Dissociations between spontaneous electroencephalographic features and the perturbational complexity index in the minimally conscious state.

The analysis of spontaneous electroencephalogram (EEG) is a cornerstone in the assessment of patients with disorders of consciousness (DoC). Although preserved EEG patterns are highly suggestive of consciousness even in unresponsive patients, moderately or severely abnormal patterns are difficult to interpret. Indeed, growing evidence shows that consciousness can be present despite either large delta or reduced alpha activity in spontaneous EEG. Quantifying the complexity of EEG responses to direct cortical perturbations (perturbational complexity index [PCI]) may complement the observational approach and provide a reliable assessment of consciousness even when spontaneous EEG features are inconclusive. To seek empirical evidence of this hypothesis, we compared PCI with EEG spectral measures in the same population of minimally conscious state (MCS) patients (n = 40) hospitalized in rehabilitation facilities. We found a remarkable variability in spontaneous EEG features across MCS patients as compared with healthy controls: in particular, a pattern of predominant delta and highly reduced alpha power-more often observed in vegetative state/unresponsive wakefulness syndrome (VS/UWS) patients-was found in a non-negligible number of MCS patients. Conversely, PCI values invariably fell above an externally validated empirical cutoff for consciousness in all MCS patients, consistent with the presence of clearly discernible, albeit fleeting, behavioural signs of awareness. These results confirm that, in some MCS patients, spontaneous EEG rhythms may be inconclusive about the actual capacity for consciousness and suggest that a perturbational approach can effectively compensate for this pitfall with practical implications for the individual patient's stratification and tailored rehabilitation.

Neocortical and medial temporal seizures have distinct impacts on brain responsiveness.

Focal epileptic seizures are characterized by abnormal neuronal discharges that can spread to other cortical areas and interfere with brain activity, thereby altering the patient's experience and behavior. The origin of these pathological neuronal discharges encompasses various mechanisms that converge toward similar clinical manifestations. Recent studies have suggested that medial temporal lobe (MTL) and neocortical (NC) seizures are often underpinned by two characteristic onset patterns, which, respectively, affect and spare synaptic transmission in cortical slices. However, these synaptic alterations and their effects have never been confirmed or studied in intact human brains. To fill this gap, we here evaluate whether responsiveness of MTL and NC are differentially affected by focal seizures, using a unique data set of cortico-cortical evoked potentials (CCEPs) collected during seizures triggered by single-pulse electrical stimulation (SPES). We find that responsiveness is abruptly reduced by the onset of MTL seizures, despite increased spontaneous activity, whereas it is preserved in the case of NC seizures. The present results provide an extreme example of dissociation between responsiveness and activity and show that brain networks are diversely affected by the onset of MTL and NC seizures, thus extending at the whole brain level the evidence of synaptic alteration found in vitro.

Where do we look for the next breakthrough in sleep research?

The wakefulness-sleep cycle sets the pace of our life. Sleep research examines the transition between wakefulness and sleep, its hormonal regulation, and its pathological disruption. Understanding sleep mechanisms would improve quality-of-life well beyond sleep itself. To this aim, we invite contributions for the Collection "sleep physiology and circadian rhythms".

Circulation of Streptococcus agalactiae ST103 in a Free Stall Italian Dairy Farm.

We report here on an outbreak of mastitis caused by Streptococcus agalactiae, or group B Streptococcus, in a northern Italy (Lombardy Region) free stall dairy farm. This outbreak was unusual because it occurred in a closed dairy herd and proved to be extremely difficult to resolve even after the application of the classical control procedures, which are specifically focused on the contagious nature of S. agalactiae. In order to better understand the potential origins of the pathogen and the critical points that could impair the eradication program and to investigate the possible presence of S. agalactiae in sources outside the mammary gland, we collected 656 individual composite milk samples, 577 samples from extramammary body sites (289 rectal, 284 vaginal, and four throat samples from milking cows, dry cows, heifers, and calves), and 81 samples from the cattle environment, including the milking parlor and the barn. Twenty-two S. agalactiae isolates were obtained from lactating cows or their environment. Of these, nine were isolated from milk, two were from rectal swabs, and two were from vaginal swabs, while nine were isolated from environmental samples. Based on molecular serotyping, pilus island (PI) typing and multilocus sequence typing, all isolates belonged to serotype III, pilus type PI-1/2b, and sequence type 103 (ST103), a type previously described to have an environmental transmission cycle and a potential human origin. Once the classical mastitis control measures were supplemented with environmental hygiene measures, herd monitoring using bulk tank milk revealed no further positive results for S. agalactiae, and the outbreak was considered resolved. IMPORTANCE Streptococcus agalactiae is an important pathogen in humans and cattle. Bovine mastitis caused by this bacterium and its control are generally associated with contagious transmission between animals. More recently, the presence of a fecal-oral transmission cycle in cattle has been proposed, linked to the ability of some S. agalactiae strains to survive in the bovine gastrointestinal tract and environment. Based on analysis of 1,316 specimens from cattle and their environment on a single dairy farm, we demonstrate the presence of sequence type 103 (ST103), which may have an environmental mode of transmission. This possibility was supported by the fact that the mastitis outbreak could not be controlled through measures to prevent contagious transmission alone and required additional environmental hygiene measures to be brought to a halt. This case study highlights that measures to control animal disease need to evolve alongside the microorganisms that cause them.

Tonic somatosensory responses and deficits of tactile awareness converge in the parietal operculum.

Although clinical neuroscience and the neuroscience of consciousness have long sought mechanistic explanations of tactile-awareness disorders, mechanistic insights are rare, mainly because of the difficulty of depicting the fine-grained neural dynamics underlying somatosensory processes. Here, we combined the stereo-EEG responses to somatosensory stimulation with the lesion mapping of patients with a tactile-awareness disorder, namely tactile extinction. Whereas stereo-EEG responses present different temporal patterns, including early/phasic and long-lasting/tonic activities, tactile-extinction lesion mapping co-localizes only with the latter. Overlaps are limited to the posterior part of the perisylvian regions, suggesting that tonic activities may play a role in sustaining tactile awareness. To assess this hypothesis further, we correlated the prevalence of tonic responses with the tactile-extinction lesion mapping, showing that they follow the same topographical gradient. Finally, in parallel with the notion that visuotactile stimulation improves detection in tactile-extinction patients, we demonstrated an enhancement of tonic responses to visuotactile stimuli, with a strong voxel-wise correlation with the lesion mapping. The combination of these results establishes tonic responses in the parietal operculum as the ideal neural correlate of tactile awareness.

A comparative study between state-of-the-art MRI deidentification and AnonyMI, a new method combining re-identification risk reduction and geometrical preservation.

Deidentifying MRIs constitutes an imperative challenge, as it aims at precluding the possibility of re-identification of a research subject or patient, but at the same time it should preserve as much geometrical information as possible, in order to maximize data reusability and to facilitate interoperability. Although several deidentification methods exist, no comprehensive and comparative evaluation of deidentification performance has been carried out across them. Moreover, the possible ways these methods can compromise subsequent analysis has not been exhaustively tested. To tackle these issues, we developed AnonyMI, a novel MRI deidentification method, implemented as a user-friendly 3D Slicer plugin-in, which aims at providing a balance between identity protection and geometrical preservation. To test these features, we performed two series of analyses on which we compared AnonyMI to other two state-of-the-art methods, to evaluate, at the same time, how efficient they are at deidentifying MRIs and how much they affect subsequent analyses, with particular emphasis on source localization procedures. Our results show that all three methods significantly reduce the re-identification risk but AnonyMI provides the best geometrical conservation. Notably, it also offers several technical advantages such as a user-friendly interface, multiple input-output capabilities, the possibility of being tailored to specific needs, batch processing and efficient visualization for quality assurance.

Implementing Smart Working in Public Administration: a follow up study.

BACKGROUND: Starting from February 2020, in Italy most organizations have had a forced transition to flexible working practice - called "smart working in emergency" - due to the Covid-19 epidemic outbreak. This allowed to continue work activities and services and contributed to contain the risk of infection in different sectors, particularly in the public administration. OBJECTIVES: This follow up study focussed on a panel of 187 workers from the Italian Workers' Compensation Authority taking part to a pilot project "Smart Working in INAIL" from January 2019 to December 2019. The aim was to investigate the effects of work organization on work attitudes, work-life balance and health outcomes before and after the introduction of the smart working. METHODS: The data were collected at two time points through a web-based questionnaire. The first wave aimed to collect information up to one month before the implementation of the smart working. The second wave aimed to collect information about potential changes occurred after one year of smart working. RESULTS: This study showed that high demands, low control and low social support might lead to reduced well-being and less satisfaction with work, and have an effect on work engagement and work-life balance. Particularly, improving social support can moderate the negative impact of high strain on well-being, preventing work-life imbalance and risk of isolation. DISCUSSION: Findings and future perspectives are discussed to support stakeholders in defining policies and practices concerning health and wellbeing at work while preserving productivity, for a successful implementation of smart working in the public administration.

Whole rat stomach decellularisation using a detergent-enzymatic protocol.

BACKGROUND: Conditions leading to reduced gastric volume are difficult to manage and are associated to poor quality-of-life. Stomach augmentation using a tissue-engineered stomach is a potential solution to restore adequate physiology and food reservoir. Aim of this study was to evaluate the decellularisation of whole rat stomach using a detergent-enzymatic protocol. METHODS: Stomachs harvested from rats were decellularised through luminal and vascular cannulation using 24-h detergent-enzymatic treatment and completely characterized by appropriate staining, DNA and Extracellular matrix -component quantifications. RESULTS: The detergent-enzymatic protocol allows a complete decellularisation of the gastric tissue, with a complete removal of the DNA with two cycles as confirmed by both quantifications and histological analysis. Extracellular matrix components, collagen, fibronectin, laminin and elastin, were optimally preserved by the treatment, while glycosaminoglycans were reduced. CONCLUSION: Gastric tissue can be efficiently decellularised. Scaffolds retained original structure and important components that could enhance integration with other tissues for in vivo transplant. The use of naturally derived material could be potentially considered for the treatment of both congenital and acquired conditions.

Economic effects of treatment of chronic kidney disease with low-protein diet.

OBJECTIVE: The most recent literature has shown extensively that a low-protein diet in patients with chronic kidney disease (CKD) delays the natural progression of end-stage renal disease and the necessary treatment of chronic dialysis. The aim of this study was to estimate the cost-effectiveness of a very low-protein diet compared with a moderate low-protein diet. DESIGN: Markov model. SETTING: The study was conducted from the perspective of the Italian National Health Service. SUBJECTS: Patients with CKD stages 4 and 5. Seven hundred and two patients were considered, and half were treated with a very low-protein diet and the other half with a moderate low-protein diet. INTERVENTION: The Markov model estimates the costs and the quality-adjusted life years (QALYs) associated with a very low-protein diet versus a moderate low-protein diet for patients with CKD Stages 4 and 5. The transition probability was estimated on data from 7 studies, which determined the efficacy of very low-protein diets in delaying the need to start maintenance dialysis. Utilities and cost were estimated from literature review and projected for the lifespan considered in the model. The annual cost of dialysis per patient was approximately €34,072. The cost of a very low-protein diet was €1,440 per patient per year in the Lazio region (conservative assumptions). Probabilistic and deterministic sensitivity analyses were performed. MAIN OUTCOME MEASURE: QALY, cost of treatment, incremental cost-effectiveness ratio. RESULTS: The model estimates that a very low-protein diet should be more effective. Dietary treatment improved 0.09 QALYs after 2 years, 0.16 after 3 years, 0.36 after 5 years, and up to 0.93 incremental QALYs after the first 10 years. After 2 years, the model estimated increment cost in favor of the most restrictive dietary treatment of €826, €2,146, €5,203, and €10,375 for 2, 3, 5, and 10 years of follow-up respectively. CONCLUSIONS: The results of these simulations indicate that the treatment of CKD patients with a very low-protein diet is cost-effective relative to a moderate low-protein diet in an Italian setting. Further studies should test this model in other countries with different dialysis costs and dietary support.

[The INSuLa Project: a knowledge survey of employers]. / Progetto INSuLa: I'indagine conoscitiva sui Datori di Lavoro.

In the framework of the INSuLa project, supported by Italian Ministry of Health, a national survey was conducted to evaluate the perceptions of employers about Occupational Safety and Health (OSH) activities. Main findings of this survey are presented in this paper. The survey was conducted on a sample of 1,010 employers weighted by economic activity sector, company size and geographic area. An ad hoc questionnaire was administered through Computer Assisted Telephone Interviewing methodology. Most of employers "mostly" or "completely" agree with the usefulness of OSH activities and the efficacy of specific prevention and protection measures. Around 37.7% of employers consider OSH management a shared responsibility with workers and 56.1% of them feel the OSH level to be increased following the implementation of Legislative Decree no. 81/08. The findings of this survey provide a picture of Italian employers' point of view about OSH and identify gaps and needs, thus contributing to choose proper actions for the improvement of OSH

Importance of an Integrated Assessment of Functional Disability and Work Ability in Workers Affected by Low Back Pain.

Background: This study examines the relationship between functional disability and work ability in workers affected by low back pain (LBP) through an analysis of correlations between the Oswestry Disability Index (ODI) and Work Ability Index (WAI). The role of personal and work factors on functional disability/work ability levels has also been studied. LBP is the most common musculoskeletal problem and a major disabling health problem worldwide. Its etiology is multifactorial. Multidisciplinary approaches may help reduce the burden of pain and disability and improve job continuity and reintegration at work. Methods: A cohort of 264 patients affected by LBP from an Italian outpatient clinic were included in a clinical diagnostic/therapeutic trial aiming at rehabilitation and return to work through an integrated investigation protocol. Data were collected during the first medical examination using anamnestic and clinical tools. The final sample is composed of 252 patients, 57.1% man, 44.0 % blue collars, 46.4% with the high school degree, 45.6% married. Results: WAI and ODI reported a negative and fair correlation (r = -0.454; p = .000). Workers with acute LBP symptoms have a higher probability of severe disability than those with chronic LBP symptoms. White collars without depressive symptoms reported higher work ability - even in chronic disability conditions-than those with depressive symptoms. Conclusion: The study found that ODI and WAI have a convergent validity and this suggests that the two tools measure capture distinctive aspects of disability related to personal, environmental, and occupational characteristics. The most important and modifiable prognostic factors found for ODI and WAI were depressive symptoms, workday absence, and intensity of back pain. The study also found a mild association between age and ODI. The study's findings highlight the importance of using a multidisciplinary approach to manage and prevent disability due to LBP.

Arbovirus screening of mosquitoes collected in 2022 in Emilia-Romagna, Italy, with the implementation of a real-time PCR for the detection of Tahyna virus.

Several Arboviruses (Arthropod-borne virus) are a concrete health risk. While some arboviruses, such as the West Nile virus (WNV) and the Usutu virus (USUV) are actively surveyed, others are neglected, including the Tahyna virus (TAHV). In this work, we tested - searching for all the three viruses - 37,995 mosquitoes collected in 95 attractive traps, baited by carbon dioxide, distributed in the lowlands of Emilia-Romagna, northern Italy, between 19 July and 12 August 2022. Among the 668 pools obtained, WNV was detected in 45 pools of Culex (Cx.) pipiens and USUV was recorded in 24 pools of the same mosquito; ten of these Cx. pipiens pools tested positive for both WNV and USUV. Interestingly, we recorded a significant circulation of both WNV lineage 1 (WNV-L1) and lineage 2 (WNV-L2): WNV-L1 strains were detected in 40 pools, WNV-L2 strains in three pools and both lineages were detected in two pools. TAHV was detected in 8 different species of mosquitoes in a total of 37 pools: Aedes (Ae.) caspius (25), Ae. albopictus (5), Ae. vexans (3), Cx. pipiens (2), Ae. cinereus (1) and Anopheles maculipennis sl (1). The significant number of Ae. caspius-pools tested positive and the estimated viral load suggest that this mosquito is the principal vector in the surveyed area. The potential involvement of other mosquito species in the TAHV cycle could usefully be the subject of further experimental investigation. The results obtained demonstrate that, with adequate sampling effort, entomological surveillance is able to detect arboviruses circulating in a given area. Further efforts must be made to better characterise the TAHV cycle in the surveyed area and to define health risk linked to this virus.

Localizing hidden Interictal Epileptiform Discharges with simultaneous intracerebral and scalp high-density EEG recordings.

BACKGROUND: Scalp EEG is one of the main tools in the clinical evaluation of epilepsy. In some cases intracranial Interictal Epileptiform Discharges (IEDs) are not visible from the scalp. Recent studies have shown the feasibility of revealing them in the EEG if their timings are extracted from simultaneous intracranial recordings, but their potential for the localization of the epileptogenic zone is not yet well defined. NEW METHOD: We recorded simultaneous high-density EEG (HD-EEG) and stereo-electroencephalography (SEEG) during interictal periods in 8 patients affected by drug-resistant focal epilepsy. We identified IEDs in the SEEG and systematically analyzed the time-locked signals on the EEG by means of evoked potentials, topographical analysis and Electrical Source Imaging (ESI). The dataset has been standardized and is being publicly shared. RESULTS: Our results showed that IEDs that were not clearly visible at single-trials could be uncovered by averaging, in line with previous reports. They also showed that their topographical voltage distributions matched the position of the SEEG electrode where IEDs had been identified, and that ESI techniques can reconstruct it with an accuracy of ∼2 cm. Finally, the present dataset provides a reference to test the accuracy of different methods and parameters. COMPARISON WITH EXISTING METHODS: Our study is the first to systematically compare ESI methods on simultaneously recorded IEDs, and to share a public resource with in-vivo data for their evaluation. CONCLUSIONS: Simultaneous HD-EEG and SEEG recordings can unveil hidden IEDs whose origins can be reconstructed using topographical and ESI analyses, but results depend on the selected methods and parameters.

High clonality of Mycobacterium avium subsp. paratuberculosis field isolates from red deer revealed by two different methodological approaches of comparative genomic analysis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the aetiological agent of paratuberculosis (Johne's disease) in both domestic and wild ruminants. In the present study, using a whole-genome sequence (WGS) approach, we investigated the genetic diversity of 15 Mycobacterium avium field strains isolated in the last 10 years from red deer inhabiting the Stelvio National Park and affected by paratuberculosis. Combining de novo assembly and a reference-based method, followed by a pangenome analysis, we highlight a very close relationship among 13 MAP field isolates, suggesting that a single infecting event occurred in this population. Moreover, two isolates have been classified as Mycobacterium avium subsp. hominissuis, distinct from the other MAPs under comparison but close to each other. This is the first time that this subspecies has been found in Italy in samples without evident epidemiological correlations, having been isolated in two different locations of the Stelvio National Park and in different years. Our study highlights the importance of a multidisciplinary approach incorporating molecular epidemiology and ecology into traditional infectious disease knowledge in order to investigate the nature of infectious disease in wildlife populations.

Humic substance/metal-oxide multifunctional nanoparticles as advanced antibacterial-antimycotic agents and photocatalysts for the degradation of PLA microplastics under UVA/solar radiation.

As a result of the accumulation of plastic in the environment, microplastics have become part of the food chain, boosting the resistance of fungi and bacteria which can frequently encounter human beings. Employing photocatalytic degradation is a possible route towards the removal of chemical and biological pollutants, such as plastics and microplastic wastes as well as microorganisms. Using biowaste materials to design hybrid nanoparticles with enhanced photocatalytic and antimicrobial features would uphold the principles of the circular bioeconomy. Here, two unexpensive semiconductors-namely titanium dioxide (TiO2) and zinc oxide (ZnO) - were synthetized through solvothermal synthesis and combined with humic substances deriving from agrifood biomass. The preparation led to hybrid nanoparticles exhibiting enhanced ROS-generating properties for simultaneous applications as antimicrobial agents against different bacterial and fungal strains and as photoactive catalysts to degrade polylactic acid (PLA) microplastics under UVA and solar irradiation. In comparison to bare nanoparticles, hybrid nanoparticles demonstrated higher antibacterial and antimycotic capabilities toward various pathogenic microorganisms as well as advanced photocatalytic activity in the degradation of PLA with a carbonyl index reduction in the range of 15-23%, thus confirming a noteworthy ability in microplastics photodegradation under UVA and solar irradiation.

Prevalence of Helminths in Small Ruminant Farms and Evaluation of Control Practices Used to Counter Anthelmintic Resistance in Southern Italy.

Anthelmintic resistance in small ruminants is a serious worldwide problem. To reduce their spread, it is essential to know the prevalence of helminths on farms and the control practices adopted. As these studies in the Calabria region of southern Italy are fragmentary and outdated, a study on the prevalence of helminths in small ruminant holdings in this area has been conducted. The measures implemented to control helminths were also evaluated through questionnaires administered to farmers. In particular, on 90 farms (45 sheep and 45 goats), 1800 faecal samples from 900 sheep and 900 goats were collected in the spring. Using the FLOTAC dual technique, parasitological examinations demonstrated the presence of gastrointestinal nematodes in 100% of sheep and goat farms, followed by Nematodirus spp. (84.44% sheep and 48.89% goats), Moniezia spp. (73.33% sheep and 35.56% goats), Trichuris ovis (48.89% sheep and 42.22% goats), lungworms (28.89% sheep and 42.22% goats), Strongyloides papillosus (40% sheep and 26.67% goats), Dicrocoelium dendriticum (13.33% sheep and 26.67% goats), Calicophoron daubneyi (6.67% sheep and 31.11% goats), Fasciola hepatica (6.67% sheep and 4.44% goats), and Skrjabinema ovis (4.44% sheep and goats). The questionnaires showed that 82% and 85% of the farmers had applied pasture rotation, and that 93.3% and 86.6% had used anthelmintics in the previous year for sheep and goats, respectively. Only 24.4% of sheep farmers and 11.3% of goat farmers had carried out parasitological tests prior to treatments. The most used classes of anthelmintics were macrocyclic lactones and benzimidazoles, and only in 21.6% and 15.6%, for sheep and goats, respectively, was drug rotation carried out. These results denote that helminths represent a health problem for small ruminants and highlight a lack of knowledge of parasite control strategies among farmers. In these conditions, anthelmintic resistance phenomena could develop over time. Therefore, it is necessary to implement all possible strategies for the control of helminths, and to prevent the spread of anthelmintic resistance phenomena on farms in southern Italy.

Thalamic feedback shapes brain responses evoked by cortical stimulation in mice and humans.

Cortical stimulation with single pulses is a common technique in clinical practice and research. However, we still do not understand the extent to which it engages subcortical circuits which contribute to the associated evoked potentials (EPs). Here we find that cortical stimulation generates remarkably similar EPs in humans and mice, with a late component similarly modulated by the subject's behavioral state. We optogenetically dissect the underlying circuit in mice, demonstrating that the late component of these EPs is caused by a thalamic hyperpolarization and rebound. The magnitude of this late component correlates with the bursting frequency and synchronicity of thalamic neurons, modulated by the subject's behavioral state. A simulation of the thalamo-cortical circuit highlights that both intrinsic thalamic currents as well as cortical and thalamic GABAergic neurons contribute to this response profile. We conclude that the cortical stimulation engages cortico-thalamo-cortical circuits highly preserved across different species and stimulation modalities.

Active surveillance of paratuberculosis in Alpine-dwelling red deer (Cervus elaphus).

Paratuberculosis (Johne's disease) is a globally widespread infectious disease affecting domestic and wild ruminants, caused by Mycobacterium avium subsp. paratuberculosis (MAP). The bacterium is excreted in the feces and is characterized by high environmental resistance. The new Animal Health Law (Regulation EU 2016/429) on transmissible animal diseases, recently in force throughout the European Union, includes paratuberculosis within the diseases requiring surveillance in the EU, listing some domestic and wild Bovidae, Cervidae, and Camelidae as potential reservoirs. Taking advantage of a culling activity conducted in the Stelvio National Park (Italy), this study investigated MAP infection status of red deer (Cervus elaphus) between 2018 and 2022, and evaluated the probability of being MAP-positive with respect to individual and sampling-level variables. A total of 390 subjects were examined macroscopically and tested for MAP, using different diagnostic tools: IS900 qPCR, culture, histopathology, and serology. Twenty-three of them were found positive for MAP by at least one test, with an overall prevalence of 5.9% (95% CI 4.0-8.7), that, respectively, ranged from 12.4% in the first culling season to 2.0 and 2.1% in the 2019-2020 and 2021-2022 culling seasons. Quantitative PCR assay on ileocecal valve and mesenteric lymph nodes detected the highest number of MAP positive animals. The results of the study showed the increased probability of being MAP-positive with increasing age and that red deer with lower body mass values were more likely to be infected with MAP. Overall, the absence of signs of clinical paratuberculosis and gross lesions together with the low level of shedding witness early phases of the disease among the positive red deer and support an improvement of the paratuberculosis status of this population, as shown by the decreased prevalence of the disease over the years.