Clonal structure, stability and dynamics of human memory B cells and circulating plasmablasts.

Memory B cells persist for a lifetime and rapidly differentiate into antibody-producing plasmablasts and plasma cells upon antigen re-encounter. The clonal relationship and evolution of memory B cells and circulating plasmablasts is not well understood. Using single-cell sequencing combined with isolation of specific antibodies, we found that in two healthy donors, the memory B cell repertoire was dominated by large IgM, IgA and IgG2 clonal families, whereas IgG1 families, including those specific for recall antigens, were of small size. Analysis of multiyear samples demonstrated stability of memory B cell clonal families and revealed that a large fraction of recently generated plasmablasts was derived from long-term memory B cell families and was found recurrently. Collectively, this study provides a systematic description of the structure, stability and dynamics of the human memory B cell pool and suggests that memory B cells may be active at any time point in the generation of plasmablasts.

Evolution and engineering of pathways for aromatic O-demethylation in Pseudomonas putida KT2440.

Biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from lignin commonly contain methoxy groups, and O-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic O-demethylation, but they are rarely compared in vivo to determine an optimal biocatalytic strategy. Here, two pathways for aromatic O-demethylation were compared in Pseudomonas putida KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in P. putida, and the strains were optimized via adaptive laboratory evolution (ALE) with vanillate as a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates ∼1.8x faster than those harboring the heterologous LigM pathway. Enzyme kinetics and transcriptomics studies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, the reductase for vanillate O-demethylase, PP_3494, a global regulator of vanillate catabolism, and fghA, involved in formaldehyde detoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms, yielding a platform strain for efficient O-demethylation of lignin-related aromatic compounds to value-added products.

Symmetry-Broken Perturbation Theory to Large Orders in Antiferromagnetic Phases.

We introduce a spin-symmetry-broken extension of the connected determinant algorithm [Riccardo Rossi, Determinant diagrammatic Monte Carlo algorithm in the thermodynamic limit, Phys. Rev. Lett. 119, 045701 (2017).PRLTAO0031-900710.1103/PhysRevLett.119.045701]. The resulting systematic perturbative expansions around an antiferromagnetic state allow for numerically exact calculations directly inside a magnetically ordered phase. We show new precise results for the magnetic phase diagram and thermodynamics of the three-dimensional cubic Hubbard model at half-filling. With detailed computations of the order parameter in the low to intermediate-coupling regime, we establish the Néel phase boundary. The critical behavior in its vicinity is shown to be compatible with the O(3) Heisenberg universality class. By determining the evolution of the entropy with decreasing temperature through the phase transition we identify the different physical regimes at U/t=4. We provide quantitative results for several thermodynamic quantities deep inside the antiferromagnetic dome up to large interaction strengths and investigate the crossover between the Slater and Heisenberg regimes.

A Longitudinal Multiinstitutional Study of Vulvar Lichen Sclerosus: From Childhood to Perimenopause.

OBJECTIVE: The main outcome of this study was the evaluation of clinical characteristics, comorbidities, and therapeutic approaches in patients with vulvar lichen sclerosus (VLS) aged from childhood to perimenopause. Secondly, it was intended to compare these characteristics according to the menarchal status. METHODS: Patients less than 45 years of age with a diagnosis of VLS from January 2002 to June 2022 in 10 referral centers were included in this retrospective longitudinal study. The univariate analysis compared the dependent variables according to menarchal status. RESULTS: One hundred eighty-six patients met the inclusion criteria. At diagnosis, between 25% and 40% of premenarchal patients reported signs related to subepithelial hemorrhage. A significantly greater presence of bleeding ( p < .005), easy bruising ( p = .028), fissures ( p = .008), petechiae/splinter hemorrhages ( p < .001), and bleeding/blistering or open sores ( p = .011) was observed in premenarchal patients with respect to the postmenarchal group. The perineum ( p = .013) and the perianal region ( p < .001) were significantly more involved in the premenarchal group. Topical calcineurin inhibitors were more used in the premenarchal population ( p = .004), whereas vitamin E oil and moisturizers were more used in the postmenarchal population ( p = .047). CONCLUSIONS: Vulvar lichen sclerosus is a chronic condition that can cause vulvar changes that result in severe morbidity and affects sexual function and quality of life, even before menopause. Vulvar lichen sclerosus continues to be misdiagnosed in this population. This may lead to an average delay from symptom onset to diagnosis. Evaluating clinical manifestations of VLS in premenarchal and postmenarchal age allowed us to find different clinical characteristics between the 2 periods suggestive of the diagnosis.

Artificial Intelligence-Powered Molecular Docking and Steered Molecular Dynamics for Accurate scFv Selection of Anti-CD30 Chimeric Antigen Receptors.

Chimeric antigen receptor (CAR) T cells represent a revolutionary immunotherapy that allows specific tumor recognition by a unique single-chain fragment variable (scFv) derived from monoclonal antibodies (mAbs). scFv selection is consequently a fundamental step for CAR construction, to ensure accurate and effective CAR signaling toward tumor antigen binding. However, conventional in vitro and in vivo biological approaches to compare different scFv-derived CARs are expensive and labor-intensive. With the aim to predict the finest scFv binding before CAR-T cell engineering, we performed artificial intelligence (AI)-guided molecular docking and steered molecular dynamics analysis of different anti-CD30 mAb clones. Virtual computational scFv screening showed comparable results to surface plasmon resonance (SPR) and functional CAR-T cell in vitro and in vivo assays, respectively, in terms of binding capacity and anti-tumor efficacy. The proposed fast and low-cost in silico analysis has the potential to advance the development of novel CAR constructs, with a substantial impact on reducing time, costs, and the need for laboratory animal use.

ADAM10 hyperactivation acts on piccolo to deplete synaptic vesicle stores in Huntington's disease.

Synaptic dysfunction and cognitive decline in Huntington's disease (HD) involve hyperactive A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10). To identify the molecular mechanisms through which ADAM10 is associated with synaptic dysfunction in HD, we performed an immunoaffinity purification-mass spectrometry (IP-MS) study of endogenous ADAM10 in the brains of wild-type and HD mice. We found that proteins implicated in synapse organization, synaptic plasticity, and vesicle and organelles trafficking interact with ADAM10, suggesting that it may act as hub protein at the excitatory synapse. Importantly, the ADAM10 interactome is enriched in presynaptic proteins and ADAM10 co-immunoprecipitates with piccolo (PCLO), a key player in the recycling and maintenance of synaptic vesicles. In contrast, reduced ADAM10/PCLO immunoprecipitation occurs in the HD brain, with decreased density of synaptic vesicles in the reserve and docked pools at the HD presynaptic terminal. Conditional heterozygous deletion of ADAM10 in the forebrain of HD mice reduces active ADAM10 to wild-type level and normalizes ADAM10/PCLO complex formation and synaptic vesicle density and distribution. The results indicate that presynaptic ADAM10 and PCLO are a relevant component of HD pathogenesis.

Distinct microRNA signatures in human lymphocyte subsets and enforcement of the naive state in CD4+ T cells by the microRNA miR-125b.

MicroRNAs are small noncoding RNAs that regulate gene expression post-transcriptionally. Here we applied microRNA profiling to 17 human lymphocyte subsets to identify microRNA signatures that were distinct among various subsets and different from those of mouse lymphocytes. One of the signature microRNAs of naive CD4+ T cells, miR-125b, regulated the expression of genes encoding molecules involved in T cell differentiation, including IFNG, IL2RB, IL10RA and PRDM1. The expression of synthetic miR-125b and lentiviral vectors encoding the precursor to miR-125b in naive lymphocytes inhibited differentiation to effector cells. Our data provide an 'atlas' of microRNA expression in human lymphocytes, define subset-specific signatures and their target genes and indicate that the naive state of T cells is enforced by microRNA.

Biological risk in Italian prisons: data analysis from the second to the fourth wave of COVID-19 pandemic.

BACKGROUND: The management of COVID-19 in Italian prisons triggered considerable concern at the beginning of the pandemic due to numerous riots which resulted in inmate deaths, damages and prison breaks. The aim of this study is to shed some light, through analysis of the infection and relevant disease parameters, on the period spanning from the second to the fourth wave of the outbreak in Italy's prisons. METHODS: Reproductive number (Rt) and Hospitalisation were calculated through a Eulerian approach applied to differential equations derived from compartmental models. Comparison between trends was performed through paired t-test and linear regression analyses. RESULTS: The infection trends (prevalence and Rt) show a high correlation between the prison population and the external community. Both the indices appear to be lagging 1 week in prison. The prisoners' Rt values are not statistically different from those of the general population. The hospitalisation trend of inmates strongly correlates with the external population's, with a delay of 2 weeks. The magnitude of hospitalisations in prison is less than in the external community for the period analysed. CONCLUSIONS: The comparison with the external community revealed that in prison the infection prevalence was greater, although Rt values showed no significant difference, and the hospitalisation rate was lower. These results suggest that the consistent monitoring of inmates results in a higher infection prevalence while a wide vaccination campaign leads to a lower hospitalisation rate. All three indices demonstrate a lag of 1 or 2 weeks in prison. This delay could represent a useful time-window to strengthen planned countermeasures.

Driving fatigue increases after the Spring transition to Daylight Saving Time in young male drivers: A pilot study.

The Spring transition to Daylight Saving Time (DST) has been associated with several health and road safety issues. Previous literature has focused primarily on the analysis of historical crash and hospitalization data, without investigating specific crash contributing factors, such as driving fatigue. The present study aims to uncover the effects of DST-related circadian desynchrony and sleep deprivation on driving fatigue, by means of a driving simulator experiment. Eighteen participants (all males, age range 21-30 years, mean = 24.2, SD = 2.9) completed two 50-minute trials (at one week distance, same time and same day of the week) on a monotonous highway environment, the second one taking place in the week after the Spring transition to DST. Driving fatigue was evaluated by analysing several different variables (including driving-based, physiological and subjective indices) and by comparison with a historical cohort of pertinent, matched controls who had also undergone two trials, but in the absence of any time change in between. Results showed a considerable rise in fatigue levels throughout the driving task in both trials, but with significantly poorer performance in the post-DST trial, documented by a worsening in vehicle lateral control and an increase in eyelid closure. However, participants seemed unable to perceive this decrease in their alertness, which most likely prevented them from implementing fatigue-coping strategies. These findings indicate that DST has a detrimental effect on driving fatigue in young male drivers in the week after the Spring transition, and provide valuable insights into the complex relationship between DST and road safety.

High inter-follicular spatial co-localization of CD8+FOXP3+ with CD4+CD8+ cells predicts favorable outcome in follicular lymphoma.

The spatial architecture of the lymphoid tissue in follicular lymphoma (FL) presents unique challenges to studying its immune microenvironment. We investigated the spatial interplay of T cells, macrophages, myeloid cells and natural killer T cells using multispectral immunofluorescence images of diagnostic biopsies of 32 patients. A deep learning-based image analysis pipeline was tailored to the needs of follicular lymphoma spatial histology research, enabling the identification of different immune cells within and outside neoplastic follicles. We analyzed the density and spatial co-localization of immune cells in the inter-follicular and intra-follicular regions of follicular lymphoma. Low inter-follicular density of CD8+FOXP3+ cells and co-localization of CD8+FOXP3+ with CD4+CD8+ cells were significantly associated with relapse (p = 0.0057 and p = 0.0019, respectively) and shorter time to progression after first-line treatment (Logrank p = 0.0097 and log-rank p = 0.0093, respectively). A low inter-follicular density of CD8+FOXP3+ cells is associated with increased risk of relapse independent of follicular lymphoma international prognostic index (FLIPI) (p = 0.038, Hazard ratio (HR) = 0.42 [0.19, 0.95], but not independent of co-localization of CD8+FOXP3+ with CD4+CD8+ cells (p = 0.43). Co-localization of CD8+FOXP3+ with CD4+CD8+ cells is predictors of time to relapse independent of the FLIPI score and density of CD8+FOXP3+ cells (p = 0.027, HR = 0.0019 [7.19 × 10-6 , 0.49], This suggests a potential role of inter-follicular CD8+FOXP3+ and CD4+CD8+ cells in the disease progression of FL, warranting further validation on larger patient cohorts.

Potential long-term effects of Covid-19 on telecommuting and environment: An Italian case-study.

To contain the sudden spread of SARS-CoV-2, many governments encouraged people to work from home, generating an unprecedented diffusion of this activity. Furthermore, Covid-19 has induced drastic changes in everyday life and travel habits, which might persist in the future. This paper aims to understand and estimate the potential long-term impacts of telework on the environment due to the pandemic, by analyzing factors affecting the frequency of telecommuting, the mode choice for traveling to work, and pollutant emissions generated by these trips. Data from a mobility survey administered in Padova (Italy) was used. Results indicate that Covid-19 could cause a rebound effect reversing the positive impacts of working from home, since, even if the number of trips could be reduced, many shifts towards non-sustainable travel modes could occur. The promotion of telework should be combined with measures fostering sustainable travel habits to pave the way towards a future green mobility.

A Weighted-LSM Method to Improve Classification and Concentration Evaluation from Laser-Induced Fluorescence Spectra.

The detection of biological agents using optical systems is an open field of research. Currently, different spectroscopic techniques allow to detect and classify chemical agents while a fast and accurate technique able to identify biological agents is still under investigation. Some optical techniques, such as Laser-Induced Breakdown Spectroscopy (LIBS) or Laser-Induced Fluorescence (LIF), are already used as classification methods. However, the presence of background, spectrum similarities and other confounders make these techniques not very specific. This work shows a new method to achieve better performances in terms of classification and concentration evaluations. The method is based on the Weighted Least Square Minimization method. In fact, by using ad hoc weights, the LSM looks at specific features of the spectra, resulting in higher accuracy. In order to make a systematic analysis, numerical tests have been conducted. With these tests, the authors were able to highlight the various advantages and drawbacks of the new methodology proposed. Then, the method was applied to some LIF measurements to investigate the applicability of the method to preliminary experimental cases. The results show that, by using this new weighted LSM, it is possible to achieve better classification and concentration evaluation performances. Finally, the possible application of the new method is discussed.

Copper Oxide Nanomaterial Fate in Plant Tissue: Nanoscale Impacts on Reproductive Tissues.

A thorough understanding of the implications of chronic low-dose exposure to engineered nanomaterials through the food chain is lacking. The present study aimed to characterize such a response in Cucurbita pepo L. (zucchini) upon exposure to a potential nanoscale fertilizer: copper oxide (CuO) nanoparticles. Zucchini was grown in soil amended with nano-CuO, bulk CuO (100 mg Kg-1), and CuSO4 (320 mg Kg-1) from germination to flowering (60 days). Nano-CuO treatment had no impact on plant morphology or growth nor pollen formation and viability. The uptake of Cu was comparable in the plant tissues under all treatments. RNA-seq analyses on vegetative and reproductive tissues highlighted common and nanoscale-specific components of the response. Mitochondrial and chloroplast functions were uniquely modulated in response to nanomaterial exposure as compared with conventional bulk and salt forms. X-ray absorption spectroscopy showed that the Cu local structure changed upon nano-CuO internalization, suggesting potential nanoparticle biotransformation within the plant tissues. These findings demonstrate the potential positive physiological, cellular, and molecular response related to nano-CuO application as a plant fertilizer, highlighting the differential mechanisms involved in the exposure to Cu in nanoscale, bulk, or salt forms. Nano-CuO uniquely stimulates plant response in a way that can minimize agrochemical inputs to the environment and therefore could be an important strategy in nanoenabled agriculture.

FOXP1 circular RNA sustains mesenchymal stem cell identity via microRNA inhibition.

Stem cell identity and plasticity are controlled by master regulatory genes and complex circuits also involving non-coding RNAs. Circular RNAs (circRNAs) are a class of RNAs generated from protein-coding genes by backsplicing, resulting in stable RNA structures devoid of free 5' and 3' ends. Little is known of the mechanisms of action of circRNAs, let alone in stem cell biology. In this study, for the first time, we determined that a circRNA controls mesenchymal stem cell (MSC) identity and differentiation. High-throughput MSC expression profiling from different tissues revealed a large number of expressed circRNAs. Among those, circFOXP1 was enriched in MSCs compared to differentiated mesodermal derivatives. Silencing of circFOXP1 dramatically impaired MSC differentiation in culture and in vivo. Furthermore, we demonstrated a direct interaction between circFOXP1 and miR-17-3p/miR-127-5p, which results in the modulation of non-canonical Wnt and EGFR pathways. Finally, we addressed the interplay between canonical and non-canonical Wnt pathways. Reprogramming to pluripotency of MSCs reduced circFOXP1 and non-canonical Wnt, whereas canonical Wnt was boosted. The opposing effect was observed during generation of MSCs from human pluripotent stem cells. Our results provide unprecedented evidence for a regulatory role for circFOXP1 as a gatekeeper of pivotal stem cell molecular networks.

Update on the Diagnostic Pitfalls of Autopsy and Post-Mortem Genetic Testing in Cardiomyopathies.

Inherited cardiomyopathies are frequent causes of sudden cardiac death (SCD), especially in young patients. Despite at the autopsy they usually have distinctive microscopic and/or macroscopic diagnostic features, their phenotypes may be mild or ambiguous, possibly leading to misdiagnoses or missed diagnoses. In this review, the main differential diagnoses of hypertrophic cardiomyopathy (e.g., athlete's heart, idiopathic left ventricular hypertrophy), arrhythmogenic cardiomyopathy (e.g., adipositas cordis, myocarditis) and dilated cardiomyopathy (e.g., acquired forms of dilated cardiomyopathy, left ventricular noncompaction) are discussed. Moreover, the diagnostic issues in SCD victims affected by phenotype-negative hypertrophic cardiomyopathy and the relationship between myocardial bridging and hypertrophic cardiomyopathy are analyzed. Finally, the applications/limits of virtopsy and post-mortem genetic testing in this field are discussed, with particular attention to the issues related to the assessment of the significance of the genetic variants.

Lacrimal bone agenesis in a dog.

A 20-month-old neutered male dachshund dog was referred because of a 10-week history of swelling close to the medial canthus of the left eye. Recurrence of the lesion and cytological appearance of the fluid content were suggestive of inflammation. Computed tomography revealed a triangular-shaped bone defect in the skull deep to the lesion. Computed tomography dacryocystography demonstrated contrast medium pooling within the maxillary recess and nasal cavity rather than filling the lacrimal duct. Lacrimal bone agenesis was diagnosed. Key clinical message: Congenital skull including lacrimal bone agenesis may be responsible for swelling of the medial canthus of the eye and computed tomography dacryocystography is helpful in making a diagnosis.

Agénésie de l'os lacrymal chez un chien. Un teckel mâle castré âgé de 20 mois a été référé en raison d'une histoire de 10 semaines d'enflure près du canthus médial de l'oeil gauche. La récidive de la lésion et l'aspect cytologique du contenu liquidien suggéraient une inflammation. La tomodensitométrie a révélé un défaut osseux de forme triangulaire dans le crâne profondément à la lésion. La dacryocystographie par tomodensitométrie a démontré une accumulation de produit de contraste dans la cavité maxillaire et la cavité nasale plutôt que de remplir le canal lacrymal. Une agénésie de l'os lacrymal a été diagnostiquée.Message clinique clé :Le crâne congénital, y compris l'agénésie de l'os lacrymal, peut être responsable de l'enflure du canthus médial de l'oeil et la dacryocystographie par tomodensitométrie est utile pour poser un diagnostic.(Traduit par Dr Serge Messier).

Improved Treatment of the Independent Variables for the Deployment of Model Selection Criteria in the Analysis of Complex Systems.

Model selection criteria are widely used to identify the model that best represents the data among a set of potential candidates. Amidst the different model selection criteria, the Bayesian information criterion (BIC) and the Akaike information criterion (AIC) are the most popular and better understood. In the derivation of these indicators, it was assumed that the model's dependent variables have already been properly identified and that the entries are not affected by significant uncertainties. These are issues that can become quite serious when investigating complex systems, especially when variables are highly correlated and the measurement uncertainties associated with them are not negligible. More sophisticated versions of this criteria, capable of better detecting spurious relations between variables when non-negligible noise is present, are proposed in this paper. Their derivation is obtained starting from a Bayesian statistics framework and adding an a priori Chi-squared probability distribution function of the model, dependent on a specifically defined information theoretic quantity that takes into account the redundancy between the dependent variables. The performances of the proposed versions of these criteria are assessed through a series of systematic simulations, using synthetic data for various classes of functions and noise levels. The results show that the upgraded formulation of the criteria clearly outperforms the traditional ones in most of the cases reported.

RUES2 hESCs exhibit MGE-biased neuronal differentiation and muHTT-dependent defective specification hinting at SP1.

RUES2 cell lines represent the first collection of isogenic human embryonic stem cells (hESCs) carrying different pathological CAG lengths in the HTT gene. However, their neuronal differentiation potential has yet to be thoroughly evaluated. Here, we report that RUES2 during ventral telencephalic differentiation is biased towards medial ganglionic eminence (MGE). We also show that HD-RUES2 cells exhibit an altered MGE transcriptional signature in addition to recapitulating known HD phenotypes, with reduced expression of the neurodevelopmental regulators NEUROD1 and BDNF and increased cleavage of synaptically enriched N-cadherin. Finally, we identified the transcription factor SP1 as a common potential detrimental co-partner of muHTT by de novo motif discovery analysis on the LGE, MGE, and cortical genes differentially expressed in HD human pluripotent stem cells in our and additional datasets. Taken together, these observations suggest a broad deleterious effect of muHTT in the early phases of neuronal development that may unfold through its altered interaction with SP1.

Eomesodermin controls a unique differentiation program in human IL-10 and IFN-γ coproducing regulatory T cells.

Whether human IL-10-producing regulatory T cells ("Tr1") represent a distinct differentiation lineage or an unstable activation stage remains a key unsolved issue. Here, we report that Eomesodermin (Eomes) acted as a lineage-defining transcription factor in human IFN-γ/IL-10 coproducing Tr1-like cells. In vivo occurring Tr1-like cells expressed Eomes, and were clearly distinct from all other CD4+ T-cell subsets, including conventional cytotoxic CD4+ T cells. They expressed Granzyme (Gzm) K, but had lost CD40L and IL-7R expression. Eomes antagonized the Th17 fate, and directly controlled IFN-γ and GzmK expression. However, Eomes binding to the IL-10 promoter was not detectable in human CD4+ T cells, presumably because critical Tbox binding sites of the mouse were not conserved. A precommitment to a Tr1-like fate, i.e. concominant induction of Eomes, GzmK, and IFN-γ, was promoted by IL-4 and IL-12-secreting myeloid dendritic cells. Consistently, Th1 effector memory cells contained precommitted Eomes+ GzmK+ T cells. Stimulation with T-cell receptor (TCR) agonists and IL-27 promoted the generation of Tr1-like effector cells by inducing switching from CD40L to IL-10. Importantly, CD4+ Eomes+ T-cell subsets were present in lymphoid and nonlymphoid tissues, and their frequencies varied systemically in patients with inflammatory bowel disease and graft-versus-host disease. We propose that Eomes+ Tr1-like cells are effector cells of a unique GzmK-expressing CD4+ T-cell subset.

Adaptive Quasi-Unsupervised Detection of Smoke Plume by LiDAR.

The early detection of fire is one of the possible applications of LiDAR techniques. The smoke generated by a fire is mainly compounded of CO2, H2O, particulate, and other combustion products, which involve the local variation of the scattering of the electromagnetic wave at specific wavelengths. The increases of the backscattering coefficient are transduced in peaks on the signal of the backscattering power recorded by the LiDAR system, located exactly where the smoke plume is, allowing not only the detection of a fire but also its localization. The signal processing of the LiDAR signals is critical in the determination of the performances of the fire detection. It is important that the sensitivity of the apparatus is high enough but also that the number of false alarms is small, in order to avoid the trigger of useless and expensive countermeasures. In this work, a new analysis method, based on an adaptive quasi-unsupervised approach was used to ensure that the algorithm is continuously updated to the boundary conditions of the system, such as the weather and experimental apparatus issues. The method has been tested on an experimental campaign of 227 pulses and the performances have been analyzed in terms of sensitivity and specificity.