Human studies employing intracerebral and transcranial perturbations suggest that the input-output properties of cortical circuits are dramatically affected during sleep in healthy subjects as well as in awake patients with multifocal and focal brain injury. In all these conditions, cortical circuits react to direct stimulation with an initial activation followed by suppression of activity (Off-period) that disrupts the build-up of sustained causal interactions typically observed in healthy wakefulness. The transition to this stereotypical response has important clinical implications, being associated with loss of consciousness or loss of functions. Here, we provide a mechanistic explanation of these findings by means of simulations of a cortical-like module endowed with activity-dependent adaptation and mean-field theory. First, we show that fundamental aspects of the local responses elicited in humans by direct cortical stimulation can be replicated by systematically varying the relationships between adaptation strength and excitation level in the network. Then, we reveal a region in the adaptation-excitation parameter space of crucial relevance for both physiological and pathologic conditions, where spontaneous activity and responses to perturbation diverge in their ability to reveal Off-periods. Finally, we substantiate through simulations of connected cortical-like modules the role of adaptation mechanisms in preventing cortical neurons from engaging in reciprocal causal interactions, as suggested by empirical studies. These modeling results provide a general theoretical framework and a mechanistic interpretation for a body of neurophysiological measurements that bears critical relevance for physiological states as well as for the assessment and rehabilitation of brain-injured patients. Suppression of cortical activity following an initial activation is a defining feature of deep sleep in healthy subjects and wakefulness in patients affected by focal and multifocal brain injuries. Experimental findings suggest that these bimodal responses disrupt the emergence of complex interactions among cortical regions, leading to loss of consciousness or functional impairments. Given their practical implications, studying the mechanisms involved within a general theoretical framework is essential. Using a neuronal network model, we provide evidence for the key role of activity-dependent adaptation mechanisms in shaping the responses to perturbation and affecting the build-up of complex cortical interactions. Overall, this work provides a mechanistic interpretation relevant to the stratification, follow-up, and rehabilitation of brain-injured patients.Studies combining transcranial magnetic stimulation (TMS) with electroencephalographic (EEG) recordings have demonstrated a dramatic impairment of the capacity of cortical networks to engage in large-scale interactions during sleep and anesthesia : while dCrucially, this dramatic change in the input-output properties of cortical circuits is not only typical of physiological sleep and anesthesia but can also occur during wakefulness in pathologic conditions. Accumulating evidence shows that cortical circuits react to perturbations with an Off-period also in awake unresponsive wakefulness syndrome patients (UWS) , previouGiven the clinical relevance of these findings, it is crucial to understand the nature of evoked Off-periods and their impact on cortical responsiveness within a mechanistic framework. An interesting hypothesis is that adaptation mechanisms play an important role in generating the Off-periods observed after cortical stimulation. Activity-dependent adaptation accounts for local fatigue mechanisms, i.e., self-inhibition, which lowers the firing rate of neuronal populations. At the level of cortical circuits, adaptation can be induced by several microscopic biophysical mechanisms involving calcium-dependent potassium channels, short-term synaptic depression  and/or Gin vivo.Here, we ask whether activity-dependent adaptation can reproduce and explain the fundamental features of the alteration of cortical responsiveness observed empirically during physiological sleep and during wakefulness in pathologic conditions. We address this question within a general formal framework resting on bifurcation analysis and in-silico simulations of cortical modules endowed with activity-dependent adaptation . We showThe neuronal network model is adapted from , which hvolution . Reset psmission . Externa10.1523/ENEURO.0435-22.2023.f1-1Extended Data Figure 1-1A, C, As in C, the differences between the probability of evoked Off-periods and spontaneous Off-periods for Cases 1–3 are as follows: Case 1: 0.08; Case 2: 0.46; Case 3: 0.14. B, Spontaneous and stimulus-evoked signals (using the same color coding as in A) for a fixed level of adaptation. Left column, Time series encompassing both spontaneous activity (up to 6000 time steps) and stimulus-evoked activity . Black triangles indicate the occurrence of the stimulation. Central column, Spontaneous activity as a function of fatigue. Right column, Three superimposed orbits due to perturbations as a function of the fatigue. Download Figure 1-1, TIF file.Bifurcation analysis of the population rate model and dynamical regimes of the spiking neuron network as in  for a fiThe second equation in pression  and slowpression  are knowWe also performed simulations of a network made of two identical modules. Each module is made by E-neurons and I-neurons described by https://github.com/annacatt/Adaptation_and_cortical_responses and available as Simulations of the spiking neuron network were performed with an open-source program relying on an event-driven numerical integration described in . To run Under mean-field approximations requiring many presynaptic contacts ates να , where  + Φ(να) . Here, tardi one :Φ current  whose fogenvalue . This fiilibrium . Here, oFinally, because of the breaking of the diffusion approximation valid only in the limit otential . Howeverotential , and thielies on .N trials . The external stimulus in each trial was delivered at time t = 0, with a prestimulus interval lasting randomly between 5000 and 5300 ms and poststimulus interval lasting 5000 ms. Stimulation artifact was reduced by applying a Tukey-windowed median filtering, as in . We used the n EEGLAB  to detecMethods and results related to human data are presented in published articles  cited thhttps://github.com/annacatt/Adaptation_and_cortical_responses and is available as The code created for this paper is freely available online at https://figshare.com/articles/dataset/Simulated_data/21112603.All the simulated data that support the findings of this study are available online at We employ a spiking network model to investigate to what extent varying the strength of adaptation and excitation can explain fundamental dynamics of cortical responsiveness empirically observed in healthy humans and brain-injured patients. Specifically, we aim to replicate and provide a mechanistic explanation of experimental data encompassing TMS-EEG recordings in healthy subjects during NREM sleep stage 2 (N2) , in UWS Activity-dependent adaptation and excitation level shape the ongoing and stimulus-evoked activity of the simulated cortical module . To fullTo check the effectiveness of this bifurcation diagram, we characterized the spontaneous dynamical regimes of equivalent networks of spiking (adaptive leaky integrate-and-fire) neurons in Crucially, we further characterized the dynamical regimes of the spiking neuron model  by exploIn Case 1, the stimulus-evoked activity and spontaneous activity do not show relevant dissociations, as both are characterized by the absence of Off-periods, as empirically observed during wakefulness in human data. Higher adaptation levels, corresponding to Cases 3–5, are all characterized by the presence of Off-periods in both the spontaneous and evoked activity. The overall time the system spends in the Off-periods increases with the adaptation level: (1) Case 3 shows Off-periods interspersed with up states, thus resembling NREM sleep stage 3 (N3); (2) Case 4 displays regular alternation between On-periods and Off-periods, thus mirroring the effect of anesthesia on brain activity; (3) Case 5 reminds of burst suppression characterized by prolonged Off-periods. Crucially, Case 2 in Considering its prevalence in real data across different physiological and pathologic conditions, we asked whether such dissociation between spontaneous and stimulus-evoked activity is a peculiar feature of the specific level of excitation, or whether it can be generalized to a large interval of excitation levels. Given the shallow slope of the lower branch of the Hopf bifurcation, changes in adaptation are expected to have more profound effects on cortical bistability than shifts in the excitation level. However, substantial changes in excitation levels for a given adaptation  still result in changes in spontaneous and stimulus-evoked dynamics similar to those reported in Empirical studies employing perturbations in humans , rodentsIn real-life experiments, the impact of the Off-period on the ability of cortical circuits to sustain causal interactions is assessed by quantifying the duration of the deterministic effects of the perturbation in the phase domain. A key empirical finding is that Off-periods not only disrupt deterministic interaction because of the associated suppression of power but that they also scramble the subsequent phase of the signal when neurons resume firing. Such stochastic reboot is characterized by oscillations with high power but low phase-locking or intertrial coherence following the Off-period. We thus performed the same analysis in the model by computing the intertrial coherence (ITC) averaged across frequencies between 5 and 30 Hz. As shown in By exploiting a spiking network model endowed with activity-dependent adaptation, the present work provides a principled mechanistic explanation for empirical results revealing common alterations of cortical responsiveness in sleeping healthy subjects, and in awake patients with multifocal and focal brain injuries. Specifically, (1) we link changes in cortical reactivity to the presence of underlying activity-dependent adaptation mechanisms, above and beyond spontaneous dynamics, (2) we explain the role of perturbations in revealing adaptation mechanisms, and (3) we expound on the impact of adaptation in disrupting causal interactions among cortical modules.The first implication of this work is that systematically varying the relationships between excitation and adaptation levels within a formal bifurcation analysis reproduces basic patterns of cortical reactivity to direct perturbations observed in humans. As shown in In this framework, the Off-periods observed after cortical stimulation in sleeping humans can be ascribed to adaptation mechanisms because of slow negative  feedback produced by calcium-gated and sodium-gated potassium currents , which aInterpreting the empirical results obtained in brain-injured and stroke patients within the theoretical framework of the bifurcation diagram discloses a more complex and interesting landscape. Indeed, brain lesions can cause global or local alterations of adaptation and excitation through different mechanisms, even in the context of preserved arousal, as assessed by eyes opening. For example, in some UWS patients, lesions, compressions, or displacements of brainstem activating systems as well as a critical load of damage to ascending fibers in subcortical white matter may enhance potassium currents , which cThe second implication of this work lies in the dissociation between observational and perturbational approaches in their ability to unveil the presence of cortical bistability and Off-periods, and bears relevance for the assessment of the aftermath of brain injury. Such dissociation is represented by the area in the excitation-adaptation bifurcation diagram identified in The third result of this theoretical and computational work is that altered levels of adaptation can have profound effects on the capability of cortical neurons to engage in reciprocal interactions, as indicated by the complex set of effects observed when changing adaptation in two reciprocally connected cortical modules without modulating their connectivity strength. Under conditions of low adaptation, the two simulated modules engage in a long-lasting series of feed-forward and feed-back interactions leading to multiple waves of activity time-locked to the stimulus, resembling the general pattern of responsiveness found in cortical slices under carbachol and norepinephrine  and in hClearly, the present theoretical framework only represents a first stepping stone on which more realistic and complex models can be built, such as those incorporating the topological organization of cortical networks . To be cSeveral computational models have been proposed in the last years to describe in detail the effect of electrical stimulations on brain circuits . Despite10.1523/ENEURO.0435-22.2023.ed1Extended Data 1https://github.com/mauriziomattia/Perseus. Also, a Python code entitled “SpikingNeuronNetwork_nest.py” allows the simulation of the spiking neuron network presented in this work using the NEST. This Python code provides an explicit and detailed implementation of the spiking neuron network we modeled. The second part is made of two MATLAB scripts entitled “main_1Module.m” and “main_2Modules.m.” These scripts were used to present and analyze the firing rate time series of the single-module and two-module spiking neuron networks having specific excitation and adaptation levels. Download Extended Data 1, ZIP file.Code accessibility statement. The included computer code is in two parts. First is a MATLAB script entitled “SpikingNeuronNetwork_Perseo.m” to simulate our networks through Perseo, which is open-source software available at


BACK app supports tailored and evidence-based self-management of nonspecific LBP. In a recent randomized controlled trial, we showed that individuals who received the selfBACK app in addition to usual care had lower LBP-related disability at the 3-month follow-up compared to those who received usual care only. This effect was sustained at 6 and 9 months.Clinical guidelines for nonspecific low back pain (LBP) recommend self-management tailored to individual needs and capabilities as a first-line treatment. Mobile health solutions are a promising method for delivering tailored self-management interventions to patients with nonspecific LBP. However, it is not clear if the effectiveness of such self-management interventions depends on patients’ initial pain characteristics. High pain intensity and long-term symptoms of LBP have been associated with an unfavorable prognosis, and current best evidence indicates that long-term LBP (lasting more than 3 months) requires a more extensive treatment approach compared to more acute LBP. The artificial intelligence–based selfThis study aims to explore if the baseline duration and intensity of LBP influence the effectiveness of the selfBACK intervention in a secondary analysis of the selfBACK randomized controlled trial., we estimated the difference in treatment effect between the strata at the 3- and 9-month follow-ups with a 95% CI.In the selfBACK trial, 461 adults (18 years or older) who sought care for nonspecific LBP in primary care or at an outpatient spine clinic were randomized to receive the selfBACK intervention adjunct to usual care (n=232) or usual care alone (n=229). In this secondary analysis, the participants were stratified according to the duration of the current LBP episode at baseline (≤12 weeks vs >12 weeks) or baseline LBP intensity (≤5 points vs >5 points) measured by a 0-10 numeric rating scale. The outcomes were LBP-related disability measured by the Roland-Morris Disability Questionnaire , average LBP intensity, pain self-efficacy, and global perceived effect. To assess whether the duration and intensity of LBP influenced the effect of selfBACKOverall, there was no difference in effect for patients with different durations or intensities of LBP at either the 3- or 9-month follow-ups. However, there was suggestive evidence that the effect of the selfBACK intervention on LBP-related disability at the 3-month follow-up was largely confined to people with the highest versus the lowest LBP intensity , but this was not sustained at the 9-month follow-up.The results suggest that the intensity and duration of LBP have negligible influence on the effectiveness of the selfBACK intervention on LBP-related disability, average LBP intensity, pain self-efficacy, and global perceived effect.https://clinicaltrials.gov/study/NCT03798288ClinicalTrials.gov NTC03798288; RR2-10.2196/14720 Low back pain (LBP) is a common reason for primary care visits globally ,2, and mPrevious studies have shown that high LBP intensity and long-term symptoms are associated with less favorable prognosis and poorer outcomes among patients in a primary care setting -14. MoreThis secondary analysis is based on data from the selfBACK multicenter RCT with two parallel arms . The trial investigated the effectiveness of evidence-based and individually tailored self-management support delivered via the selfBACK mobile app as an adjunct to usual care for adults with nonspecific LBP . The metWe recruited adults (18 years or older) with nonspecific LBP who had consulted a primary care clinician  in the Trondheim Municipality in Norway or in the region of Southern Denmark, or who had undergone a clinical examination at an outpatient spine clinic (Spine Centre of Southern Denmark). The inclusion criteria were to have experienced LBP within the preceding 8 weeks, a score of mild to moderate LBP-related disability rated as 6 points or higher on the Roland-Morris Disability Questionnaire (RMDQ), a smartphone, and access to email. The 6-point cutoff on RMDQ defines mild to moderate disability due to LBP and is considered to have the potential for a clinically meaningful improvement. The exclusion criteria were the inability to carry out the intervention , fibromyalgia, previous spinal surgery, current pregnancy, or current participation in other studies related to LBP. Participants were recruited from March 8 to December 14, 2019. After giving informed consent, the participants completed a web-based questionnaire and were then randomized to the selfBACK intervention or usual care (control group) in a 1:1 ratio using a computer-generated sequence stratified by country (Norway or Denmark) and type of care provider .The intervention was delivered as an adjunct to usual care and has been described in detail in a previous study . In brieThe outcomes were LBP-related disability measured by the RMDQ  , averageSubgroups were defined according to baseline reporting of the duration of the current LBP episode (≤12 weeks vs >12 weeks) and the average LBP intensity in the preceding week (≤5 vs >5 NRS points). The duration of LBP was assessed by the question “What is the length of time you have had LBP during this episode?” with four response options “less than 1 week,” “1-4 weeks,” “5-12 weeks,” and “more than 12 weeks,” whereas the average LBP intensity was measured by the NRS .P values. All analyses were adjusted for the two variables used to stratify the randomization  as well as the level of education , gender , and age (years). Additionally, estimates stratified by LBP duration were adjusted for baseline LBP intensity in the preceding week , whereas estimates stratified by pain intensity were adjusted for the duration of the current LBP episode measured at baseline  as well as the average baseline LBP intensity .An intention-to-treat analysis was used to estimate mean group differences with 95% CIs from constrained longitudinal data analysis using a linear mixed model ,27. In tAll analyses were performed using Stata, version 16.1 (StataCorp).All participants provided written informed consent before participation in the study; this also covered the secondary analyses performed in this study. There was no financial compensation for participants, but all participants got a ticket in a raffle for an iPad. The selfBACK RCT was approved by the Regional Committee for Medical and Health Research Ethics in Central Norway (No. 2017/923-6) and the Danish Data Protection Agency (201-57-0008) and regional ethics committee in Denmark (S-20182000-24). All study data was processed and analyzed without any personal information that could identify the participants.The flow of participants through the trial is shown in  The results stratified by LBP duration (≤12 weeks vs >12 weeks) at baseline are shown in Pinteraction=.03). These differences were not sustained at 9 months.Overall, the baseline LBP intensity was not found to influence the effectiveness of the selfBACK intervention . HoweverThe duration and intensity of LBP did not have any major influence on the effectiveness of individually tailored self-management support as delivered via the selfBACK app on LBP-related disability, LBP intensity, pain self-efficacy, and global perceived effect. For participants with high LBP intensity at baseline, there was a larger difference between the control and the selfBACK group than for those with lower pain intensity. However, the numerical differences were small and with CIs that included the null value. These findings suggest that tailored and evidence-based self-management support along with usual care can be a useful supplement to usual care regardless of the duration and intensity of LBP.Clinical practice guidelines recommend tailored self-management support as a first-line treatment for LBP regardless of symptom severity and duration ,28. To oFurthermore, the lack of heterogenic effects between the LBP duration and intensity strata may in part be explained by the individually tailored intervention. The selfBACK system provides evidence-based and personalized recommendations for self-management by using information on personal characteristics, individual goals, and symptom progression assessed by weekly follow-up questions in the app . AccordiWe also observed a small increased effect on LBP-related disability for those with high LBP intensity at baseline; however, the clinical significance of this finding is questionable . It is possible that the effect would have been larger among those with high LBP intensity and long-term LBP if the self-management intervention was combined with other components suggested by the guidelines, such as cognitive behavioral therapy . UnfortuAssessing if symptom severity such as the duration and intensity of LBP influences the effect of a given treatment is clinically relevant and may have important implications for implementing an intervention in clinical practice. Our results did not find an increased effect of the selfBACK intervention for a specific subgroup in this study, indicating that primary care clinicians should not restrict individually tailored and evidence-based self-management support to certain subgroups of patients based on the duration of the current LBP episode or level of LBP intensity.The randomized design and the repeated measures of the outcomes are important strengths of this study. However, there are some limitations that should be considered when interpreting the results. First, the participants were stratified after randomization, which may potentially create an imbalance in baseline characteristics between the subgroups. However, by assessing the means and proportions of the sociodemographic variables and LBP characteristics, they remained largely equally distributed between the selfBACK and control groups for both the LBP duration and LBP intensity strata. Second, the number of comparisons performed in this study was large due to the two stratification variables and four outcome measures, which increases the risk of chance findings. Third, the RCT was not specifically powered for subgroup analyses, resulting in somewhat imprecise estimates with wide CIs. Fourth, the choice of the cutoff value for the stratification of LBP intensity was based on a pragmatic approach . Using other cutoff values may yield different results. Finally, missing data at the 6- and 9-month follow-ups (~24% missing at both time points) is a possible source of bias. Although the analyses were conducted according to an intention-to-treat principle using a linear mixed model, the underlying and nonverifiable assumption is that data are missing at random.The baseline duration and intensity of LBP had no or minor influence on the effectiveness of tailored and evidence-based self-management support as delivered via the selfBACK app. Our results, therefore, suggest that primary care clinicians should not restrict the use of tailored and evidence-based self-management support based on the duration and intensity of LBP.


Joint-Tissue Imputation; JTI) developed to predict gene expression from genotype data. We tested for changes in the predicted expression of 17,384 protein coding genes over a time transect of 4500 years using 91 present-day and 616 ancient individuals from Britain. We identified 28 genes at seven genomic loci with significant (FDR < 0.05) changes in predicted expression levels in this time period. We compared the results from our transcriptome-wide scan to a genome-wide scan based on estimating per-SNP selection coefficients from time series data. At five previously identified loci, our approach allowed us to highlight small numbers of genes with evidence for significant shifts in expression from peaks that in some cases span tens of genes. At two novel loci (SLC44A5 and NUP85), we identify selection on gene expression not captured by scans based on genomic signatures of selection. Finally we show how classical selection statistics (iHS and SDS) can be combined with JTI models to incorporate functional information into scans that use present-day data alone. These results demonstrate the potential of this type of information to explore both the causes and consequences of natural selection.Ancient DNA can directly reveal the contribution of natural selection to human genomic variation. However, while the analysis of ancient DNA has been successful at identifying genomic signals of selection, inferring the phenotypic consequences of that selection has been more difficult. Most trait-associated variants are non-coding, so we expect that a large proportion of the phenotypic effects of selection will also act through non-coding variation. Since we cannot measure gene expression directly in ancient individuals, we used an approach ( Ancient DNA (aDNA) time-series can provide direct evidence of natural selection on specific variants, avoiding confounding factors associated with inferring selection using modern data . HoweverOne problem is that, similar to genome-wide association studies, selection signals often span multiple genes due to linkage disequilibrium (LD), making it difficult to identify the loci targeted by selection. Indeed, long haplotypes due to selective sweeps make this problem even more challenging. One approach that has been promising in the GWAS context is to try to incorporate functional information, for example expression quantitative loci (eQTL), which have been used to link significant GWAS hits to functional consequences in transcriptome-wide association studies (TWAS) . SimilarJoint Tissue Imputation (JTI); Changes in gene expression are expected to underlie many complex traits relevant to recent human evolution , particuLCT, for example) has substantially affected the expression of several nearby genes. Our work demonstrates the utility in incorporating functional information relevant to specific hypotheses into genome-wide scans for selection.This approach allows us to perform a gene-level test for selection on gene expression, identifying four novel signals of selection on gene expression resulting from small shifts in allele frequency that were not captured by genome-wide scans for selection. We are also able, for regions identified to have been under selection in this or other analyses, to identify which genes are likely to have changed their expression due to this selection—in several cases showing that selection at known loci (beagle4 (We assembled a dataset of 91 present-day and 616 ancient (4500–1000 BP) individuals from Britain. Our approach assumes that the sample population is closed and homogeneous. We thus chose this population from Britain due to its small geographical spread, relatively continuous demographic history, and large aDNA sample size. Modern individuals were from the GBR population of the 1000 Genomes project . Ancientbeagle4 . We thenbeagle4 .bmws software to estimate time varying selection coefficients based on the time series of allele frequencies, and reports P-values based on fitting a gamma distribution to the root mean squared selection coefficient, averaged in 20-SNP sliding windows. The results are largely consistent with those of LCT, DHCR7, SLC22A4, OAS1, the HLA region and other loci  for significant shifts in predicted expression  of European ancestry, compared to African ancestry. However British (GBR) LCLs show the lowest expression  and the singleton density score (SDS) . The tesMCM6, TMEM163, P4HA2, CXCR4, and SLC22A5) were significant in both the gene-level SDS and the transcriptome-wide selection scan. 13 genes that were significant in the transcriptome-wide scan were filtered out of the gene-level SDS analysis due to missing SDS scores. Based on iHS scores, 48 genes had FDR < 0.05, of which 5 were also significant in the transcriptome-wide scan . 1 gene that was significant in the transcriptome-wide scan was filtered out of the gene-level iHS analysis due to missing iHS scores.Based on SDS scores, 34 genes had significant evidence for selection (FDR < 0.05). Five of these (ρ=0.0438).Given that the SDS scan detects selection in the last ~ 2000 years, we expected it to be more correlated with the transcriptome-wide scan compared to iHS which captures the last ~ 10000 years. In terms of shared significiant signals, 5/34 is not significantly different than 5/48. However, we note that all 15 genes that achieved significance in the transcriptome-wide scan and had gene-level SDS available had the same predicted direction of change in gene expression in the SDS analysis. On the other hand, the predicted direction of changes in gene expression in the iHS analysis were uncorrelated with those predicted by the transcriptome-wide scan  with evidence for selection, of which 24 were identified by a SNP-based genome-wide selection scan on the same data. Importantly, the transcriptome-wide scan identified significant shifts in predicted expression of genes that were not captured by scans based on SNP-based genomic signatures of selection that do not incorporate functional information. Though we focused here on the application to ancient DNA data, we also demonstrated how eQTL data can be incorporated into selection scans based on present-day data, with complementary results.SLC45A2, where we find no significant genes in the transcriptome-wide scan). Nonetheless, we find several examples where the most significant gene in the transcriptome-wide scan is the targeted gene at a genome-wide scan peak. Second, the transcriptome-wide scan can identify genes (for example SLC44A5) that are not identified in the genome-wide scan because the selection is relatively polygenic. Finally, the transcriptome-wide scan can identify the effects of selection on genes that are not themselves the target of selection. For example, selection on the expression of LCT affects the expression of several other genes which may themselves have functional consequences. More generally, although the interpretation of selection scans tends to focus on a single causal gene at a locus, the transcriptome-wide scan makes it clear that the linked and co-regulated genes can be important and the phenotypic changes that selection acts on reflect the composite effect of many genes. For example, we predict that selection on lactase persistence significantly changed the expression of at least five other genes and the fitness consequences of the selected allele would depend on the aggregate effects of these changes.The results of the transcriptome-wide scan can be interpreted in multiple ways. First, where significant genes overlap with peaks from SNP-based genome-wide scans, the transcriptome-wide scan can be used to prioritize genes that are targets of selection. This is analogous to the way in which eQTL colocalization is helpful but not a complete solution to identifying causal genes at genome-wide association peaks. The most significant gene may not be the most important, or may not have a JTI model, or the target of selection may be a coding variant  Second,Overall, this study demonstrates the potential of incorporating functional predictive models in the analysis of ancient DNA to explore the phenotypic drivers and consequences of selection. Our transcriptome-wide scan for selection provides a broad overview of the regulatory shifts associated with recent human evolution in Britain, and shows how the TWAS workflow can be used to better understand the molecular basis and consequences of selection.beagle4  as those with evidence for significant changes in predicted expression.We generated gene-level selection statistics from SNP-level classical selection statistics, the integrated haplotype score (iHS) and the singleton density score (SDS) . We retrβi). SDS and iHS were not available for every SNP included in our prediction models, so we removed genes for which less than half of the SNPs had scores available. We retained a total of 13,588 genes for the SDS analysis, and 15,123 genes for the iHs analysis. As the SNP-level SDS and iHS scores were normalized, we re-normalized the gene-level selection scores by dividing by square root of the sum of the squared effect sizes:i in the model for each gene. We treated the normalized gene-level statistics as z-scores to generate p-values, to which we applied genomic control (We calculated gene-level selection statistics by taking the sum of the SDS/iHS values of each SNP included in the predictive model for each gene multiplied by its effect size on normalized gene expression ( control .Supplement 1Supplement 2


Policy development to manage new off-label uses of medications is an issue relevant to health policy stakeholders internationally. Retrospective drug utilization analyses may be useful to identify practice trends in the use of drugs for unapproved (off-label) uses. Since drug use evaluations (DUE) are generally performed for expensive medications or when safety concerns warrant increased scrutiny, patterns of off-label use of inexpensive drugs will probably be undetected. Tamoxifen citrate, an estrogen receptor antagonist, is indicated in Israel exclusively for palliative [sic: meaning adjuvant] treatment of breast cancer. This DUE was motivated by observations that tamoxifen may be used off-label for indications without evidence of safety or efficacy.To assess the extent of off-label prescribing of tamoxifen and ascertain what evidence is available supporting the use of the drug for the off-label indications observed.A retrospective DUE of tamoxifen was performed for the 12 months of calendar year 2008 in a 650,000-member HMO in Israel for patients who received at least 1 prescription for tamoxifen. All patients for whom tamoxifen was dispensed in 2008 were identified from pharmacy claims data. The HMOs electronic patient record (EPR) was subsequently queried to identify the diagnoses of patients who received tamoxifen and exclude those patients who had a diagnosis code (ICD-9-CM 174.x or 175.x) for breast cancer. An EPR chart review was also performed to identify and exclude patients who had a diagnosis or treatment of breast cancer that was recorded in free text. For the patients who did not have a diagnosis code or free-text description of breast cancer, the recorded off-label diagnoses and ICD-9-CM codes in the EPR during the visit when tamoxifen was first prescribed were tabulated. A literature search was conducted to collect information supporting the use of tamoxifen for the observed offlabel indications. We defined the use as "supported" if studies were found in PubMed, Cochrane database, or Micromedex that supported the clinical decision to use the drug for the off-label indication.877 patients were treated with tamoxifen in 2008 of whom 826 (94.2%) had a diagnosis of breast cancer, and 51 patients (5.8%) received the drug from 41 physicians in 7 medical specialty categories for 25 different off-label diagnoses. Of these 25 diagnoses, 33 patients (64.7% of 51 patients with off-label use) received tamoxifen for 8 diagnoses that were associated with some evidence to support their off-label use. Malignant neoplasm of ovary (n = 13) and female infertility (n = 13) were the most commonly recorded indications with some evidence to support off-label use. Of the 13 women treated for infertility, 9 (69.2%) had been treated with clomiphene citrate prior to being treated with tamoxifen, suggesting that some physicians may be experimenting with tamoxifen as second-line treatment.Tamoxifen was found to be used off-label in only 5.8% of the patients who received the drug in 2008 in this HMO, and only 18 patients (2.1%) received tamoxifen for a diagnosis that did not have some supporting evidence of efficacy. Since off-label prescribing of tamoxifen was found to be relatively rare, this HMO did not impose a prior authorization requirement for this drug and instead added an edit in the EPR to block off-label prescribing by requiring the physician to register a diagnosis of breast cancer.


The prevention measures undertaken due to the COVID-19 pandemic, such as lockdown and social distancing may have caused changes in the population’s lifestyle, including physical activity (PA). The current study aimed to investigate the PA level according to the perceived COVID-19 impact in youth in Luxembourg.This representative cross-sectional study was based on the Health Behaviour in School-aged Children (HBSC) Luxembourg 2022 survey. The population includes 8018 participants aged 11 to 18 years old. The PA was measured on how many days per week they practice 60 or more minutes of moderate-vigorous (MVPA) and vigorous PA (VPA). The perceived COVID-19 impact on PA was measured by the question “What impact did the Covid-19 measures have on your PA?”, with 5 answers categories ranging from very negative to very positive. A chi-square test was performed to observe the association between the perceived COVID-19 impact on PA and gender. In addition, a linear regression was performed to analyse the relation between MVPA and VPA and the perceived COVID-19 impact, adjusted by age, splited by gender.An association was found between gender and the perceived COVID-19 impact on PA. 28.2% of boys perceived a very positive impacted of COVID-19 on PA, compared to 15.5% of girls. There was a relationship between MVPA, and the perceived COVID-19 impact on PA, in both genders. Participants who perceived a positive impact of the COVID-19 on PA practiced MVPA and VPA more frequently than the others, for both boys  and girls .Findings suggest a rather positive impact of the COVID-19 on PA, especially for boys. In addition, those who perceived a positive impact practiced significantly more days of MVPA and VPA, in both gender. This study highlights adolescent’s perception of COVID-19 impacts on their PA and draw attention to the positive impact perceived and its relation to an increased PA practice. Future studies should further investigate the reasons behind the perception of a positive impact, in order to mimic the contextual factors favouring the increased practice of PA.


BOK, we focused on the identification of novel BOK circRNAs in human ovarian and prostate cancer cells. Total RNA was extracted from ovarian and prostate cancer cell lines and reversely transcribed using random hexamer primers. A series of PCR assays utilizing gene-specific divergent primers were carried out. Next, third-generation sequencing based on nanopore technology followed by extensive bioinformatics analysis led to the discovery of 23 novel circRNAs. These novel circRNAs consist of both exonic and intronic regions of the BOK gene. Interestingly, the exons that form the back-splice junction were truncated in most circRNAs, and multiple back-splice sites were found for each BOK exon. Moreover, several BOK circRNAs are predicted to sponge microRNAs with a key role in reproductive cancers, while the presence of putative open reading frames indicates their translational potential. Overall, this study suggests that distinct alternative splicing events lead to the production of novel BOK circRNAs, which could come into play in the molecular landscape and clinical investigation of ovarian and prostate cancer.CircRNAs have become a novel scientific research hotspot, and an increasing number of studies have shed light on their involvement in malignant progression. Prompted by the apparent scientific gap in circRNAs from apoptosis-related genes, such as  Cancers of the reproductive organs, including human ovarian and prostate cancer, account for a big percentage of cancer morbidity and mortality worldwide, while their incidences have gradually increased in many countries . AccordiApoptosis is a significant component of numerous physiological processes, including normal cell turnover, proper tissue development, and maintenance of organismal homeostasis . ImpairmBOK, also known as BCL-2-related ovarian killer, is an insufficiently understood member of the BCL2 family [BOK was first found in ovarian tissue. Further investigation has revealed that it is also present in other tissue types, but that reproductive tissues are where it is prominently expressed [BOK transcript variants that might contribute to its diverse functions in cancer. However, there have been no studies so far regarding the potential of BOK to produce circular transcripts, either in normal or pathological conditions.2 family . The terxpressed ,10. Althxpressed . Currentxpressed . AlternaBOK in prostate and ovarian cancer cells allows us to explore potential connections between circRNAs and apoptosis in these cancer types.CircRNAs constitute a subpopulation of non-coding RNAs, characterized by a covalent loop structure, which are produced by back-splicing . Driven For the elucidation of circRNA structure and alternative splicing events, previous studies have presented a workflow using algorithms based on short sequencing reads . HoweverBOK circRNAs deriving from alternative splicing events, in ovarian and prostate cancer cells, a rather unexplored area of scientific inquiry. In addition, this is the first study that utilized a targeted approach for the BOK gene, significantly increasing the sensitivity for circRNA identification. For this purpose, targeted third-generation sequencing with nanopore technology was implemented. Overall, this approach benefited from the advent of high-throughput sequencing and an extensive data-processing bioinformatics pipeline and led to the discovery of 23 novel BOK circRNAs in ovarian and prostate cancer cells. Besides the revelation of the full-length sequences of BOK circRNAs, their putative interactions with miRNAs and their coding potential were also examined.Based on the above, the primary focus of this research study was the identification of novel BOK circRNAs, which were submitted to GenBank of NCBI , and this way intronic regions of the gene were incorporated into its circular transcripts. Both 5′ or 3′ ends of BOK exons could be extended or truncated. We also observed that exon 2 was rarely represented in the novel circRNA sequences. In addition, the splicing of the non-coding exon between the 1st and 2nd coding exons—a rather uncommon exon that was previously found mainly in expressed sequence tags (ESTs)—was detected in circular BOK transcripts expressed only in DU 145 and MDAH-2774 cells  or the start of the upstream exon (acceptor) of the back-splice junction.Furthermore, interesting facts about the back-splice junction emerged. Most back-splice sites were non-canonical, meaning that there was neither GT at the donor site nor AG at the acceptor site. In fact, the generation of multiple distinct BOK circRNAs are characterized by distinct expression patterns among the seven cancer cell lines (BOK circRNAs were detected in the ovarian cancer cell lines and 9 BOK circRNAs in prostate cancer cell lines. A representative read proving the expression of each circRNA in the respective cell line(s) is provided in the The novel ll lines . In partBOK circRNAs are expressed in only one cell line, their expression pattern does not appear to correlate with the epithelial subtypes of ovarian cancer. For instance, circ-BOK-20 is expressed in cells from both endometrioid adenocarcinoma and high-grade ovarian serous adenocarcinoma. Interestingly, out of the full catalog of the identified circRNAs, only circ-BOK-17 was detected in the DU 145 prostate cancer cell line.Although we did not detect an obvious cancer-specific circRNA expression pattern, we noticed that the features of each cancer cell line may influence the expression of circRNAs, to some extent. For instance, circ-BOK-10 was exclusively expressed in LNCaP and OVCAR-3, an androgen- and an estrogen-dependent cancer cell line, respectively. Moreover, circ-BOK-20 was detected in two out of three estrogen-dependent cell lines (Ishikawa and OVCAR-3), while it was not detected in the estrogen-independent MDAH-2774 cell line. Additionally, although most BOK are differentially represented among the cell lines. Specifically, extensions of the known BOK exons were mainly present in ovarian cancer cell lines. Regarding the non-coding exon between the first and second coding exons, it is highly represented in the DU 145 prostate cancer cell line, while shorter versions of it are also represented in the MDAH-2774 ovarian cancer cell line  in our circRNA dataset. Indeed, we found 16 the NCBI . Althoug as well ; howeverBOK. Recent studies point towards a wide complexity of BOK in cancer, which can be attributed to the variety of alternative transcripts of this gene. Although most of the studies so far paid more attention to alternative splicing as a key modulator of mRNA production, increasing evidence has shown that alternative splicing of ncRNAs is even more common [BOK in ovarian and prostate cancer in such depth that could be elucidated only by a high-throughput sequencing approach [The precise and accurate identification of circRNA sequences is vital for a comprehensive understanding of circRNA functions and mechanisms. These molecules have rightfully been in the spotlight in recent years, and the ongoing elucidation of their role in ovarian and prostate cancer onset and progression has established circRNAs as potent disease regulators and biomarkers . This cae common ,36,37. Bapproach .BOK gene and decipher its transcriptional landscape in reproductive cancers. This methodology circumvents the length restriction of 200 nt near the back-splice junction of second-generation paired-end sequencing approaches, which is insufficient to read the majority of circRNAs. In fact, many novel BOK circRNAs had a length of more than 400 nt, and we would not have been able to identify these sequences through different approaches.The circular transcriptome assembly during the RNA sequencing data analysis is complex and challenging, mainly due to the high sequence similarity with linear cognates, especially with short-read approaches . In thisBOK circRNAs, intriguing features of the back-splice junction also emerged. The exons that participate in the back-splice junction were in most cases truncated forms of known BOK exons. Moreover, most back-splice sites were non-canonical; these non-canonical splicing events, particularly when they involve transposable element-derived sequences, are a significant source of newly emerging transcripts during evolution [BOK circRNAs with non-canonical splicing sites disrupt normal gene expression and alter the ratio of pathological to normal transcript variants in malignant conditions. Another interesting aspect of the back-splice junction of BOK circRNAs is the sequence identity that was usually observed in exonic and/or intronic regions forming the back-splice junction. This finding seems to be characteristic of circRNAs deriving from other genes as well. For example, novel PRMT1 circRNAs in breast cancer and BCL2L12 circRNAs in colorectal cancer share this feature, where several nucleotides at the exons forming the back-splice junction are identical [During the annotation of the novel volution . Non-canvolution ,41. Thusdentical ,39,42.BOK circRNAs exhibit cell line-specific expression profiles, with some circRNAs being unique to particular cancer cell lines. For instance, circ-BOK-10 demonstrates exclusive expression in LNCaP and OVCAR-3, cell lines that are androgen- and estrogen-dependent, respectively, suggesting a potential link between hormone dependency and circRNA expression. Furthermore, the detection of circ-BOK-20 in estrogen-dependent cell lines but its absence in an estrogen-independent cell line underscores the potential involvement of hormonal regulation in circRNA expression. Moreover, the observation of specific genomic regions of BOK being differentially represented in various cell lines underscores the dynamic nature of BOK gene expression and warrants further investigation to elucidate the functional significance of these variations in different cancer contexts.Notably, our research reveals that these novel BOK exons in the novel circRNA structure was also prevalent in our dataset. These events are in agreement with similar studies that report alternative splicing events in circRNAs [BOK exon between the first and second coding exons—which so far has been an outcome of computational analysis of expressed sequence tags (ESTs)—is evidenced by our results, considering that it participates in the structure of three novel circRNAs either as a full-length exon or as a 5′-truncated one. This finding increases the scientific questions around the complexity and purpose of alternative splicing events of BOK. Moreover, four single-exon BOK circRNAs were identified in our dataset. Interestingly, these circRNAs comprise extended BOK exons, which were not found either in multi-exonic circRNAs, or linear transcripts of the gene. According to the relevant literature, single-exon circRNAs derive from unusually long exons while multi-exon circRNAs are mostly generated from exons of regular length [BOK pre-mRNA produces a variety of transcripts and is expected to result in protein isoforms with a different pattern of BCL2-homology domains or in transcripts with distinct regulatory functions. The same could apply to the intense alternative splicing events found in circular BOK transcripts, implying a potentially different role of the detected circRNAs in the cell. This fact would be interesting to examine in parallel with normal ovarian and prostate cells.Additionally, the use of exon skipping and extended circRNAs ,44. Inter length , which wBOK. This finding adds to the evidence that intronic regions within this gene are functionally active and present in circular transcripts. This observation suggests that competitive splicing between circular and linear BOK transcripts occurs [BOK. Furthermore, miR-545-3p is proven to be sponged by other circRNAs and affect cancer progression through signaling regulation, as well as proliferation, migration, and invasion of cancer cells [Our data enabled a full-length sequence analysis of circRNAs containing previously intronic regions of s occurs . It is wer cells ,48,49. TBOK circRNAs in ovarian and prostate cancer, particularly focusing on their miRNA sponging potential. Potential miRNA target sites were predicted in all BOK circRNAs. Interestingly, some of these miRNAs with high prediction target scores, including miR-3158-5p, miR-466, and miR-214-5p, have been previously implicated in the onset and progression of reproductive cancers [BOK circRNAs might play critical roles in altering the expression levels of tumor-promoting and tumor-suppressing genes that are relevant to these malignancies. Moreover, the functional in silico analysis of this study sheds light on the potential interactions of the novel  cancers ,50. By sBOK circRNAs possessing putative ORFs were predicted to harbor m6A sites as well, strengthening the possibility that these transcripts might be translationally active. This modification is associated with the initiation of circRNA translation in a cap-independent manner, and a single m6A site is sufficient to drive translation initiation [BOK exons. The presence of putative ORFs and m6A sites in BOK circRNAs suggests that they could produce functional peptides or proteins. This contributes to a better understanding of the functional diversity of circRNAs, which were previously assumed to be non-coding RNA molecules; however, these findings are so far only based on computational predictions and require further experimental validation.In addition, 15 out of the 16 itiation . MoreoveBOK circRNAs could not be annotated due to mismatches at the back-splice junction. Additionally, our data does not distinguish between nuclear and cytoplasmic circRNAs, and it is yet unknown how much intron retention can influence the compartmentalization of circRNAs within cells or whether it serves any regulatory purposes. Moreover, while our study provides valuable insights into the BOK circRNAs in human ovarian and prostate cancer cells, we acknowledge the limitation of not having access to patient samples. Although the experimental pipeline that was followed in this study allowed several unknown circRNA characteristics to come to the surface, there are a few limitations as well. Firstly, the relatively high error rate of nanopore sequencing reads influences the accuracy of circRNA detection, and some BOK gene in human ovarian and prostate cancer cells, it is imperative to highlight potential avenues for future research in this field. Primarily, the validation of our findings in patient samples remains a critical endeavor to assess the clinical relevance of these circular RNAs as diagnostic or therapeutic targets. Obtaining patient samples and conducting further investigations in this context would be essential to bridge the gap between our cell line-based study and clinical applications. Furthermore, functional studies, including siRNA knockdown experiments and mechanistic investigations, are essential to elucidate the precise roles of these circular RNAs in tumorigenesis and apoptosis regulation. Additionally, the exploration of potential interactions between these circular RNAs and other biomolecules, such as proteins or miRNAs, could provide a more comprehensive understanding of their regulatory networks. While our study has unveiled a comprehensive profile of novel circRNAs associated with the apoptosis-related 2 concentration adjusted to 5%.For the implementation of the current workflow, 4 human ovarian and 3 human prostate cancer cell lines were used. In particular, the MDAH-2774 (ATCC CRL-10303), ES-2 (ATCC CRL-1978), OVCAR-3 (ATCC HTB-161), and Ishikawa (ECACC 99040201) ovarian cancer cell lines, and the DU 145 (ATCC HTB-81), PC-3 (ATCC CRL-1435), and LNCaP (ATCC CRL-1740) prostate cancer cell lines were propagated. The ovarian cancer cell lines are of distinct epithelial subtypes: MDAH-2774 and Ishikawa were established from ovarian endometrioid adenocarcinomas, ES-2 from a poorly differentiated ovarian clear cell carcinoma, and OVCAR-3 from high-grade ovarian serous adenocarcinoma. Regarding the prostate cancer cell lines, DU 145 and LNCaP were established from prostate carcinoma, while PC-3 was established from grade IV adenocarcinoma. All cell lines were cultured following each supplier’s guidelines. Cells were incubated at 37 °C, in a humidified atmosphere with COTotal RNA was isolated from each of the 7 cell lines, when cells reached 80% confluency, using the TRItidy G™ Reagent  according to the manufacturer’s protocol. The purity and concentration of the extracted RNA were evaluated spectrophotometrically at 260 and 280 nm. Next, 2 μg of each RNA sample were reversely transcribed using random hexamer primers , with the M-MLV reverse transcriptase . The first-strand cDNA synthesis was conducted as previously described, resulting in 20 μL of total cDNA volume .BOK exons : one pair for the first-round PCR and a second, internal pair for a nested PCR assay  or diluted PCR product at a ratio of 1:50 in nuclease-free H2O (nested PCR), with a final reaction volume of 25 μL. The thermal protocol was conducted according to the manufacturer’s guidelines: a denaturation step at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, an annealing step at 61 °C for 1 min, and an extension step at 72 °C for 30 s. The final elongation was carried out at 72 °C for 1 min. Then, agarose gel electrophoresis of the PCR products was performed and followed the purification of the specific amplicons. The PCR products of each cell line were mixed and purified at equal volumes using spin columns . The concentration of the purified products was evaluated using a Qubit 2.0 Fluorometer .Two pairs of gene-specific divergent primers were designed for the 2nd, 3rd, and 4th coding CR assay . First-rA DNA library for barcoding and adapter ligation was prepared using the Native Barcoding Expansion Kit 13–24 and the Ligation Sequencing Kit , following the manufacturer’s protocols. Briefly, the purified, pooled PCR products derived from each cell line were used as input for the construction of a barcoded library. A Qubit 2.0 Fluorometer was used for the quantification of the barcoded libraries. Nanopore sequencing was performed on the MinION Mk1C platform with the Flongle adapter . A FLO-MIN106D flow cell with R9.4.1 chemistry was used, following the manufacturer’s instructions for priming and loading the flow cell.The passed raw sequencing reads contained in the FASTQ files were mapped against the human reference genome (GRCh38) with the Minimap2 aligner . Alignme® file for the BOK gene as input [https://github.com/pkarousi/ASDT_remodeler.git accessed on 15 March 2023) to discern the reads representing BOK circRNAs. Then, extended manual annotation was conducted for each cancer cell line (https://github.com/pkarousi/Read_catcher.git accessed on 10 June 2023) was used to examine the presence of the novel identified BOK circRNAs across the 7 cell lines, based on the sequence of all splice junctions, including the back-splice junction itself  algorithm was used, providing a modified GenBankas input . Followiell line . Finallyn itself . The steBOK circRNAs. Particularly, all circRNAs were evaluated using the miRDB custom prediction web tool [In silico analysis was performed to predict interactions of the novel web tool , regardihttps://www.ncbi.nlm.nih.gov/orffinder accessed on 10 September 2023) was employed to search for putative open reading frames (ORFs) within the nucleotide sequences the novel identified circRNAs. Then, ORFs were queried in all reading frames starting with the ATG codon, and only those ORFs producing putative peptides exceeding 30 amino acids were considered. Then, the prediction of the N6-methyladenosine (m6A) modification sites on the BOK circRNA sequences that possess a putative ORF followed, via the SRAMP prediction server [BOK circRNAs translational potential, as post-transcriptional methylation is largely implicated, in a cap-independent manner, in the regulation of the circRNA translation process. Particularly, the circRNA sequences were submitted in the SRAMP prediction server in FASTA format, and the analysis of RNA secondary structure was also chosen so that the m6A sites would be visualized. Finally, the existence of IRES was also predicted, providing a FASTA file with the sequences of the 17 circRNAs with putative ORFs to the IRESpy bioinformatics tool [Then, further investigation of our circRNA dataset was performed regarding their potential to be translated. For this purpose, the ORF Finder bioinformatics tool (n server . The preics tool .BOK is a gene with abundant transcriptional activity that generates a wide range of circRNAs, which contain numerous complex alternative splicing events. It would be interesting to investigate the alterations of the expression levels of the identified circRNAs, not only in cancerous cells but also in normal ones, under the effect of therapeutic drugs. Moreover, the potential of BOK circRNAs as diagnostic and prognostic molecular biomarkers could be elucidated in the future by assessing their expression in ovarian and prostate cancer patients’ tissue samples. The comprehensive understanding of the function of the novel BOK circRNAs function will aid in elucidating the possible need for such a wide range of circRNAs and their involvement in ovarian and prostate cancer.In conclusion, it is evident that


CACNA1C is implicated in numerous neuropsychiatric disorders, in which key characteristics include deficits in social functioning and communication. Recently, we reported sex-dependent impairments in social behavior and ultrasonic vocalizations (USV) in juvenile heterozygous Cacna1c+/− (HET) rats. Specifically, HET females displayed increases in rough-and-tumble play that eliminated the typically observed sex difference between male and female rats. Interestingly, female wild-type Cacna1c+/+ (WT) pairs also showed a similar increase in social play when housed with HET females, suggesting their behavior may be influenced by HET cage mates. This indicates that the genetic makeup of the social environment related to Cacna1c can influence social play, yet systematic studies are lacking.Healthy brain development depends on early social practices and experiences. The risk gene In the present study, we housed juvenile females in MIXED- or SAME-genotype cages and tested them in a social play paradigm with a same- and opposite-genotype partner.The results show that the early social environment and the genotype of the play partner influence social play and 50-kHz USV emission. Experience with a WT play partner appears necessary for HET females to show comparable levels of play and 50-kHz USV emission. Same-genotype HET pairs played less and emitted fewer 50-kHz USV than same-genotype WT or opposite-genotype pairs; however, we found that the decrease in social play and 50-kHz USV in HET pairs can be rescued by playing with a WT partner. The effect was particularly prominent when the first play partner was WT, as we found it increased play and 50-kHz USV emission in all subsequent interactions with ensuing partners.Cacna1c+/− haploinsufficient rats. Specifically, our results show that WT peers can rescue behavior and communication alterations in Cacna1c female rats. Our findings have important implications because they show that the genetic makeup of the social environment can divulge phenotypic changes in genetic rat models of neuropsychiatric disorders.These findings suggest that the genetic makeup related to the social environment and/or social peers influences social play in  CACNA1C has been linked through genome-wide association studies (GWAS) to neuropsychiatric disorders with prominent social deficits, such as autism spectrum disorder (ASD)  . The CACc target . In humac target . In gene in mice  and rats in mice .Due to rats’ gregarious nature, they are a widely used model species for studying social behavior and communication . As juveAcoustic communication through ultrasonic vocalizations (USV) is another essential factor in the social repertoire of rats. Juvenile and adult rats emit two main types of USV: aversive 22-kHz and appetitive 50-kHz calls. Aversive 22-kHz calls are associated with negative affective states and occur mainly in aversive contexts, such as exposure to predators or pain, but also during defensive and submissive displays in intermale fighting . On the Social behavior and USV in combination convey considerable information on the animal’s affective state and social motivation and how they respond to various social peers. Therefore, using social behavior in conjunction with USV seems to be a particularly suitable tool for studying genetic and environmental risk factors in rodent models for neuropsychiatric disorders, such as ASD. Regarding genetics, studies have shown that different rat strains display differences in play . MoreoveCacna1c model exhibits several characteristics reminiscent of ASD-like social and communication deficits in juveniles and adults  rats compared to Cacna1c wildtype (WT) controls. In contrast, female HET pairs played much more than WT controls, but 50-kHz USV emission was comparable. Sex-dependent effects are not altogether surprising as there has been ample evidence for sex-specific effects of the risk gene CACNA1C in humans  rats (N = 40) and Cacna1c+/+ (WT) littermate controls (N = 40) by systematically manipulating the genotype of cage mates and the genotype of play partners, respectively.The interplay between acute and long-term effects of the genetic makeup related to the social environment and social peers was assessed using rough-and-tumble play and concomitant emission of 50-kHz USV in a group of juvenile female As described in preceding studies , 2020, HCacna1c Cel-1 F).50-GCTGCTGAGCCTTTTATTGG-30 (Cacna1c Cel-1 R).50-CCTCCTGGATAGCTGCTGAC-30  and male HET rats . The dayRats were identified by paw tattoo, using non-toxic animal tattoo ink . The ink was inserted subcutaneously through a 30-gauge hypodermic needle tip into the center of the paw on PND 3 ± 1. All procedures were conducted in strict accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and the relevant local or national rules and regulations of Germany and were subject to prior authorization by the local government .Cacna1c haploinsufficient females.For our design we manipulated the genotype of the cage mate and the genotype of the play partner to fully assess the interplay between acute and long-term effects of the genetic makeup related to the social environment and social peers on rough-and-tumble play behavior and concomitant 50-kHz USV emission in juvenile On the day of weaning (PND 21), juvenile females were separated into experimental housing conditions in which the genotype of the cage mate was manipulated. Experimental housing consisted of social housing in groups of 4–6 with same-sex littermates in either SAME-genotype cages  or MIXED-genotype cages . All females were housed in polycarbonate Macrolon Type IV cages  and kept under standard laboratory conditions (22 ± 2°C and 40–70% humidity) with free access to standard rodent chow and water.Forty WT and HET females from SAME- and MIXED-genotype cages were selected as focus rats and were matched with at first unfamiliar non-littermate WT and HET play partners. For each focus rat the genotype sequence of the play partners was counterbalanced so that they either played first with a WT play partner or with a HET play partner. Each focus rat played with both genotypes. Play partners were always from the same experimental housing condition as the focus rat. Play partners did not differ more than 10 g in weight.Testing consisted of two play sequences, each composed of three play sessions that took place over consecutive days . During Testing took place between PND 31 and 36. All rats were isolated the day prior to their first play session in a Makrolon type III cage  to enhance social motivation. In line with previous experiments , 2020, pUltrasonic vocalizations recordings were taken during the play phase using UltraSoundGate Condenser CM16 Microphones connected to an UltraSoundGate 416H USB audio device (Avisoft Bioacoustics) placed 35 cm above the floor of the center of the observation arena. Briefly, acoustic data was recorded with a sampling rate of 250 000 Hz  by Avisoft RECORDER USGH and transferred to Avisoft SASLab Pro for acoustical analysis. Start of behavioral recording was acoustically labeled by an audible beep signal from a stopwatch to synchronize audio and video recordings.The following behavioral measures were scored as play components or non-play components by a trained observer blind to the experimental conditions using the Observer XT : Play components included pinning, wrestling, chasing, evasion, self-pinning, crawl-over, biting and nape attack. Non-play components consisted of sniffing, physical contact, self-grooming and grooming of the partner  for repeated measurements were calculated with the between-subject factor genotype of the cage mates (H), genotype of the focus rat (G), genotype sequence of the play partner  (SQ) and the within-subject factors genotype of the play partner  (P), play session (SE), rough-and-tumble play component  (C). Cacna1c rat model and manipulated the genotype of the cage mates (SAME- vs. MIXED-genotype cages) and the genotype of the play partner (WT vs. HET play partner). Effects of Cacna1c haploinsufficiency were assessed in female constitutive heterozygous Cacna1c+/– rats (HET: N = 40) and compared to wildtype Cacna1c+/+ littermate controls (WT: N = 40).In the current study, we explored the interplay between acute and long-term effects of the genetic makeup related to the social environment and social peers on rough-and-tumble play and concomitant emission of 50-kHz USV during the critical developmental period in juvenile female rats. To this aim, we used a well-established genetic F = 0.000, p = 0.990]. Because the genotype of the cage mate had no effect on play, the following results  = 4.316, p = 0.046; For juvenile WT and HET females, the amount of time spent engaged in rough-and-tumble play averaged across all play sessions was not dependent on the genotype of the cage mates, i.e., housing . A prominent rescue effect was found within same-genotype HET pairs. For same-genotype HET pairs the duration of play was higher when the first play partner was a WT compared to a HET  = 26.584, p < 0.001]. For juvenile female WT and HET rats, the increase in play duration across sessions was dependent on the genotype and sequence of the play partners . Genotype of the play partner  did not affect the prevalence of the individual play components , yet there was an effect for the sequence of the play partner . Moreover, an interaction between genotype of the play partner and genotype of the focus rat and sequence of play partner was also found . Importantly, the duration for specific play components was dependent on the sequence of the play partner .Detailed analysis of the specific play components showed that time spent pinning was most prevalent, followed by wrestling, nape attacks and chasing  but no main effect for the genotype of the focus rat or sequence of the play partner was observed .Between subjects, effects for rough-and-tumble play components between same- and opposite-genotype pairs showed an interaction effect of focus rat genotype and sequence of the play partner . However, with a closer look at the duration of non-play social behavior between pairs playing first with a same- or an opposite-genotype partner, no differences were found and are therefore not reported  and WT females playing with an opposite-genotype partner (WT-HET), more time was spent in non-play social interactions when the first play partner was HET compared to WT ”’. No otF = 4.610, p = 0.039]. However, the main effects for the genotype of the focus rat, the genotype of the cage mates and the genotype and sequence of the play partner did not affect non-play social interactions  = 0.717, p = 0.404, F = 20.460, p < 0.001]. The genetic composition of the cage mates had no effect on the total averaged number of 50-kHz USV emissions across all play sessions. Accordingly, the post-hoc t-test results , the decrease in 50-kHz USV was rescued when the first play partner was WT compared to the HET  = 6.785, p = 0.014], with fewer 50-kHz calls associated with HET females compared to WT females. Furthermore, an interaction effect was observed between the cage mates’ genotype and the play partner’s sequence .Between-subject comparisons showed a main genotype effect .Detailed investigation of the emission of 50-kHz USV across individual play sessions between same- and opposite-genotype pairs showed an increase of 50-kHz USV across all play sessions . In particular, the effect appears to be driven by higher emission rates of 50-kHz USV when the first play partner was the opposite-genotype in females with SAME-genotype cage mates. The effect is most prominent in the same-genotype HET pairs, which produced more rough-and-tumble 50-kHz calls when the first play partner was a WT  = 8.647, p < 0.001]. In same-genotype pairs (WT-WT and HET-HET), the emission of 50-kHz USV appears to be higher when the first play partner was the opposite-genotype compared to the same-genotype. Specifically, when the first play partner was a WT, the HET females playing with same-genotype partners showed higher 50-kHz USV emissions across three all play sessions  showed higher 50-kHz USV emissions across each individual play session when the first play partner was the same-genotype. Within the pair constellations, the HET females playing with an opposite-genotype partner showed higher 50-kHz USV emissions only during the first play session, when the first play partner was a HET compared to a WT HET-WT: ”. In WT Cacna1c haploinsufficient rats and wildtype littermate controls. In contrast to what we expected based on our previous study . It is understandable, therefore, that they might be accustomed to playing with the HET partner in such cases. The reason for this is that the difference in behavior only occurs during the first play session. We can speculate that WT females may produce more 50-kHz USV initially to create a playful atmosphere possibly out of habitual tendency, even when they are paired with a new play partner. However, in subsequent sessions, they learn that this behavior is not necessary. Regarding the opposite-genotype WT pairs, the difference in 50-kHz USV emissions occurs only in the second play session. This might also be attributed to the influence of the previous play partner. In half of the pairs, the first play interaction involves a HET partner, whereas in the other half, they have already played with a WT partner for 3 days. As a result, they may still be highly motivated to play and, therefore, emit more 50-kHz USV to encourage their new HET play partner to engage in higher levels of playful behavior.We know from devocalization studies that a vocal rat playing with a devocalized partner will increase the emission of 50-kHz USV to be comparable to pairs of two vocal rats leading to an increase in the playful mood and higher rates of play . AlthougInterestingly, while the genotype of cage mates did not affect play behavior, we found an interaction between the genotype of the cage mate and the sequence of the first play partner within 50-kHz USV emissions. In the females from SAME-genotype cages, the emission of 50-kHz USV was higher when the first play partner was the opposite genotype. Sequence effects in SAME-genotype cages are interesting as they reinforce the impression that WT females emit more 50-kHz USV when paired with HET play partners and that the HET females may adapt not only their behavior but also their social communication to complement the play partner leading to an increase in play and 50-kHz USV emissions in the subsequent play partners. In the MIXED-genotype housing condition, we see only an effect in the WT-HET pairs with higher 50-kHz USV when playing with a same-genotype partner first, which is likely driven by the increase in 50-kHz USV during the second play session. Thus, while not a primary effect, the genotype of the cage mate is not wholly unimportant as it provides the initial play experiences with and for both genotypes. For HET females, this is particularly important as it may rescue deficits that would be more prominent if housed with only other SAME-genotype HET cage mates.There is strong evidence for social learning from conspecifics in various species, including rats . This inCacna1c juvenile HET and WT females. It provides further evidence to support findings in mice (Studies in mice have shown that social deficits in an ASD mouse model can be rescued by rearing them with a highly social mouse strain . From ou in mice  that eleCacna1c adult females reared in mixed-genotype cages, we found reduced 50-kHz USV in same-genotype HET compared to same-genotype WT control and opposite-genotype pairs (In contrast to the current study, pe pairs . Althougpe pairs , speakinWhile we did not see any main effect of manipulating the cage mate’s genotype on the play behaviors, we did see an effect on 50-kHz USV emissions. Thus, the effects on behavior may be more subtle and become prominent when looking at the individual rats in each pair and how they respond to one another when initiating or responding to a playful attack. Social dominance in juvenile rats is typically measured through pinning behavior, with the more dominant rat pinning more . In the CACNA1C gene has been linked through GWAS to an increased risk of ASD (Cacna1c haploinsufficient rats appears to manifest differently in males and females. It has been suggested that in humans autistic females may have fewer social impairments and show higher levels of social motivation than males (Cacna1c haploinsufficient rats.In humans, the k of ASD , partly k of ASD , 2021 yek of ASD . As adulk of ASD . Late emk of ASD , ASD-likan males , which iv1.2 protein levels may exert similar sex differences regarding ASD-like phenotypes in Cacna1c haploinsufficient juvenile rats, which may be supplemented by housing them with WT and HET cage mates in females. Furthermore, mixed-genotype housing in males may mask behavioral social deficits but lead to more noticeable deficits in social communication through USV, resulting in decreased social motivation and reduced 50-kHz USV emissions (Additionally, in female children a diagnosis of attention-deficit-hyperactivity disorder is often diagnosed years before a correct or accompanying diagnosis of ASD is recognized, due to the masking of social deficits . Thus, tmissions . The houCacna1c haploinsufficiency. Our study emphasizes the crucial role of genetic factors and social peers in shaping early social environments and their significant impact on the development of social behavior and communication in a rat model exhibiting ASD-like deficits.While the anticipated influence of the genetic composition of the social environment on rough-and-tumble play behavior was not observed, our findings demonstrate that MIXED-genotype housing provides an optimal context for HET females to engage in natural rough-and-tumble play interactions with WT partners. This valuable interaction effectively mitigates social deficits resulting from The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Tierschutzbehörde, Regierungspräsidium Gießen, Germany.TK conceived the study with the help of MW. RK and TK performed the experiments. RB and TK analyzed the data. RB and TK wrote the manuscript with the help of MW, RS, and RK. RS and MW acquired funding. All authors contributed to the article and approved the submitted version.


The SARS-CoV-2 pandemic challenged health systems worldwide, thus advocating for practical, quick and highly trustworthy diagnostic instruments to help medical personnel. It features a long incubation period and a high contagion rate, causing bilateral multi-focal interstitial pneumonia, generally growing into acute respiratory distress syndrome (ARDS), causing hundreds of thousands of casualties worldwide. Guidelines for first-line diagnosis of pneumonia suggest Chest X-rays (CXR) for patients exhibiting symptoms. Potential alternatives include Computed Tomography (CT) scans and Lung UltraSound (LUS). Deep learning (DL) has been helpful in diagnosis using CT scans, LUS, and CXR, whereby the former commonly yields more precise results. CXR and CT scans present several drawbacks, including high costs. Radiation-free LUS imaging requires high expertise, and physicians thus underutilise it. LUS demonstrated a strong correlation with CT scans and reliability in pneumonia detection, even in the early stages. Here, we present an LUS video-classification approach based on contemporary DL strategies in close collaboration with Fondazione IRCCS Policlinico San Matteo’s Emergency Department (ED) of Pavia. This research addressed SARS-CoV-2 patterns detection, ranked according to three severity scales by operating a trustworthy dataset comprising ultrasounds from linear and convex probes in 5400 clips from 450 hospitalised subjects. The main contributions of this study are related to the adoption of a standardised severity ranking scale to evaluate pneumonia. This evaluation relies on video summarisation through key-frame selection algorithms. Then, we designed and developed a video-classification architecture which emerged as the most promising. In contrast, the literature primarily concentrates on frame-pattern recognition. By using advanced techniques such as transfer learning and data augmentation, we were able to achieve an F1-Score of over 89% across all classes.  The SARS-CoV-2 virus, which originated in China in 2019, has spread globally and is highly contagious [Lung UltraSound testing aids in visualising and quantifying pulmonary involvement, typically retaining the white lung pattern or bilateral submantellar-subpleural consolidations ,4. The pIn the literature, the first line diagnosis of pneumonia exploits X-rays (CXR) , which aDifferent studies compared these techniques highlighting that CT and LUS outperform CXR ,10,11. TIn this context, academia evaluated different Deep Learning (DL) models to automatically expose the presence of SARS-CoV-2 from medical images . SeveralOn the other hand, another investigation  conceiveAnalysis of different key-frame selection strategies to perform LUS clip summarisation and extract meaningful content, resulting in fewer data to be elaborated and faster diagnosis. We evaluated the key-frame selection approaches, training benchmark architectures on the selected frames and testing on frames that experienced physicians extracted, containing pneumonia scoring patterns that the end-to-end video classification network should highlightAssessment of diverse DL architectures to identify the most promising one. The architectures varied on structure topology and video assessment strategyThe data used to train the networks was collected from the emergency department (ED) of the Fondazione IRCCS Policlinico San Matteo Hospital in Pavia. The medical staff at the ED collected 12 clips for each patient and assigned each clip a standardised score using standard scales ,17. In tThree different ranking scales to assess the severity of lung involvement, whereas the literature proposes investigations whose classifications mainly retain whether or not there is a viral pneumoniaRobustness to noise and adversarial attacks assessed through a data augmentation process applied to the training setEventually, we assessed the X3D architecture concerning t-SNE, PCA, and Grad-Cam strategies to demonstrate the trustworthiness of the resultsHere, we propose an investigation comparing different video classification methodologies, resulting in the X3D network as the most performing one. Accordingly, this manuscript’s main contributions are:We organised the paper as follows: Materials and Methods describe the AI methodologies, algorithms, and data used to conduct the experiments in detail. Results and Discussion retain the essential results to compare our study with the literature, thus emphasising their significance. Eventually, the last section offers the main scientific advancements that extend the field based on current knowledge and our achievements.In this section, we provide a detailed description of the dataset we used for SARS-CoV-2 end-to-end video classification and the selection, design, and training of the CNN architecture we employed. Specifically, we focus on data augmentation, transfer learning, training options, and the hyperparameters used to train and fine-tune our video classification networks.In particular, we addressed end-to-end video classification, since the proposed system takes as input the original clip acquired by the medical instrumentation. Then, the classification system elaborates this video and gives as output the pneumonia severity classification. The elaboration consists of two main steps: the former is the video summarisation, which produces a shorted clip containing only the most informative frames, while the latter is the classification of this summarised video adopting a suitable deep learning architecture.The first step to analysing the LUS dataset is understanding the manuscript’s scoring methodology. Remarkably, this investigation employed the ranking scale we introduced in our prior study that laid the foundations for this improvement. This manuscript operates at most four classes to indicate the severity of lung involvement. The pneumonia severity classification scale comprises scores ranging from 0 to 3, where Score 3 describes a lung almost incapable of breathing. A lung rated as Score 3 indicates that the illness affects the pleural line, namely the interface between the fluid-rich soft tissues of the wall and the gas-rich lung tissue , whilst Since March 2020, medical personnel at the San Matteo Hospital’s ED have been gathering LUS tests to examine the health of patients with suspected SARS-CoV-2 infection. The doctors operated the Aloka Arietta V70 ultrasound device , which works with convex and linear probes at frequencies of 5 MHz and 12 MHz, respectively. They standardised the procedure by focusing on the pleural line at a depth of 10 cm with the convex probe, and adjusting the gain to optimise the imaging of the pleural line, vertical artifacts, and peripheral consolidations with or without air bronchograms. Longitudinal and transversal scans were performed to examine the full length of the pleural line, with all harmonics and artifact-erasing software disabled.LUS was performed on patients with suspected SARS-CoV-2 infection due to the potential presence of false negatives in rRT-PCR testing. Specifically, the artefacts observed in the earlier section of the manuscript may be caused by either pulmonary edema or non-cardiac causes of interstitial syndromes . Even ifIn this study, a “clip” refers to the result of an LUS test, consisting of a set of frames or images. The medical personnel at the hospital collected 12 clips for each patient, all assigned a standardised LUS score according to Nonetheless, different doctors scored the clips, so the ED staff conducted a review to validate the classifications and avoid incorrect severity-scoring issues. This process ensured that each clip had a standardised rank value and that there were no discrepancies in the scores associated with different clips at the same severity stage, as emphasised in other studies .Accordingly, the dataset operated in this manuscript consisted of 624 clips randomly selected from the initial 5400 clips distribution.Fondazione IRCCS Policlinico San Matteo Hospital’s Emergency Department physicians oversaw the methodical procedure and ensured that the labeling was accurate. During the first part of the data collection and annotation process, they manually selected all clips from each patient, assessed the quality of each clip, and either proceeded to evaluate it based on the two scoring methodologies or discarded it. They reviewed each clip to assign a score and verify that SARS-CoV-2 pneumonia patterns, as described in Finally, this study randomly split the data into training (80%), validation (10%), and testing (10%) sets, following standard deep learning practice  and keepSimilarly, we increased the statistical variance in the training set by applying various data augmentation strategies listed in We repeatedly applied augmentations to the training data to exponentially expand the training set.According to the AI act established by the European Commission, ensuring cybersecurity is crucial in guaranteeing that artificial intelligence applications are resistant to attempts to alter their service, behavior, and performance, or compromise their safety properties through malicious interference by third parties exploiting system vulnerabilities. Cyberattacks on AI systems can leverage assets specific to AI, such as introducing adversarial attacks on trained models, namely providing the optimised architectures with slightly different inputs and confuse their behavior. Consequently, providers of high-risk AI systems should take appropriate measures to ensure an appropriate level of cybersecurity in relation to the risks, taking into account the underlying ICT infrastructure as necessary.At the end of the training settings management stage, with transfer learning and data augmentation, we collected 29,952, 171, and 171 clips for the training, validation, and test sets.Histogram : a histoRelative entropy : a methoResNet + K-means clustering : a technThere are several ways to summarise video data, including selecting the most important frames, reducing the memory needed for video processing and storage, and simplifying the structure of the video information. This paper used three different methods for extracting key frames:All these key-frames selection algorithms have been used to summarise the clips. These summarised clips are then used to train the networks described in CNN + LSTM : we desiCNN + Transformer : the secR(2+1)D : researcMultiscale Vision Transformer (MViT) : can claSlow-fast architecture : the arcX3D : it is aThis research evaluated various architectures to perform the end-to-end video classification of the data described in the earlier section. This investigation aims to determine the severity of lung involvement in an end-to-end fashion operating three different hierarchical ranking scales. All the architectures receive the summarisation of clips evaluated through the three methodologies we described in First, we assessed the quality of the key-frame video summary algorithms. This research represents a follow-up of the first study concerning frame classification . Hence, Similarly, we evaluated the following similarity indexes (Equations (1)–(3)) to compare the datasets originating from the key-frame algorithms  and the Equation (1) reports the Structural Similarity Index (SSIM) where ominator .Equation (2) is the Kullback-Leibler Divergence  defines the Jensen-Shannon divergence as a symmetrised and smoothed version of Equation (2).It is crucial to decrease false negatives to the maximum extent feasible, particularly when treating an infectious disease such as SARS-CoV-2. A patient’s incorrect diagnosis introduces a false negative, which causes improper care, lack of necessary treatment that reflects cross-contamination among subjects with additional pathologies, and faulty medications that may harm an infected person.TP) refers to correct classifications, False Negative (FN) refers to incorrect classifications, True Negative (TN) refers to correct classifications, and False Positive (FP) refers to incorrect classifications.In this research, the performance of the network classifications was evaluated using the validation and test sets. The focus was not only on accuracy, but also on precision, recall, and F1-Score (Equations (4)–(7)) and ROC-AUC . These mResearchers often place a particular emphasis on recall in order to reduce false negatives. Recall measures the performance of correctly identifying frames that do not contain SARS-CoV-2 pneumonia patterns and that belong to either of the considered classes or that display a healthy lung. Precision tells the reader about the classification performance in detecting the considered patterns. Therefore, the F1-Score is considered as a function of the previous two metrics. This parameter provides a more accurate measurement in terms of accuracy, taking into account the trade-off between precision and recall in unbalanced class distributions. Therefore, we need to evaluate recall and F1-Score in order to minimise false negatives while maintaining high precision.Eventually, this study assessed the quality and robustness of the classification performance through explainable AI strategies. Accordingly, we operated the gradient class activation mapping (Grad-CAM) algorithm and the statistical analysis of features deriving from deep architectural layers to evaluate whether we could clearly identify patterns from LUS clips and how dividable such patterns are in architectural encoded features. The former enables the interpretation of the architecture’s decision-making. Indeed, it emphasises the decisive parts to assign a rank through a heat map. Concerning the latter strategies, we performed PCA and t-SNE over the inner layers of the X3D architecture. Hence, we coloured the clusters according to the clips’ original classes.The study assessed the quality of video summaries produced by the key-frame selection algorithms. Likewise, we report in Hence, this research reports key-frame selection as a promising methodology to deliver the LUS clips summarisation and reduce memory footprint, enabling faster training.Being that the three similarity indexes are very close, we also adopt the three key-frames selection algorithms to enlarge the size of our dataset.We employed a ResNet-50 to measure the closeness between the frames extracted by the doctors and the automatic algorithms. Namely, we trained the architecture only with the automatically extracted frames to classify the manually chosen frames by the doctor , containing B-lines (Score 1 or Score 2) or consolidations (Score 3)Four-way classification: we considered all the scores in Table 1Hence, this manuscript reports the X3D architecture’s results according to the three classification tasks mentioned above. The designated architecture steadily approached optimisation convergence concerning the hyperparameters and training options in The results of the network’s weights at the end of each training process are reported, regardless of the number of epochs chosen for optimisation. We did not perform early stopping, which involves evaluating the epoch that shows promising performances with the validation set during optimisation, because the training process converged when the number of epochs elapsed. In addition, the training settings in We used Grad-CAM to validate the network’s decision-making. The physicians evaluated whether the X3D correctly identified B-lines, pleural line abnormalities, or other patterns examined in the LUS scoring section, which is the procedure doctors use to assess patients’ health. Eventually, we further validated the results by analysing the features extracted from the X3D architecture related to each clip coming from the test set. We reduced the order of each feature operating t-SNE and PCA algorithms in 2D. Furthermore, the first investigation is the only one assessing the clips operating one of the tasks we mentioned in this manuscript, namely the three-way classification. All the others discriminate between bacterial or viral (SARS-CoV-2) pneumonia and healthy lungs.Hence, our results in In conclusion, previous research on SARS-CoV-2 LUS clip assessment has some limitations. Some studies only utilised transfer learning and relied on low quality data sources that were not assessed by a qualified physician. Additionally, only the first study in In summary, we developed a reliable AI diagnostic tool to provide overworked medical personnel with an efficient and affordable SARS-CoV-2 detection system. The close collaboration with the Fondazione IRCCS Policlinico San Matteo ED allowed us to conduct our research using highly reliable and validated LUS data. We employed modern DL strategies, including video classification architectures, data augmentation, transfer learning, and key-frame selection algorithms, to assess the severity of lung involvement in SARS-CoV-2 infected individuals.This study used three different scoring scales to measure accurate and robust diagnostic performance. We addressed the issue of data heterogeneity, including low sensitivity leading to inadequate treatment and cross-contamination. We improved existing state-of-the-art diagnostic methods ,35,36 foThis study provides an end-to-end approach for classifying and scoring LUS clips. The Fondazione IRCCS Policlinico San Matteo ED reviewed every exam to uniformly assign the same score to lungs with the same disease stage.The proposed AI diagnostic tool features the advantage of providing a tool to detect and diagnose SARS-CoV-2 severity only considering LUS. Therefore, this analysis produced a DL-based system to automatically detect SARS-CoV-2 pneumonia patterns in LUS clips and rate their severity based on three standardised scoring scales with impressive, reliable, and promising results. The main drawback is that ultrasound imaging technologies require specialised expertise to achieve diagnostic reliability, including high sensitivity and overall accuracy. Moreover, the validated results are lacking an external cohort of patients, but this problem could be easily solved in the future by involving other hospitals into the research.