A group of 207 participants was available for analysis. Homozygosity for the Glu301-Glu 303 deletion was detected in 4 participants; 76 participants were heterozygote while 127 were homozygote reference.PCR reactions for the polymorphisms analysis were carried out as previously reportedA > G) were determined by High Resolution Melting (HRM) analysis18 , the distribution of our sample  the allelic distribution observed here is similar to European  and American  populations.Genotypes for the rs2180619 , verbal fluency and current mood . All p ≥ 0.995.Following genotyping, participants were grouped as follows: 24 (11.6%) presented only the ADRA2B variant, 92 (44.4%) presented only the CB1 variant, 92 (27.1%) presented both ADRA2B and CB1 variants, 35 (16.9%) presented no variants. Groups matched for age and education, general short-term memory , both (ADRA2B + CB1) or none, we ran a mixed-model analyses of variance (ANOVAs) on affective O-span data (old words remembered regardless of order) with Valence  and span length (short and long) as repeated measures factor, and Genotype (ADRA2B vs CB1 vs. ADRA2B vs none) as the between-subjects factor.Second, we ran a Bayesian Profile Regression to identify high and low performance clusters of individuals and to test the influence of ADRA2B or CB1 variants, isolated or in combination, on Affective Working Memory in older adults. We also investigated whether these clusters showed particular behavioural profiles.44 and carried out statistical analyses with the freely available package PReMiuM42 for R statistical software (R Core Development Team 2018). This method allows us to explore the link between a response variable and a set of associated covariate data through cluster membership, so that the outcome and the clusters mutually inform each other45. It also allows us to include different neuropsychological and demographic characteristics potentially involved in the processing and maintenance of affective information in the O-span test variables in the analysis, in addition to those related to genetic variability.To examine the genotype-related effects on affective WM, we adopted a statistical technique known as Bayesian Profile Regression (BPR)X and the response vector (Y). In this manner, the approach allows us to take potential multicollinearity among exploratory variables into account, which would be very difficult to capture in a standard regression framework.The BPR assigns participants to clusters, based on the similarity of their covariates, and then associates clusters to affective O-span performance and supervises the clustering assignment in a unified fashion. Clusters are determined by both covariate data  methodsSupplementary Figures 1, 2, 3, 4


Subsequently, artificial pools of Anopheles gambiae (An. gambiae) specimens with known genotypes of L1014F, L1014S, and N1575Y were created, nucleic acids were extracted, and kdr mutations were detected. These data were then used to define a linear regression model that predicts the allelic frequency within a pool of mosquitoes as a function of the measured ΔCt values (Ct mutant − Ct wild type probe). Polynomial regression models showed r2 values of >0.99 (p < 0.05). The method was validated with populations of variable allelic frequencies, and found to be precise (1.66–2.99%), accurate (3.3–5.9%), and able to detect a single heterozygous mosquito mixed with 9 wild type individuals in a pool of 10. Its pilot application in field-caught samples showed minimal differences from individual genotyping (0.36–4.0%). It allowed the first detection of the super-kdr mutation N1575Y in An. gambiae from Mali. Using pools instead of individuals allows for more efficient resistance allele screening, facilitating IRM.Bioassays and molecular diagnostics are routinely used for the monitoring of malaria vector populations to support insecticide resistance management (IRM), guiding operational decisions on which insecticides ought to be used for effective vector control. Previously developed TaqMan assays were optimised to distinguish the wild-type L1014 from the knockdown resistance ( Anopheles gambiae (An. gambiae) mosquitoes kdr genotiewed in .However, is it necessary to genotype individual mosquito specimens, or could pooled mosquitoes be used instead to measure allele frequencies for vector control monitoring activities?The presence and frequency of resistance in mosquito populations  is the primary outcome in global databases like the IR Mapper , and thiAn. gambiae samples and discuss its advantages, limitations, and perspectives for application in the framework of IRM.Here, we developed and validated a novel approach for monitoring the frequency of pyrethroid resistance mutations in pooled An. gambiae Kisumu (insecticide susceptible), VK7 (1014F and 1575Y control), and RSP-ST (1014S control) laboratory strains were used for the initial development of the method  until analysis.The e method . Field pkdr point mutations L1014F and L1014S and the Taqman assay of Jones et al. [kdr point mutation N1575Y.Initially the allelic frequency of the corresponding mutations was determined for the three colonies . Artificial pools of 10 adult mosquitoes with known allelic frequency were created. Three replicates for each defined allelic frequency were prepared. To do so, 3–5 days old female mosquitoes (laboratory strains and field populations) were first individually genotyped by extracting genomic DNA (gDNA) from single legs following the DNAzol protocol according to the manufacturer’s instructions, with the following modifications: 200 μL DNAzol reagent , 100 μL of 100% ethanol for DNA precipitation, 10 μL of DEPC treated water for DNA solubilization. Genotyping was performed using the TaqMan assays of Bass et al. [s et al.  for kdr https://dmc-malvec.eu) with the aim of developing an automated multiplex diagnostic platform (LabDisk) for malaria vectors able to concurrently assess DNA and RNA markers within the same sample [Protocols for nucleic acid (NA) extraction were designed in the framework of the interdisciplinary research project DMC-MALVEC , 440 µL binding buffer , 200 µL of each wash buffer . Nucleic acid elution was performed with 180 µL elution buffer for 10 min at 50 °C. The integrity of NA was assessed via agarose gel electrophoresis (1.0% w/v). Mosquito tissue from 10 specimens was mechanically disrupted using a cordless mortar  and pestle , with the addition of 200 µL TE buffer . Subsequently, NA  was extracted using the MagnaMedics magnetic bead-based protocol with the following modifications to minimise time and materials needed. A total of 150 µL lysis buffer was added to the mechanically disrupted tissue, followed by 10 min incubation at room temperature and a centrifugation step at 16,000 × kdr 1014F, 1014S [kdr mutations 1014F and 1014S in the same reaction. Each probe was labelled with a different fluorescent dye . The 1575Y assay was used in the duplex format (HEX for wildtype N1575 and FAM for 1575Y), as previously described in Jones et al. [Previously published primers and probes were used for the F, 1014S , and 157F, 1014S  assays  gene for normalisation (ΔCt = Ct mutant probe − Ct EF) or using solely the Ct values of the mutant probe were also tested. All oligonucleotides were optimized using primer/probe matrices for efficiency, sensitivity and specificity  in terms of final concentration  in the mkdr mutation status was used to fit linear regression models that predict allelic kdr frequencies as a function of measured ΔCt values. Both linear and polynomial (quadratic) regression models were fitted and the best model chosen on the basis of adjusted R squared and residual plot analysis (goodness of fit), standard error of estimate (accuracy), and p values (significance). An independent validation set of artificial populations was used to corroborate the predictive value of the selected models from the training set, and also to calculate the average margin of error. For this reason, the equations developed were applied to calculate % allelic frequencies from ΔCt values and the average difference between predicted and expected values was calculated for each trait.The SPSS statistical software was used . A training set of pools of 10 mosquitoes composed of different numbers of individuals from the lab colonies with known 2pf) was calculated as described in [p values < 0.05 were considered statistically significant.Variance due to pooling formation  for Ct values are presented in kdr mutation status of each strain was initially determined . In all cases, quadratic models showed superior performance compared to models with a single term in terms of goodness of fit, estimated by adjusted R squared values and residual plot analysis , and by using the Ct value of the mutant probe only, were also explored but showed suboptimal results when compared to the ΔCt approach . TherefoThe developed models show a good fit for all three mutations with adjusted R squared values in the range of 0.996–0.997 , and no All models were able to reach as low as 5.0% in allelic frequency  with theThe developed regression models were applied to calculate allelic frequencies in an independent series of samples with known allelic frequencies for each mutation . The margin of absolute error in determination (accuracy) between true and estimated allelic frequencies was in the range of 3.26–5.9%, depending on the locus . Variabip < 0.001). When looking at individual populations covering different scales of allelic frequencies, the average differences between actual and measured mutant allelic frequency values were 7.7% for high- (60–80%), 3.7% for mid- (20–50%) and 2.1% for low- (5–15%) allelic frequency populations  case where populations are comprised exclusively of a mixture of mutant 1014F and 1014S mosquitoes . By usinAn. gambiae field populations from Mali (Gounkan area). In a first step, we genotyped the legs of N = 30 individuals to obtain the accurate allele frequency. Next, we genotyped three pools of each 10 randomly chosen remaining bodies to estimate the allele frequency with our new model. Results from individuals showed a 1014F allelic frequency of 75.0%  compared to 71.0%, as assessed by the average of the three pools, indicating a difference of only 4.0 % between the two methods. The 1575Y mutation was detected at a frequency of 30.0% in N = 30 individuals , compared to 29.64% when analysing the corresponding samples in pools. The difference between the two methods in this case was negligible .In order to further validate our approach, we applied it to previously collected An. gambiae kdr mutations  in pooled mosquito samples. Firstly, optimisation of previously developed TaqMan assays now allows the detection of kdr 1014F and 1014S and L1014 alleles in a single triplex reaction; this was previously performed in two separate reactions. Secondly, the presence and frequency of resistant alleles was accessed in pooled mosquito samples, and the outcomes were compared with the outcomes derived by “gold standard” kdr assessment in individual mosquitoes. The developed method allows the detection of allelic frequencies of as low as 5.0%. This means that a single heterozygous mosquito can be reliably detected in a pool of 10 mosquitoes in which all the remaining individuals are wild type. The method is both precise (Precision = 1.66–2.99%) and accurate (Accuracy = 3.3–5.9%). The variance in pool formation was smaller than 5%, which is reasonable and not unexpected, considering that the natural variability in body size will affect the allele frequency measured per mosquito.Reducing the time and cost of determinations is essential for vector control monitoring in the frame of IRM applications. Here, we developed and validated a novel approach to reliably calculate allelic frequencies of major kdr N1575Y mutation was detected for the first time in An. gambiae mosquitoes from Mali.Allelic frequencies measured in field caught samples using individual mosquitoes or pools were very similar; for 1014F, the difference between the two approaches was 4.0%, and for 1575Y, the corresponding difference was 0.36%. Both these figures are practically negligible in the frame of monitoring applications for evidence based IRM decisions. In practice, very often fixation levels or the absence of certain mutations are recorded, and intermediate levels of resistance alleles are rather rare. Moreover, in the pilot application, the super-www.irmapper.com), a tool that currently drives decision making for IRM at various stakeholder levels. Required entry fields in this data base are “test method used”, “mechanism tested”, “number of mosquitoes used per assay”, “outcome of the assays (detected/ not detected)” and “frequency of mutations”. All this information is readily available by using a pool strategy, yet at a substantially reduced cost and time. An approximately 10-fold reduction in cost  for analysing mosquitoes for kdr mutations is possible with the new approach, while throughput and analysis time are also substantially reduced.The novel approach we describe here does not distinguish between homozygotes and heterozygotes. This is a limitation, as the operationally relevant pyrethroid resistance phenotype is primarily expressed when mosquitoes are at the homozygote RR stage. However, given that the frequencies commonly detected in the field are often very high or very low, this information might be more relevant in research projects but not for decision making in IRM. Moreover, the outcome of the assay is fully compatible with the widely used IR Mapper database , with only minimal requirements for optimization and assay development. A quick overview on how to setup the experimental procedure to calculate allelic frequencies from mosquito pools is presented in Plasmodium detection assays on mosquito pools has been also introduced in many low transmission malaria countries to save time and resources [Plasmodium falciparum infective mosquitoes [later  solutions, sets the basis for the development of more holistic and automated approaches to vector population monitoring [https://dmc-malvec.eu, https://infravec2.eu) [The application of esources . A novelsquitoes ; with hinitoring , as the vec2.eu) , and arevec2.eu) .kdr mutations, we optimised existing TaqMan qPCR assays and developed reliable regression models to analyse data from pooled specimens. The novel approach is able to measure allele frequencies from pools of ten mosquitoes with very high accuracy, i.e., equivalent to genotyping individual mosquitoes, but requiring substantially less time and lower cost. Vector control monitoring activities in the frame of IRM programmes may greatly benefit from the described approach.We tested a simple research question: "Can we genotype pools of mosquitoes and still retrieve the same information on point mutations as we would using single individuals?”. Working with the example of


The impact was applied on the right tibiae, 5 days/week for 8 weeks mimicking walking (450 µε), uphill running (850 µε) and jumping (1250 µε) conditions. Trabecular and cortical parameters were determined by micro-CT, bone growth rate by calcein labeling and toluidine blue staining followed by histomorphometry. Bio-mechanical properties were evaluated from bending tests. HI group reduced rat body weight and food consumption compared to shams. Bone growth rate also decreased in MI and HI groups despite developing thicker hypertrophic and proliferative zone heights. HI group showed significant increment in bone mineral density, trabecular thickness, cortical and total surface area. Ultimate load and stiffness were also increased in MI and HI groups. We conclude that impact loading during adolescence reduces bone growth moderately but improves bone quality and biomechanics at the end of the growing period.Physical activity is beneficial for skeletal development. However, impact sports during adolescence, leading to bone growth retardation and/or bone quality improvement, remains unexplained. This study investigated the effects of  It has been hypothesized by “Wolff’s law”3 that this dynamic adaptive process, termed bone remodeling, occurs in bone mass and architecture due to stimuli obtained from its mechanical environment5. Bones can receive stimuli in the form of mechanical loading resulting from various intensities in physical activities and sports. Ground reaction forces, which generate stresses and strains on our weight-bearing bones, are determinant factors of bone remodeling. They are greater when we move faster and/or more intensively, hence vary according to the type of physical activities6. Low impact sports such as swimming, can produce lower ground reaction forces than high impact sports such as gymnastics or cross-country running8. A regular and sufficient amount of impact loading can prove to be very effective for bones at all ages9.A fundamental tenet of bone biomechanics is the adaptation phenomenon of bone microstructure under regularly applied mechanical loadingin vivo bone loading models such as tibial bending, ulnar and tibial axial loading14. Compared to other models, the tibial compression model has the potential to generate cortical and trabecular bone adaptation under applied mechanical load16. Axial loading in mouse tibia has been used for several years to investigate the effects of loading as a function of age18, sex19, disease20 and strain level16. However, still, there is a lack of data on the loading mechanism and effects of non-invasive loading in bone formation for the rat tibial axial compression model.Well-controlled and physiologic mechanical loading models are essential to successfully define and identify the anabolic and catabolic mechanisms involved in bone remodeling. To date, many loading models have been implemented in different animal studies, ranging from whole body rodent vibration, exercise models, and 21. In this period, regular impact loading in sufficient amount can ensure a proper bone accrual and also contribute to building up a strong skeleton22. It has been reported that23 performing a high impact physical activity, such as jumping, can effectively contribute to improving the hip bone strength in adolescents24. There are a few clinical and animal studies investigating the effects of impact loading on adolescent growth, but results are inconsistent. In some published clinical studies26, researchers have not provided a clear, distinct separation between the nutritional and mechanical factors. The studies expressed the physical activities in hours per week whereas the intensity of the activity  have not been considered. As a result, it becomes challenging to infer about the isolated effect of impact loading on the growing bones from these studies. In animal studies, the effects of exercise on long bone growth are also inconsistent, resulting in either no change27, minimal change28, or significant reduction30 in growth, measured as changes in long bone length. Hence, if any loading induced changes occur during the adolescence and whether they modify the bone microstructure are clinically relevant questions which remain still unresolved. We hypothesize that bone morphometry and biomechanics can be improved, but bone growth would remain unaffected under the controlled impact loading applied during the adolescence period. Moreover, among three different impact loading levels, we suggest that the higher impact intensity influences more importantly bone morphometry and biomechanics. This study aimed to use an animal model (rat tibia) to investigate the effects of well controlled in vivo low, medium and high impact loadings applied during puberty on bone growth, morphometry and biomechanics.Adolescence is a dynamic period for bone growth and developmentThe control and sham groups gained body weight whereas the HI group lost 13–16% of body weight compared to the shams at the end of the loading period Fig. . Food inBoth HI and MI groups exhibited a reduction in proximal tibial growth rate Fig. , resultiHypertrophic (HZ) and proliferative (PZ) zone thicknesses Fig. , as wellThe effects of impact loading exercise on bone microstructure were assessed by comparing the impact groups with the shams after 4 and 8 weeks of repeated loading regime. For trabecular bone, the HI group showed an increase in BV/TV and a decrease in Tb.Sp Table  after 4 From three-point bending tests, significant differences were found among the groups for some structural and intrinsic mechanical properties Table . The meaIn this study, the effects of eight weeks controlled impact loading during the adolescence on bone growth, quality and mechanics have been investigated using a rat tibial compression loading model. Our findings could be used as a basis for future investigation on the impact loading effects during the adolescence for finding a suitable loading protocol which would be beneficial for the overall bone microstructure during the growing period.31. Our findings showed that the HI group has less body weight and reduced food intake despite receiving the maximum intensity of the exercise regime. Part of this weight loss could be ascribed to the decreased appetite of the impact groups32, which was evidenced by a significantly lower caloric intake for the HI group 51. Osteochondroses are considered to be disorders of primary and secondary growth centers, or lesions at the apophyseal or epiphyseal growth areas of bones52. Active young athletes are prone to osteochondroses52, although it is not considered to be a permanent disability for diagnosed patients53. In most cases, conservative treatment for such symptoms includes sufficiently long rest52. In one form of such condition known as Scheuermann’s disease54, regular physical exercises are even recommended. In our study, the rats have been sacrificed immediately after the repeated loading regime. So, it remains unclear whether a sufficient amount of unloading period would affect the growth plate thickness and change it accordingly or not.Bone growth rate is generally correlated to the overall growth plate thickness55, which can eventually slow down longitudinal bone growth. Our observations can be compared with the studies using rat57 and swine55 models, where a reduction in body weight coupled with reduction in longitudinal bone growth has been observed. Our overall findings indicate that the generalized claim of the linear relationship between bone growth and growth plate height58 might not always be implemented. However, our findings are supportive of other studies47, where a contrary relationship between the growth rate and height of the growth plate was also observed.The change in longitudinal bone growth could also be associated with the caloric intake and body weight of the rodents. It has been reported that the reduction of body weight due to reduced caloric intake can affect cell production in the proliferative zone in a negative manner59. Also, higher loading intensity is generally associated with an increased BMD60. In fact, BMD in athletes is elevated under high impact training conditions61. The increase of BMD in our study could be related to hormones triggering mechanisms. Indeed, an increased BMD is controlled by a decreased parathyroid hormone response coupled with an increased calcitonin response63, both of which take time to react under favorable loading conditions. Hence, this could explain the significant increment of BMD assessed after 8 weeks instead of 4 weeks of loading. In similar studies, where adult rats have been tested for repetitive jumping exercise64 or treadmill running exercise65, an increased BMD was observed in the loaded limbs.Compared to the MI group, the HI group showed load adaptive changes in trabecular microstructure at an earlier (after 4 weeks) period and affecting more structural parameters Table . BMD was66, as found in this study. The significant BV/TV in HI group after 8 weeks of loading can be explained with the increased BMD for the same group  indicated an additional improvement in trabecular structure under impact loading conditions. The significant reduction in Tb.Sp is often considered as the concomitant increase with BV/TV and Tb.Th72. The significant reduction in Tb.Sp indicates the occurrence of a loading induced bone gain through increased connectivity and gradual thickening of the trabecular structure73. The significant change in Tb.Sp in HI group at an earlier stage can be associated with the significant increase in BV/TV from the same group  are highly correlated to each other76. Hence, the increased ultimate loads for MI and HI groups can be justified from their significantly increased cortical area. Our findings agree with other studies on adult rodents, where an increased ultimate strength have been associated with exercised tibiae in 15 and 10 week old rats31 and mice74, respectively. Bone stiffness can be related to its morphology and cross-sectional geometry in growing rats77. More specifically, stiffness can be associated with the increase or decrease in cortical thickness (Ct.Th), total area (Tt.Ar) and cortical area (Ct.Ar)79. Ct.Ar has been increased significantly for both MI and HI groups  and failure stress (σfail) are significantly increased and decreased respectively in both MI and HI groups  daily exercise of medium (850 µε) and high (1250 µε) impact physical activity during adolescence can influence bone growth, and improve the quality and mechanics of bone microstructure at the end of the growing period. This loading model provides the scope to fully understand the role of controlled mechanical loading during the adolescent period and will be used in future for the design of non-invasive loading models to modify the bone microstructure and mechanical strength for building up a healthy and robust skeleton. Future work will investigate whether these impact loads applied during puberty are determinant factors for bone quality and strength in adult life.in vivo loading. Animal care and use conformed to the guidelines of the Canadian Council on Animal Care (CCAC), and the Institutional Animal Care Committee approved the experimental protocol at the Sainte-Justine University Hospital, Montreal, Canada. Rats were housed two per cage at 25 °C with a 12-h light/dark cycle. Standard laboratory diet and water ad libitum were provided. Three days prior to the beginning of loading, rats were randomly divided into five groups (n = 6/group): control, sham, low impact (LI), medium impact (MI), and high impact (HI). Body weights and food intakes were monitored weekly from the beginning (4th week) of in vivo loading until the end (11th week) to monitor overall health.Animals  were obtained from Charles River Laboratories  at approximately 3 weeks of age and allowed to acclimate for one week before the start of 2), the cyclic impact loading was applied to the right tibia with a custom-built impact loading device  were applied at 2 Hz and characterized by symmetric active loading/unloading with a 0.10 sec of rest insertion between load cycles95  loading period of 10 min16 , zig-zag uphill running (850 µε), and vertical jumping (1250 µε) conditions99. Animals  were obtained from Charles River Laboratories  at approximately 3, 7 and 11 weeks of age (n = 6/age group) and allowed to acclimate for one week before the strain calibration experiment. After CO2 asphyxiation, followed by decapitation, right tibiae were collected for each age group of rats. Tibiae were isolated from the middle of the femur to the toes. After tibial collection, an incision was made near the medio proximal surface of the tibia. Overlying skin and muscles were retracted to expose the bonding surface, polished with an abrasive paper and cleaned with ethanol solution. A single element strain gauge  was bonded with cyanoacrylate  at 35% of the tibial length (L)  Fig. . A compr(L) Fig. . Haversi(L) Fig. . Using tin-vivo micro-CT  scanner with rotatable X-ray source and detector. Following the weekly loading regime, rats were anesthetized , positioned on the carbon fiber half-tube bed of the Skyscan 1176, and maintained on anesthetic gasses for the duration of the scanning process (5–12 min). A cylindrical shape Styrofoam holder was used to position the right tibia to ensure the placement of the tibia along the scanner midline. All scans were performed with 18 μm voxel resolution, 65 kV, 384 μA, 350 ms exposure time, 0.65° rotation step, and a 1-mm Al filter93. A Phantom calibration was performed for each scanning day, prior to bone imaging using two cylindrical hydroxyapatite phantoms (0.25 and 0.75 g/cm3 CaHA). Images were reconstructed using NRecon software 93.A micro-CT scanner was used to perform eight weekly CT scans of the right tibia of the rats, from 4 to 11 weeks of age. The imaging system was a Skyscan 1176 93  was applied93. CTAn software v.1.16 was used for performing morphometric analysis for the selected VOIs of trabecular bone and allowed evaluating the following parameters: bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular spacing (Tb.Sp), and connectivity density (Conn.Dn)100  for trabecular bone was selected as a percentage of the entire tibial length (L) Fig. . The VOI93 Fig. . The vol100 Fig. .Figure 593. A global gray threshold value of 65 was set for all analyses93. Cortical microarchitectural measurements, including tissue mineral density (TMD), cortical bone area (Ct.Ar), total area (Tt.Ar), medullary area (Ma.Ar), cortical thickness (Ct.Th), endocortical perimeter (Ec.Pm), periosteum perimeter (Ps.Pm), and mean eccentricity (Ecc) were evaluated from the cortical bone VOIs100 , rats were sacrificed using CO2 asphyxiation, followed by decapitation. Right tibiae (n = 30) from all groups of rats were cleaned of soft tissues and tested to failure in three-point bending under displacement control at 0.20 mm/sec using a Mach-1 V500C . A custom made bending setup was used to place the rat tibiae horizontally with the anterior surface upwards and centered between the supports , a is the width of the bone cross-section in the mediolateral direction (mm), b is the width of the bone cross-section in the anteroposterior direction (mm), and t is the average of the cortical thickness (mm)31. Assuming linear elastic bone material103 and using the beam bending theory, Young’s modulus and ultimate stress were determined by the following equations:ult is the ultimate stress (MPa), k is the stiffness (N.mm−1), L is the span length (mm), Fult is the ultimate load (N), and c is the distance from the cross-section centroid to outermost point on the cross-section (mm), which was approximated as bone width/287. Moreover, energies to ultimate stress (mJ/mm3) and failure stress (mJ/mm3) were assessed by calculating the respective areas under the stress-strain curve.Following the last micro-CT scan  were made intraperitoneally at a dosage of 15 mg/Kg43. After polymerization, tibial blocks were cut in 6 µm sections using a microtome (Leica SM2500). The longitudinal bone axis was used to cut the tibiae for 36 slides, six series of six slides, containing two sections per slide. For each tibia, the first slide of each series was set aside from light for growth rate measurements, for a total of 6 slides . Microscopic observations were performed with a microscope (Leica DMR with Retina Qimaging Camera) using 2.5x magnification.Proximal sections (~10 mm) from each tibia were fixed for 48 h using the formalin solution . For dehydration and clarification, graded alcohol solutions and xylene were used respectively. Embedding process was performed using methylmethacrylate (MMA) 104  between injections104 Fig. . Measure 44 Fig. .43. These parameters included the PZ and HZ heights, hypertrophic cell height, as well as the number of proliferative cells per column44  was used to perform statistical analyses. To determine time effects, loading effects, and their interaction on body weight and food consumption during the experimental period, a repeated ANOVA test  was performed. The sham and control groups were compared to isolate any handling and manipulation effects, whereas, the impact groups were compared with the sham group to infer on the impact loading effects. For assessing any significant differences in average bone growth rates and histomorphometric parameters measured at the 11Measurement process of bone growth rate using calcein image


Among 115,869 Veterans ≥65 years old with a history of moderate to severe musculoskeletal pain  admitted to Veterans Health Administration hospitals in fiscal year 2013, we tested whether medication use is potentially inappropriate  or other drugs) according to Alzheimer’s Disease and Related Dementias (ADRD) status based on ICD-9 codes. We used Poisson regression to estimate the association of the number of CNS Beers Criteria drugs  and other Beers Criteria drugs according to ADRD status. The mean age of the cohort at the index hospital admission was 74.5 (SD 8.2), with 19.0% having ADRD and a Charlson comorbidity index of 3.9±3.0. After adjusting for age, sex, race/ethnicity, marital status, body mass index, 16 diagnostic criteria for pain and chronic conditions, mental health conditions and use of other system Beers Criteria drugs, we found that the mean number of CNS Beers Criteria drugs were 25% higher among Veterans with ADRD, with an adjusted risk ratio (aRR) of 1.25 . On the other hand, the mean number of other Beers Criteria drugs appeared to be 2% lower among ADRD group compared with inpatients without ADRD , p<0.004). Clinicians need to be aware of potential side effects of using CNS Beers drugs in people with ADRD and whether there are alternatives to their use.


R = 210 ± 44 N vs. 340 ± 48 N, respectively, p < 0.001, and L = 207 ± 46 N vs. 303 ± 35 N, respectively, p < 0.001). Similarly, lower MEHS was observed in U15 male skiers compared to male elite skiers for both limbs . Correlations between MEHS and chronological age were modestly significant only for the U15 group (r = 0.37 and p < 0.001). When the correlations for the U15 group were performed between MHES and maturity offset , statistical significance was reached by all the correlations run for 3 variables . This cross-sectional description of MEHS in alpine skiers from youth to elite level highlights the importance of biological maturation for MEHS values in youth athletes and presents novel data that may offer insights into new approaches for injury prevention.Competitive alpine skiers are subject to substantial risks of injury, especially concerning the anterior cruciate ligament (ACL). During “landing back weighted” episodes, hamstrings may partially counteract the anterior shear force acting on the tibia by eccentrically resisting the boot-induced drawer of the tibia relative to the femur. The aim of the present study was to provide novel descriptive data and sport-specific reference values on maximal eccentric hamstrings strength (MEHS) in competitive alpine skiers from youth to elite level, and to explore potential relationships with sex, age and biological maturation. 170 competitive alpine skiers were investigated: 139 youth athletes  and 31 elite athletes . MEHS was assessed by the . U15 female skiers presented lower MEHS compared to female elite skiers for both limbs ( Competitive alpine skiers are known to be subject to substantial risks of injury . AlthougPhysical aspects of the athlete have been suggested to be among the top 5 key injury risk factors in alpine ski racing  and fitnConsidering that both quadriceps and hamstrings muscle groups are significantly activated during jump landings , it is rEccentric muscle actions are an inherent part of skiing ,b, and sAccordingly, the sub-goals of the present study were: (1) to screen two distinct populations of competitive alpine skiers (including youth athletes and elite athletes) by assessing MEHS during Nordic Hamstrings Exercise (NHE), which has extensively been used in different sports such as Australian football, rugby, soccer and sprinting ; (2) to In total 170 competitive alpine skiers participated in the study: 139 U15 youth athletes  and 31 adult athletes . The maximal eccentric hamstring strength was assessed by using a NHE measurement device ; its reliability and application on athlete populations is reported in several previous studies . BrieflySeca, Hamburg, Germany), body height and sitting height , as well as chronological age at the time of measurement and sub-ischial leg length as the difference between body height and sitting height. Based on the collected data the prediction of an individual maturity offset is enabled, which marks a point in time before or after peak height velocity (PHV). The estimated APHV is given by subtracting the maturity offset from the actual chronological age  and coefficient of determination (2r). The level of significance was set at p < 0.05.Data were reported as mean ± SD. Differences between groups were statistically analyzed for MEHS values using an unpaired Student’s n = 51) presented a significantly lower eccentric hamstrings strength compared to female elite skiers (n = 19) for both right and left limbs . Similarly, N = 88) compared to male elite skiers (n = 12) for both limbs . Male skiers always presented significantly higher values of eccentric hamstrings strength irrespective of limb, age, or sportive level compared to female skiers (p < 0.001).A total of 170 competitive alpine skiers performed a maximal NHE-test. A comprehensive snapshot of the differences in strength between females and males for different limbs and at different age/sportive level is presented in p = 0.45); in a very similar fashion, U15 male skiers showed no significant differences of asymmetry for eccentric hamstrings strength production when compared to male elite skiers . However, it is worth mentioning that compared to elite skiers, U15 skiers showed a higher variance in the asymmetry values observed.Data for asymmetry of force production between right and left limb during NHE (difference between stronger and weaker leg expressed as percentage), are presented in r and r2-values, together with statistical significance, are shown in Correlations between MEHS and chronological age are presented in r = 0.30, r2 = 0.09, p = 0.1) nor for female and male groups . Conversely, the correlations for the U15 group were observed to be statistically significant when the data were grouped  and when the data were expressed by sex . When the correlations for the U15 group were performed between MEHS and maturity offset .Pearson’s correlation between MEHS and chronological age did not reach any statistical significance in the elite group, neither for the grouped data . Accordingly, a relative MEHS value-based ranking among the skiers is apparently different than an absolute value-based ranking . However, when strength was correlated to maturation offset for the latter group, this association showed moderate to strong linear relationships in a gender dependent manner . (3) In the U15 athletes, a body weight normalization of the MEHS values removes any relations to maturity offset.The present investigation aimed to provide a cross-sectional observation of MEHS values in 170 competitive skiers (139 U15 athletes vs. 31 elite athletes). The main findings were the following: (1) greater MEHS during NHE was observed by the elite skiers compared to the U15 group and greater strength was developed by male compared to female skiers for both groups. (2) While no correlation was found between strength and chronological age in elite skiers, a weak to moderate association was found in the U15 group (n = 159) (n = 158) (n = 105)  , and forn = 158) , and forn = 105) . It mustn = 105) , a priorF = 11.91 ± 8.3% vs. 10.46 ± 1.47%, respectively; M = 9.88 ± 7.67% vs. 11.31 ± 1.89%, respectively)  for force developed during NHE was similar between the U15 and Elite groups and between sexes (ctively) . The prectively) . MoreoveAn indirect  justification of why MEHS may be of importance for the purpose of ACL injury prevention in alpine ski racing can be found in the following theoretical considerations. During typical ACL-injury mechanisms, such as the “landing back weighted” mechanism, hamstring muscles may functionally counteract the anterior shear force acting on the tibia . This hypothesis is further supported by the simulation study findings of , the facHowever, irrespective of these future aims, it is important to know about sport-specific reference values, potential age/maturity related influences and asymmetry problems in the corresponding populations, as being explored in the current study. Such information is essential for the interpretation of forthcoming longitudinal studies, and to better understand how to implement MEHS related prevention strategies effectively.It was no surprise that elite skiers (ranging from 17 to 28 years old) showed greater MEHS compared to the younger cohort (ranging from 12 to 15 years old): however, we further aimed to clarify if such discrepancy was just due to the age difference or to the fact that the two groups belonged to two distinct sportive levels –5. MEHS r = 0.37) and when divided for sex . However, these correlations presented very low r2-values , thus explaining, in the best of the cases , only up to 16% of the variability of strength vs. age. Accordingly, we decided to investigate the relationship of eccentric hamstrings strength for female and male skiers in function of maturity offset . Interestingly, these relationships resulted in higher r2-values (r2 = 0.35) and after (r2 = 0.49) peak height velocity, while females showed and r2-value of 0.22 after peak height velocity. It must be specified that all of the female subjects have already reached their peak height velocity, so we could not present any relationship between maximal eccentric strength and maturity offset in the months/years before 0 value . This is due to the fact that in this and other studies females reached their peak height velocity earlier than males (possibly around 11–12 years old)  ,b.The male subjects who already reached their peak height velocity were the ones that presented the higher absolute values for MEHS: while this was an expected finding, it is worth highlighting that few athletes within a year before PHV showed similar, if not greater, values of MEHS compared to other skiers which already passed PHV. In our opinion, these observations could be potentially regarded as selection criteria for either successive injury risk or general athletic performance, as young skiers that present such values of MEHS before complete maturation may start from a better overall condition compared to their peers, in a future perspective.The aforementioned correlations between biological maturation and MEHS suggest that in younger cohorts is important to consider if an athlete has already reached her/his peak height velocity point, in order to better interpret force values in key of injury prevention and performance. In fact, the lower force values observed in the U15 male skiers’ group were identified for athletes that were between 2 and 1 years before peak height velocity: if one would have considered just strength and/or chronological age for such subjects, potential misinterpretations could have been made for conclusions on risks of injury and/or selection criteria for high performance paths. One potential solution to better address the influence of biological maturation on MEHS in future might be found in a normalization with body weight . On the current study population such a normalization removed any relations between maturity offset and MEHS . HoweverThis study aimed on a cross-sectional description of MEHS in competitive alpine skiers from youth to elite level. It may provide reference values and background knowledge for the interpretation/implementation of future ACL-injury prevention and athletic conditioning studies/interventions in the sport of alpine ski racing. Moreover, it highlighted the importance of considering biological maturation for meaningful interpretations of force values of youth athletes that are close to their growth spurts. Future investigations of MEHS in the context of ACL-injury prevention and/or athletic conditioning should focus on longitudinal observations of the same athletes during their sportive career. More integrative approaches should be implemented, such as combining muscle function testing with ultrasound-based assessment of hamstrings muscle mechanical behavior, its architectural adaptations to longitudinal training and the investigation of potential underlying molecular mechanisms.JS and WF conceptualized the study. JS, LE, and MJ conducted the data collection. MF and LE contributed to the analysis and interpretation of the data. MF, LE, and JS drafted the manuscript. All other authors revised it critically, and approved the final version and agreed to be accountable for all aspects of this work.MJ and BB were professionally affiliated with Swiss-Ski. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sida hermaphrodita), is yet another potential substitute for alfalfa in animal diets. The aim of this study was to evaluate the efficacy of dehydrated Virginia fanpetals meal as a substitute for dehydrated alfalfa meal in rabbit diets. The results of this study indicated that Virginia fanpetals meal can be included in rabbit diets at up to 20% as a substitute for alfalfa without compromising the production performance of animals, nutrient digestibility, nitrogen retention, carcass quality, or meat quality parameters.Alfalfa is an important forage crop in rabbit nutrition. Despite the numerous advantages of alfalfa, efforts have been made to find its potential substitutes. Virginia fanpetals (Medicago sativa) is an important forage crop in rabbit nutrition. Despite the numerous advantages of alfalfa, efforts have been made to find its potential substitutes. The aim of this study was to evaluate the efficacy of Sida hermaphrodita meal as a substitute for alfalfa meal in rabbit diets. The experiment was performed on 90 New Zealand White rabbits divided into three groups. DA group was fed a diet containing 20% dehydrated alfalfa. In the DA/DS group, rabbits received a diet containing 10% dehydrated alfalfa and 10% dehydrated Sida. The diet administered to the DS group contained 20% dehydrated Sida. The results of this study indicate that the dietary supplementation with Sida contributed to an increase in the final body weight of rabbits and improved the feed-conversion ratio. Experimental diets had no influence on nutrient digestibility, nitrogen retention, and selected carcass characteristics of rabbits, except for the proportion of the hind part. The content of dry matter, total protein, and monounsaturated fatty acids in the hind leg muscles of rabbits was higher in the DA group than in the experimental groups. The concentrations of saturated fatty acids were higher in the tissues of animals fed diets supplemented with Sida.Alfalfa ( Medicago sativa) is an important forage crop in rabbit nutrition. Both alfalfa hay and dehydrated alfalfa meal are used as ingredients of complete diets. Alfalfa is a rich source of fiber and protein in rabbit diets [Amaranthus hypochondriachus), bambara groundnut (Vigna subterranean), perennial peanut (Arachis glabrata), artichoke leaves (Cynara cardunculus), bermudagrass (Cynodon dactylon), berseem hay , birdsfoot trefoil (Lotus corniculatus), pigeon pea leaves (Cajanus cajan), kudzu (Pueraria spp.), orchard grass (Dactylis glomerata), water hyacinth (Eichhornia crassipes), moringa leaves (Moringa oleifera), and many other [Alfalfa , a member of the Mallow  family native to North America, is yet another potential substitute for alfalfa in animal diets. Virginia fanpetals has many advantages. Due to its specific biochemical composition, Sida in the green stage can be used as a highly nutritious feed with a protein content of up to 30%. When harvested in the dry stage, the species has various applications in the bioenergy, insulation, and cellulose-paper industries. When harvested in the green stage, it can be used in the pharmaceutical, animal feed, honey, and biogas industries. Sida is also a profitable energy crop considered a future feedstock for biofuel production [Virginia fanpetals  meal in diets for growing meat-type rabbits.The objective of this study was to evaluate the efficacy of dehydrated The animal protocol and the number of animals used in this study were consistent with regulations of the Local Institutional Animal Care and Use Committee , and the study was carried out in accordance with EU Directive 2010/63/EU on the protection of animals used for scientific purposes .The experimental animals were 90 New Zealand White rabbits . The rabbits were randomly allocated to three groups. When the experiment began, the animals were 35 days old and had average body weight (BW) of 908.97 ± 104.11 g (mean ± SD). The animals were 90 days old when the experiment ended. The experiment was performed between September and November, in a separate facility on a rabbit farm. All rabbits were kept in wire-mesh flat-deck cages measuring 0.5 × 0.6 × 0.4 m . Rabbits had ad libitum access to feed served once a day via automatic feeders and water from nipple drinkers. The animals were housed under standard conditions with a temperature of 16–18 °C, relative air humidity of 60–75%, forced room ventilation, and a controlled photoperiod (12 h light with intensity of 25 lx and 12 h dark).Control group (DA) rabbits were fed a diet containing 20% dehydrated alfalfa meal. In the first experimental group (DA/DS), rabbits received a diet containing 10% dehydrated alfalfa and 10% dehydrated Sida meal (DS). The diet administered to the second experimental group (DS) contained 20% dehydrated Sida meal. The ingredients of diets are presented in 80 P20K60 kg·ha−1. The plants were cut before budding, at a height of 30 cm, they were chopped with an electric chaff cutter and dried at a temperature of 60 °C in BINDER dryers. After 24 h, dried biomass was ground in a mill  to a 2-mm particle size. The feed was mixed in the Zuptor 300 horizontal feed mixer  and formulated in the MAGNUS 100 compact feed pelletizer (Ożarów Poland) with a matrix diameter of 100 mm and a pellet diameter of 6 mm.All diets were isonitrogenous and their nutritional value corresponded to the requirements of growing meat-type rabbits . DehydraDuring the experiment, the rabbits were weighed on an electronic scale within an accuracy of 1 g, and their BW were determined at the beginning of the experiment (35 days of age) and at the end of the experiment (90 days of age). Average daily weight gains, total feed intake and the feed conversion ratio (FCR) were calculated.A digestion and balance trial was performed on 10 rabbits  selected randomly from each group. The animals were 45 days old when the digestion and balance trial began, and 65 days old when the trial ended. They were placed individually in metabolic cages fitted with a system for quantitative collection of feces and urine. During the 10-day adaptation period that preceded the experiment proper, the rabbits were allowed to adapt to the new environment and feed. Pelleted feed in the amount of 150 g was served once a day at 10:00. The rabbits had free access to drinking water. Non-ingested feed residues and feces were collected every day and weighed within an accuracy of 1 g. The collected feces were frozen, and feces and feed samples were dried and ground. Urine was preserved with 20% sulfuric acid to calculate the total volume of the collected urine at the end of the experiment. The chemical composition and energy value of feed samples were determined.The balance method used in studies of the type supports the calculation of nutrient and energy digestibility coefficients (DC) and nitrogen (N) retention. Nutrient digestibility was calculated from the following formula: DC = (a − b)/a × 100%, where a is the nutrient content of feed, and b is the nutrient content of feces.At the end of the feeding trial, the animals were fasted for 24 h and sacrificed according to the standard guidelines for euthanizing experimental animals. The carcasses were skinned and eviscerated. The head was dissected along the occipital joint, the forepart was dissected between the 7th and 8th thoracic vertebrae, and the loin was dissected between the 6th and 7th lumbar vertebrae. The hind part with the perisacral area and hind legs was the remaining part of the carcass after dissection . The fol® C2000 basic, Germany, Staufen). To determine fatty acid composition, all fat samples were methylated by the modified Peisker method [3 of a methanol:chloroform:concentrated sulfuric acid mixture, 100:100:1 v/v, was added to ca. 150 μL fat, thermostat −80 °C, 3 h), and fatty acid methyl esters were obtained. Fatty acids were separated and determined by gas chromatography: VARIAN CP–3800 gas chromatograph-Netherlands, flame-ionization detector (FID), capillary column , split injector, split ratio 50:1, 1 μL sample, detector temperature −250 °C, injector temperature −225 °C, column temperature −200 °C, carrier gas—helium, flow rate −1.2 cm3/min. Fatty acids were identified by comparing the retention times of individual fatty acid methyl ester standards  and the retention times of peaks in the analyzed samples. The relative content of each fatty acid was expressed as a percent of the total peak area of all fatty acids in the sample. The concentrations of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) were calculated.Hind leg muscles were collected from 5 males and 5 females from each group for analyses of the chemical composition and fatty acid profile of meat after 24 h of chilling at +4 °C. Feed and feces samples were also subjected to chemical analyses. The content of dry matter, crude ash, total protein, ether extract, acid detergent fiber (ADF), and acid detergent lignin (ADL) was determined by standard methods . Neutralr method  . The results were analyzed statistically by one-way analysis of variance (ANOVA), and the significance of differences among groups was determined by Duncan’s multiple range test at software .The initial and final BW of rabbits from the control group and experimental groups did not differ significantly . HoweverNutrient digestibility  was simiSelected carcass characteristics, presented in The content of dry matter and total protein in hind leg muscles was significantly higher in the control group than in the experimental groups , whereas14:0 and C16:0 was higher in group DS than in group DA , the content of C18:0 was higher in groups DA/DS and DS than in group DA, and the content of C16:1, C17:1 and C18:1 was higher in group DA (control) than in the experimental groups. No significant differences in PUFA levels were found between groups.The fatty acid profiles of hind leg muscles are shown in Mortality or serious diseases symptoms that could affect the results of this study were not observed during the experiment.Sida hermaphrodita in livestock nutrition, and there appears to be no published information on rabbits. Therefore, it was difficult to compare the results of this study with the findings of other authors. In general, fresh and dehydrated alfalfa is regarded as a valuable feed ingredient for rabbits [Sida hermaphrodita, similarly to other species of the genus Sida , Sida acuta, Sida cordifolia, Sida spinosa, Sida rhombifolia, and Sida veronicaefolia, has health-promoting properties which positively affect the health status and, indirectly, productivity of animals [Malvaceae, Sida acuta, revealed that experimental diets had no adverse effects on performance or overall health in rabbits [The performance parameters of rabbits from all groups  were wit rabbits ,34,35,36 rabbits ,38. In t animals . However rabbits ,24,25.Sida hermaphrodita on nutrient digestibility in rabbits or other animal species has not been examined to date, except for a study investigating gilts, which revealed an increase in protein and fiber digestibility with increasing levels of Sida meal (up to 10%) in the ration [The values of nutrient digestibility coefficients and nitrogen retention, calculated in this experiment, are typical of growing broiler rabbits ,32. Despe ration . Howevere ration , alfalfae ration , who anaThe lower average weights of carcass and valuable cuts in group DA resulted from lower slaughter weight of rabbits . The valThe proximate chemical composition of meat  was typiThe results of numerous studies show that feeding high levels of alfalfa meal has a positive effect on the quality of rabbit meat. It may increase the concentrations of total essential amino acids, taste-related amino acids, and alpha-linolenic acid, thus improving the nutritional and organoleptic quality of meat ,42. CaprSida hermaphrodita can be included in rabbit diets at up to 20% as a substitute for alfalfa without compromising the production performance of animals, nutrient digestibility, nitrogen retention, carcass quality, or meat quality parameters.The results of this study indicate that


Ecological significance of trees growing in urban and peri-urban settings is likely to increase in future land-use regimes, calling for better understanding of their role as potential reservoirs or stepping stones for associated biodiversity. We studied the diversity of fungal endophytes in woody tissues of asymptomatic even aged pedunculate oak trees, growing as amenity trees in a peri-urban setting. The trees were classified into three groups according to their phenotypic vitality . Endophytes were cultured on potato dextrose media from surface sterilized twigs and DNA sequencing was performed to reveal the taxonomic identity of the morphotypes. In xylem tissues, the frequency and diversity of endophytes was highest in oak trees showing reduced vitality. This difference was not found for bark samples, in which the endophyte infections were more frequent and communities more diverse than in xylem. In general, most taxa were shared across the samples with few morphotypes being recovered in unique samples. Leaf phenolic profiles were found to accurately classify the trees according to their phenotypic vitality. Our results confirm that xylem is more selective substrate for endophytes than bark and that endophyte assemblages in xylem are correlated to the degree of host vitality. Thus, high vitality of trees may be associated with reduced habitat quality to wood-associated endophytes. Trees growing in urban and peri-urban settings have high amenity value and support a variety of ecological and environmental benefits, including improvement of air quality and catchment of stormwater runoff . Urban aA functionally significant part of tree-associated biodiversity is made up by the internal microfungi, called endophytes. These fungi colonize all tissues of trees, and generally remain asymptomatic. As a part of the “ecosystem microbiome”  they conUlmus spp.) that were differentially susceptible to Ophiostoma novo-ulmi, a vascular pathogen causing Dutch elm disease (DED). They found less diverse and frequent infections by culturable endophytic fungi in xylem of elms with low susceptibility to DED, as compared to xylem of elms showing high DED susceptibility. Moreover, xylem phenolic composition differed between trees in different susceptibility classes, suggesting that defensive responses could explain the observed pattern in endophyte assemblages , a common tree species both in woodlands and green spaces, with relatively well characterized endophyte communities . The seedlings were planted in six groups of eight trees for a total of 48 trees at 4 m × 4 m spacing. Given the close proximity of trees to their neighbors, effects of surrounding vegetation, precipitation, temperature and soil conditions were expected to be rather similar, as well as their exposure to ambient inoculum.Endophyte diversity was studied in 15–year-old Figure 1). Low vitality was not found to be associated with signs of specific pathogen or pest attacks. To investigate whether the phenotypic differences correlate with internal quality of the trees, we also analyzed phenolic compounds in oak leaves (where the metabolism of these compounds is active), as a proxy for the stress level and defensive capacity of the trees.Tree vitality was estimated on the basis of a visual assessment of the crown transparency  and the Figure 1) were selected for sample collection . From each tree, four asymptomatic twigs (approximately 30 cm in length) were collected between 1 and 2 m height, one from each of the four cardinal points. The twigs were transported in plastic bags to the laboratory where they were kept at 4°C degrees until further processing (within 24 h). From each twig, leaves were first removed and air-dried and then stored for analysis of phenolic compounds  and a 5 cm long section was cut from the previous year’s growth. The section was surface sterilized using 70% ethanol and 4% sodium hypochlorite under aseptic conditions as described by At the end of September 2014, eight trees representing each vitality class  if they shared several common macro-morphological characteristics: coloration, shape, elevation, edge, texture, presence or absence of extrolites on the surface, effect on medium, and growth rate. A colony was defined as a singleton MT if it could not be grouped with any other colony.Table 1). Plugs of pure cultures growing on MEA were transferred to Petri dishes containing malt extract broth and incubated for 2–4 weeks at room temperature in the dark. Mycelia were then filtered, placed in 50 mL Falcon tubes and lyophilized for 48 h. Samples were pulverized in a FastPrep®-24 homogenizer . Genomic DNA was extracted with E.Z.N.A. SP Plant DNA kit  following manufacturer’s instructions. DNA quantity and quality was measured using a NanoDrop® ND-1000 spectrophotometer . The internal transcribed (ITS) region of rDNA was amplified by PCR using general primers ITS1 and ITS4 , 0.4 μM of each primer, and 10 ng μL-1 of DNA. The PCR program started with denaturation at 95°C for 5 min, followed by 33 cycles of 95°C for 30 s, annealing at 57°C for 30 s, and 72°C for 30 s, followed by a final extension step at 72°C for 7 min. To check if PCR was successful, the PCR products were visualized under UV light by gel electrophoresis on 1.5% agarose gel dyed with GelRedTM. PCR products were purified with Agencourt AMPure XP  following manufacturer’s instructions. After quantification of amplified products with Qubit fluorometer 3.0 , samples were sent for Sanger sequencing at National Genomics Infrastructure (NGI) at Science for Life Laboratory .Two representative specimens of each non-singleton MTs were selected for DNA extraction, with the exception of those that had only one or no viable specimen left , and those that were identified from a previous study  (MT 1). and ITS4 . The PCRTM  software. Putative taxa were assigned by comparing the acquired sequences to those in the reference database at NCBI1 through BLASTN search. The ITS sequence homology for delimiting fungal taxa was set to >98.5% for presumed fungal species) and 94–97% for determination at the genus level. ITS sequence information was deposited in GenBank (NCBI) with accession numbers KX815458-KX815496 (Table 1).The raw sequence data were visualized with Chromas . Among the samples sequenced, one was excluded from further analyses because of contamination. All sequences were aligned and edited with SeqMan Pron = 24) and each sample was milled into a homogenous powder using an MM301 ball mill (Retsch GmbH). The extraction and HPLC analysis was done following the procedure described in g, and the clear supernatant was transferred into another vial. The procedure was repeated with another 500 μL solvent and the supernatants were combined. The solvent was evaporated to dryness in a vacuum concentrator and the extract was stored at 4°C until analysis (within 1–3 days from extraction). Before the analysis, the extracts were dissolved in 400 μL methanol:water  and passed through disposable filters (0.45 μ pore size) to remove any particles. The HPLC system consisted of a Merck Hitachi LaChrom device with a D-7100 pump, D-7200 autosampler, D-7300 column oven at 40°C, and a D-7455 DAD detector scanning the absorbance between 220 and 400 nm. Injection volume was 20 μL and separation was achieved on a 100 mm HyPurity C18 (Thermo Scientific) column using a gradient made of water  and methanol (B) as follows: 10% B (0–1 min); 10–70% B (1–20 min); 70% B (20– 23 min); 70–100% B (23–30 min); followed by flushing and equilibration to initial conditions (flow rate 0.8 mL min-1). The peak area (AU) was normalized toward dry weight of each sample (mg).To gain information about the defensive status of the trees, leaf material that was removed from the twigs was air-dried for analysis of phenolics compounds. Four healthy-looking leaves from each tree were pooled to one sample (MT)  of bark and xylem samples with at least one emerging colony. To estimate endophyte diversity, MT richness (SMT)  was calcU (W) test was used to test the difference in CF and SMT values between bark and xylem. Kruskal–Wallis (KW) test was used to determine differences in CF and SMT values among the orientation and the vitality classes  (adonis function) based on Bray-Curtis dissimilarity matrix (vegan package). Homogeneity of variance among groups was tested using permutational test of multivariate group dispersions implemented as betadisper in the vegan package  . Venn dipackage) . The varpackage) . Variabi package .® Pro 13, SAS Institute Inc., Cary, NC, United States).Nestedness (N) analysis (R package bipartite) was used to measure the structure of the morphotype system in the tissue and vitality class of pedunculate oak. The associations among morphotypes, vitality classes and tissue types were then visualized with bipartite network graphs (R package bipartite). Logistic regression analysis was then performed to predict the probability of finding a given morphotype based on tissue type, vitality class, and tissue–vitality interaction .To examine differences in phenolic profiles between the trees classified to different vitality classes, the peak area values (AU/mg) of a total of 10 UV-absorbing peaks and vitality class were used as input variables in quadratic canonical discriminant analysis (JMPTable 1). Seven MTs were not sequenced because there were no viable cultures or they did not grow on malt extract broth. All remaining MTs belonged to Ascomycota that were putatively assigned to Dothidiomycetes, Sordariomycetes, Leotiomycetes, and Eurotiomycetes and two unassigned Ascomycota classes. Of those, eight were putatively identified to species level and four to genus level. The singletons (MT30) were not sequenced (Table 1).A total of 569 fungal isolates were recovered from 768 bark and xylem samples of pedunculate oak. The emerged colonies were initially grouped into 29 MTs and one mixed group of unidentified singletons [(MT 30) which comprised 54 specimens in total]. After DNA analysis, the number of MTs was reduced to 27 since sequence similarity putatively indicated the same species for MT 6 and MT 14 as well as MT 22 and MT 23 . The CF of bark samples did not differ among the three vitality classes . In contrast, the CF of xylem samples differed among the vitality classes, being highest in low vitality trees . Dunn’s post hoc test results showed a significant difference in xylem CF between low and high vitality class (p = 0.0001) and low and medium vitality class (p = 0.0004). No significant difference was found between high and medium class (p = 0.38). Sample orientation  and tree  had no significant effect on CF  and were not considered as factors in further analyses.Bark tissue yielded more endophyte isolates than xylem ; 79% of the MTs were found in bark, whereas 21% were found in xylem (Table 1). Similarly to CF, SMT of bark samples did not differ among the vitality classes , whereas that of xylem samples did; low vitality samples yielded a higher number of MTs than medium and high vitality samples . Dunn’s post hoc test showed a significant difference between low and high vitality class (p = 0.0001) and low and medium vitality class (p = 0.0005). No significant difference was found between high and medium class (p = 0.34). Sample orientation  and tree  had no significant effect on S  and were not considered as factors in further analyses. Individual-based rarefaction curves constructed for tissue type and vitality class confirmed these results for tissue type and vitality class. Only the curve of low vitality bark samples approached the asymptote . When the singletons were removed from the analysis, the shape of all curves improved .Bark and xylem differed in the number of different MTs they hosted (Mann-Whitney p-a = 0.82), though their abundance differed (Jab = 0.34) . Based on PERMANOVA (abundance), bark and xylem fungal communities differed significantly . Most MTs recovered in bark were found in all three vitality classes, resulting in similar fungal compositions . However, according to the MTs similar abundance the differences among classes increased  , even though PERMANOVA (abundance) analysis did not provide statistically significant differences . Most MTs were found in low vitality xylem, however, the xylem communities of the three vitality classes resulted to be rather similar considering the MTs presence/absence  and MTs similar abundances  . Based on PERMANOVA (abundance), xylem-associated fungal communities did not differ between high and low vitality classes  nor between high and medium vitality class . Instead, there was a difference in fungal community composition between the medium and low vitality class .Most MTs were found in both tissue types . The web structure between tissue and vitality class was more complex in xylem of low vitality trees (N = 37.2) than in medium (N = 15.9), or high (and N = 4.5) vitality trees . The logistic regression showed that tree vitality (P > χ2 = 0.0002), tissue type (P > χ2 = 0.0001), and the interaction between vitality and tissue type (P > χ2 = 0.0029) influenced the endophytic community composition (Table 2). The presence of Pyrenochaeta cava (MT14) was positively correlated to low vitality class. The presence of some MTs was positively correlated with tissue type (Tubakia dryina and MT4 in bark) or with the combination of tissue type and vitality class  (Table 2).The MTs’ associations with tissue types and vitality classes were visualized using bipartite graphs  and kaempferol derivatives . One compound  remained unclassified . The canonical discriminant analysis of oak leaves showed a clear division among the phenolic profiles of the leaves of the vitality classes with each class separating from the other two . Among the ten dominating phenolics’ peaks, highest levels were detected in low vitality trees, while lowest levels were observed in medium vitality trees .The phenolic compounds detected in oak leaves were putatively identified as quercetin derivatives . Interestingly, this species was recently described by Carpinus betulus, which was not found in the immediate vicinity of the oaks we studied. More research would be needed to clarify the frequency of this fungus on oaks.The taxonomic diversity detected in our study resembled that described in previous studies on oak endophytic communities . The MTs studies . We recoand Asia . Tubakiaerma sp.  were notP. cava has been found to be primary associated with elms resistant to Ophiostoma novo-ulmi (A. leiphaemia, C. quercinus, T. dryina, Pezicula spp., and Phoma spp. have been reported in the literature as latent pathogens or saprotrophs (Pezicula species, despite being possible latent pathogens, can produce secondary metabolites with anti-fungal properties (Pezicula species recovered in our study caused discolouration of the MEA suggesting that these species might be actively producing extracellular secondary metabolites in their environment. Despite some endophytes are known to positively affect their host, it is also known that fungal endophytes are ubiquitous fungi whose lifestyle might change under certain circumstances (Because trees are long-living organisms and are subjected to multiple endophytic infections along their life span, it has been difficult to address how endophytes might influence tree characteristics, i.e., the phenotypic vitality. Moreover, it has also been difficult to understand the functions of endophytes, most of which are still unknown. Some of the identified morphotypes may contribute to the health of the oaks as antagonist to pathogenic fungi . For insovo-ulmi . Among trotrophs . Peziculoperties . The twomstances . The funmstances . It is tmstances . This memstances .Because all trees sampled in our study were planted at the same time in the same, limited area, we can assume that all trees were exposed to a similar inoculum pressure of horizontally spreading endophytes . Thus, wIn conclusion, our results suggest that the xylem in trees with decreased vitality is a good substrate for endophytic diversity. The role of dead wood as a supporter for fungal diversity is well established , but ourThe raw data supporting the conclusions of this manuscript will be made available by authors, without undue reservation, to any qualified researcher.MA and JW designed the experiments and conducted the sampling. MA conducted the laboratory work. MA, JM, BA, and JW analyzed the data. MA wrote the first draft of the manuscript. JW wrote sections of the manuscript. MC, JM, and BA provided valuable comments on the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.