Harvesting the Power of Green Synthesis: Gold Nanoparticles Tailored for Prostate Cancer Therapy.

Biosynthetic gold nanoparticles (bAuNPs) present a promising avenue for enhancing bio-compatibility and offering an economically and environmentally responsible alternative to traditional production methods, achieved through a reduction in the use of hazardous chemicals. While the potential of bAuNPs as anticancer agents has been explored, there is a limited body of research focusing on the crucial physicochemical conditions influencing bAuNP production. In this study, we aim to identify the optimal growth phase of Pseudomonas aeruginosa cultures that maximizes the redox potential and coordinates the formation of bAuNPs with increased efficiency. The investigation employs 2,6-dichlorophenolindophenol (DCIP) as a redox indicator. Simultaneously, we explore the impact of temperature, pH, and incubation duration on the biosynthesis of bAuNPs, with a specific emphasis on their potential application as antitumor agents. Characterization of the resulting bAuNPs is conducted using ATR-FT-IR, TEM, and UV-Vis spectroscopy. To gain insights into the anticancer potential of bAuNPs, an experimental model is employed, utilizing both non-neoplastic (HPEpiC) and neoplastic (PC3) epithelial cell lines. Notably, P. aeruginosa cultures at 9 h/OD600 = 1, combined with biosynthesis at pH 9.0 for 24 h at 58 °C, produce bAuNPs that exhibit smaller, more spherical, and less aggregated characteristics. Crucially, these nanoparticles demonstrate negligible effects on HPEpiC cells while significantly impacting PC3 cells, resulting in reduced viability, migration, and lower IL-6 levels. This research lays the groundwork for the development of more specialized, economical, and ecologically friendly treatment modalities.

Carbapenem-resistant Klebsiella pneumoniae bloodstream infections in haematological malignances and hematopoietic stem cell transplantation: Clinical impact of combination therapy in a 10-year Brazilian cohort.

Bacterial bloodstream infections (BSI) are a common threat among patients with haematological malignancies (HM) and hematopoietic stem cell transplant recipients (HSCT). The purpose of this research was to describe clinical and microbiological aspects of BSI caused by carbapenem-resistant Klebsiella pneumoniae (CRKp) and assess risk factors associated with 30-day mortality in a 10-year cohort of haematological patients. A total of 65 CRKp-BSI episodes occurring in HM patients and HSCT recipients and CRKp-BSI between January 2010 and December 2019 were retrospectively studied. Acute leukemias were the most frequently observed underlying disease (87.7%) and 18 patients (27.7%) received HSCT. Mucosal barrier injury in the gastrointestinal tract was the primary cause of bacteremia (86.1%). Also, 14 individuals (21.6%) had an Invasive Fungal Disease (IFD) throughout the episode. Regarding treatment, in 31 patients (47.7%) empirical therapy was deemed appropriate, whereas 33 (50.8%) patients received a combination therapy. Microbiological data revealed that the majority of isolates (53-58%) had the Polymyxin B co-resistance phenotype, while amikacin resistance was less common (16 samples, or 24.7%). The mortality rates at 14 and 30 days were 32.3% and 36.9%, respectively. In a multivariate Cox regression analysis, prompt appropriate antibiotic administration within three days was associated with a better outcome (Adjusted Hazard Ratio [aHR]: 0.33; 95% Confidence Interval [CI]: 0.14-0.76; p = 0.01), whereas hypotension at presentation (aHR: 3.88; 95% CI: 1.40-10.74; p = 0.01) and concurrent IFD (aHR: 2.97; 95% CI: 1.20-7.37; p = 0.02) were independently associated with death within 30 days. Additionally, a favorable correlation between combination therapy and overall survival was found (aHR: 0.18; 95%CI: 0.06-0.56; p = 0.002). In conclusion, 30-day mortality CRKp-BSI was elevated and most of the isolates were polymyxin B resistant. Early appropriate antimicrobial treatment and the use of combination therapy were linked to a better outcome.

Betacoronavirus Infection Outbreak, São Paulo, Brazil, Fall 2023.

We report a human coronavirus OC43 infection outbreak in hospitalized patients and healthcare workers in São Paulo, Brazil, occurring after SARS-CoV-2 cases disappeared. Infection was associated with healthcare workers in 5 (29.4%) patients. Routine surveillance including a respiratory virus panel can improve coronavirus detection in both healthcare professionals and patients.

Candida albicans chitinase 3 with potential as a vaccine antigen: production, purification, and characterisation.

Chitinases are widely studied enzymes that have already found widespread application. Their continued development and valorisation will be driven by the identification of new and improved variants and/or novel applications bringing benefits to industry and society. We previously identified a novel application for chitinases wherein the Candida albicans cell wall surface chitinase 3 (Cht3) was shown to have potential in vaccine applications as a subunit antigen against fungal infections. In the present study, this enzyme was investigated further, developing production and purification protocols, enriching our understanding of its properties, and advancing its application potential. Cht3 was heterologously expressed in Pichia pastoris and a 4-step purification protocol developed and optimised: this involves activated carbon treatment, hydrophobic interaction chromatography, ammonium sulphate precipitation, and gel filtration chromatography. The recombinant enzyme was shown to be mainly O-glycosylated and to retain the epitopes of the native protein. Functional studies showed it to be highly specific, displaying activity on chitin, chitosan, and chito-oligosaccharides larger than chitotriose only. Furthermore, it was shown to be a stable enzyme, exhibiting activity, and stability over broad pH and temperature ranges. This study represents an important step forward in our understanding of Cht3 and contributes to its development for application.

Prognostic value of flow-status in severe aortic stenosis patients undergoing percutaneous intervention.

PURPOSE: Low-flow status is a mortality predictor in severe aortic stenosis (SAS) patients, including after transcatheter aortic valve implantation (TAVI) treatment. However, the best parameter to assess flow is unknown. Recent studies suggest that transaortic flow rate (FR) is superior to currently used stroke volume index (SVi) in defining low-flow states. Therefore, we aimed to evaluate the prognostic value of FR and SVi in patients undergoing TAVI. METHODS: A single-centre retrospective analysis of all consecutive patients treated with TAVI for SAS between 2011 and 2019 was conducted. Low-FR was defined as < 200 mL/s and low-SVi as < 35 mL/m2. Primary endpoint was all-cause five-year mortality, analyzed using Kaplan-Meier curves and Cox regression models. Secondary endpoint was variation of NYHA functional class six months after procedure. Patients were further stratified according to ejection fraction (EF < 50%). RESULTS: Of 489 cases, 59.5% were low-FR, and 43.1% low-SVi. Low-flow patients had superior surgical risk, worse renal function, and had a higher prevalence of coronary artery disease. Low-FR was associated with mortality (hazard ratio 1.36, p = 0.041), but not after adjustment to EuroSCORE II. Normal-SVi was not associated with survival, despite a significative p-trend for its continuous value. No associations were found for flow-status and NYHA recovery. When stratifying according to preserved and reduced EF, both FR and SVi did not predict all-cause mortality. CONCLUSION: In patients with SAS undergoing TAVI, a low-FR state was associated with higher mortality, as well as SVi, but not at a 35 mL/m2 cut off.

Digital flashcards and medical physiology performance: a dose-dependent effect.

Use of digital flashcards promotes active recall, spaced repetition, and self-assessment academic principles. This work explores the association and dose-dependent effect of this study method and locomotor (LP) and cardiovascular physiology (CP) grades. A single-faculty cohort study of medical LP and CP students was conducted, and 155 and 676 flashcards, respectively, were created through Moodle. An exploratory analysis examined three exam results (2019), and a confirmatory study used a fourth exam (2021) in another CP cohort. Of 685 students enrolled, 558 participated in the exploratory analysis: 319 (69%) for LP and 311 (84%) for CP, of which 203 LP and 267 CP students were flashcard users. Median grades were higher among flashcard users, and the number of cards reviewed was positively correlated with grades (r = 0.275 to 0.388 for LP and r = 0.239 to 0.432 for CP, P < 0.001). Multiple linear regression models confirmed a positive dose-dependent association between results and the number of flashcards studied: for every 100 LP cards reviewed, exam grades increased 0.44-0.75 on a 0-20 scale range (P < 0.001), and for every 1,000 CP flashcards, results raised 0.81-1.08 values (P < 0.05). These findings were confirmed in the 2021 CP cohort of 269 participants, of whom 67% were flashcard users. Digital flashcard revision has a consistent positive dose-dependent association on LP and CP grades.NEW & NOTEWORTHY Implementing flashcard-based strategies is a feasible way to promote active recall, spaced repetition, and self-assessment, and students are highly adherent to these initiatives. There is a positive dose-dependent association between the number of flashcards reviewed and physiology grades. These results are consistent across different physiology subjects, under different cohorts, over short and medium terms.

Phenotyping patients with ischaemic heart disease at risk of developing heart failure: an analysis of the HOMAGE trial.

AIMS: We aim to characterize the clinical and proteomic profiles of patients at risk of developing heart failure (HF), with and without coronary artery disease (CAD) or prior myocardial infarction (MI). METHODS AND RESULTS: HOMAGE evaluated the effect of spironolactone on plasma and serum markers of fibrosis over 9 months of follow-up in participants with (or at risk of having) CAD, and raised natriuretic peptides. In this post hoc analysis, patients were classified as (i) neither CAD nor MI; (ii) CAD; or (iii) MI. Proteomic between-group differences were evaluated through logistic regression and narrowed using backward stepwise selection and bootstrapping. Among the 527 participants, 28% had neither CAD or MI, 31% had CAD, and 41% had prior MI. Compared with people with neither CAD nor MI, those with CAD had higher baseline plasma concentrations of matrix metalloproteinase-7 (MMP-7), galectin-4 (GAL4), plasminogen activator inhibitor 1 (PAI-1), and lower plasma peptidoglycan recognition protein 1 (PGLYRP1), whilst those with a history of MI had higher plasma MMP-7, neurotrophin-3 (NT3), pulmonary surfactant-associated protein D (PSPD), and lower plasma tumour necrosis factor-related activation-induced cytokine (TRANCE). Proteomic signatures were similar for patients with CAD or prior MI. Treatment with spironolactone was associated with an increase of MMP7, NT3, and PGLYRP1 at 9 months. CONCLUSIONS: In patients at risk of developing HF, those with CAD or MI had a different proteomic profile regarding inflammatory, immunological, and collagen catabolic processes.

Guild Vertical Stratification and Drivers of Bat Foraging in a Semi-Arid Tropical Region, Kenya.

Africa faces significant challenges in reconciling economic and social development while preserving its natural resources. Little is known about the diverse bat community on the continent, particularly in drier ecosystems. A better understanding of the bat community will help improve and inform the management of these ecosystems. Our study aimed to provide detailed information on the main drivers of bat richness and activity at three different heights above the ground in a semi-arid region of Kenya. We assessed how bat activity varied with space and height using acoustic sampling and complementary methods. We sampled 48 sites at ground level and two sites on meteorological masts at 20 m and 35 m above the ground. We recorded more than 20 bat species, including one species of concern for conservation. Our models showed that the use of space varies with bat guild, creating trade-offs in the variables that affect their activity. Low-flying bat species are mostly associated with habitat variables, whereas high-flying species are more dependent on weather conditions. Our study highlights the richness of bat assemblages in semi-arid environments and emphasizes the need for management measures to protect bat diversity in the face of habitat degradation caused by climate change, land management, and development projects.

Antimicrobial stewardship programs in seven Latin American countries: facing the challenges.

BACKGROUND: Studies have shown that more than 50% of the antibiotics used in hospitals are unnecessary or inappropriate and, that antimicrobial resistance may cost up to 20 billion USD in excess medical costs each year. On the other hand, Antimicrobial Stewardship Programs (ASP) significantly reduce inappropriate antimicrobial use, emergence of antimicrobial resistance, healthcare associated infections, and costs in hospital settings. OBJECTIVE: To evaluate the development of ASP and antibiotic savings in 7 Latin American hospitals using standardized quantitative indicators in all the participating health care institutions. METHODS: An interventional study was conducted, where pre- and post- evaluations were performed using a standardized score tool adapted from the Joint Commission International accreditation standards and, the Colombian Institute of Technical Standards and Certification. We evaluated ASP from 7 Latin American hospitals between 2019 and 2020. A pre-intervention evaluation was done in each hospital to quantify the degree of development of the ASP (ASP Development score). Based on these results, tailored on-site training was implemented in each hospital, followed by a post-intervention evaluation to quantify improvement of ASP-development indicators. In addition, monetary savings in antimicrobials derived from the ASP intervention were estimated. RESULTS: In the pre-intervention evaluation, the average ASP development score for the 7 institutions was 65.8% (40-94.3%). The items with the lowest development score were those related to monitoring and communicating the ASP progress and success. For the post-intervention evaluation, 2 institutions couldn't participate due to the pressure imposed by the COVID-19 pandemic. For the remaining 5/7 hospitals, the average ASP development score was 82.3% with an increase of 12.0% when compared to the pre-intervention measurement of the same institutions (average pre-intervention score 70.3% (48.2%-94.3%) The items with a significant increase were key performance indicators, AMS education and training of the prescribers. Three of the seven (3/7) hospitals reported antibiotic monetary savings associated to the ASP intervention. CONCLUSIONS: The use of the tool described shown to be useful to evaluate specific areas of ASP-development that were lacking and tailor interventions for the participating hospitals, consequently, it helped improve ASP-development in the institutions that underwent pre- intervention and post-intervention analysis. In addition, the strategies showed monetary savings on antimicrobial costs when measured.

BNT162b2 mRNA COVID-19 against symptomatic Omicron infection following a mass vaccination campaign in southern Brazil: A prospective test-negative design study.

BACKGROUND: Evidence regarding effectiveness of BNT162b2 mRNA COVID-19 vaccine against Omicron in Latin America is limited. We estimated BNT162b2 effectiveness against symptomatic COVID-19 in Brazil when Omicron was predominant. METHODS: This prospective test-negative, case-control study was conducted in Toledo, Brazil, following a mass COVID-19 vaccination with BNT162b2. Patients were included if they were aged ≥12 years, sought care for acute respiratory symptoms in the public health system between November 3, 2021 and June 20, 2022, and were tested for SARS-CoV-2 using RT-PCR. In the primary analysis, we determined the effectiveness of two doses of BNT162b2 against symptomatic COVID-19. RESULTS: A total of 4,574 were enrolled; of these, 1,758 patients (586 cases and 1,172 controls) were included in the primary analysis. Mean age was 27.7 years, 53.8 % were women, and 90.1 % had a Charlson comorbidity index of zero. Omicron accounted for >97 % of all identified SARS-CoV-2 variants, with BA.1 and BA.2 accounting for 84.3 % and 12.6 %, respectively. Overall adjusted estimate of two-dose vaccine effectiveness against symptomatic COVID-19 was 46.7 % (95 %CI, 19.9 %-64.6 %) after a median time between the second dose and the beginning of COVID-19 symptoms of 94 days (IQR, 60-139 days). Effectiveness waned from 77.7 % at 7-29 days after receipt of a second dose to <30 % (non-significant) after ≥120 days. CONCLUSION: In a relatively young and healthy Brazilian population, two doses of BNT162b2 provided protection against symptomatic Omicron infection. However, this protection waned significantly over time, underscoring the need for boosting with variant-adapted vaccines in this population prior to waves of disease activity. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov number, NCT05052307 (https://clinicaltrials.gov/ct2/show/NCT05052307).

Fear of Covid-19, anxiety, and depression during the pandemic: A network analysis

Introduction: Covid-19 has generated several psychological impacts on people's lives and brought new challenges for dealing with the pandemic's repercussions. Objective: The objective was to analyse the relationship between the symptoms of fear of Covid-19, anxiety, and depression using different samples extracted at three distinct time-points. This study performed a network analysis of the fear of Covid-19 and anxiety- and depression -related symptoms based on data collected during the Covid-19 pandemic. Method: Three yearly cross-sectional surveys were conducted based on independent samples (2020-2022) of Brazilian adults (Time-point 1, Time-point 2, and Time-point 3 with 1,520, 1,523, and 1,517 participants, respectively). The Fear of Covid-19 Scale, the Generalised Anxiety Disorder Scale (7 items), and the Patient Health Questionnaire (9 items) were applied. The network structure invariance, the global strength invariance, and the strength of centrality index between the samples were compared. Results: Items from the three instruments had high values (>70%) in all samples (excluding the fear of Covid-19 at Time-point 3). Depression and anxiety symptoms increased, while fear-related behaviours decreased. Network analysis indicates that networks became progressively more parsimonious, and few edges connected different scales. Network comparisons revealed similar structures, but key differences were found: Time-point 1 showed significantly higher global connectivity, and changes in edge weights were observed only within the scales. Conclusions: Our findings suggest that the pandemic had a greater disorganising impact at its beginning, with a continuous increase in the mental health symptomatology and a reduction in the ratio between fear and anxiety and/or depression.

Introducción: El COVID-19 generó varios impactos psicológicos en la vida de las personas imponiendo nuevos desafíos para confrontar la pandemia. Objetivo: Analizar la relación entre los síntomas de miedo al COVID-19, ansiedad y depresión utilizando diferentes muestras extraídas durante tres períodos de tiempo distintos. Fueron analizadas redes del miedo y los síntomas de ansiedad y depresión usando datos recolectados durante la pandemia de COVID-19. Método: Tres encuestas transversales anuales basadas en muestras independientes (2020-2022) de adultos brasileños (punto temporal 1, punto temporal 2 y punto temporal 3 con 1520, 1523 y 1517 participantes, respectivamente). Se aplicaron la Escala de Miedo al COVID-19, la Escala de Trastorno de Ansiedad Generalizada (7 ítems) y el Cuestionario de Salud del Paciente (9 ítems). Se compararon la invariancia de la estructura de la red, la invariancia de la fuerza global y la fuerza del índice de centralidad entre los tres puntos temporales. Resultados: Los ítems de los tres instrumentos tuvieron valores altos (> 70 %) en todos los puntos temporales (excluyendo el miedo al COVID-19 en el punto temporal 3). A lo largo de los tres puntos temporales, los síntomas de depresión y ansiedad aumentaron, mientras que el miedo disminuyó. Las redes se volvieron progresivamente más parsimoniosas y pocas aristas conectaron diferentes escalas, con estructuras similares. Conclusiones: La pandemia tuvo un mayor impacto desorganizador en su inicio, con un aumento continuo de la sintomatología de salud mental y una disminución de la relación entre miedo y ansiedad o depresión.

Identifying the most important, frequent, and physically demanding tasks of Brazilian firefighters.

This study aimed to identify the most important, frequently performed, and physically demanding tasks performed by Brazilian firefighters and to identify tasks that could be used to assess physical fitness. A subjective task analysis was conducted. Five hundred twenty-four firefighters (84% male; 16% females) responded to an online survey and rated 37 tasks across three domains (most important, most frequent, and most physically demanding). A dichotomous decision analysis was used to inform the proposed physical fitness tests. Wildland firefighting tasks presented the highest overall mean rate. Traffic control was considered the most important and frequently performed task. Lifeguard rescue was considered the most physically demanding task. The dichotomous analysis identified 14 essential tasks (seven structural firefighting and seven automobile accidents). The tasks identified may be helpful in developing criterion physical fitness tests and training programs related to firefighters' demands.Practitioner summary: The unpredictability, variability, and dangerousness of firefighting make it challenging to observe the physical demands imposed on firefighters. A subjective task analysis was conducted to identify essential tasks performed by Brazilian firefighters. Wildland firefighting, lifeguard rescue, automobile accidents, and structural firefighting tasks were the most important, frequent, and physically demanding.

Patient Satisfaction with Healthcare Services and the Techniques Used for its Assessment: A Systematic Literature Review and a Bibliometric Analysis.

Patient satisfaction with healthcare provision services and the factors influencing it are be-coming the main focus of many scientific studies. Assuring the quality of the provided services is essential for the fulfillment of patients' expectations and needs. Thus, this systematic review seeks to find the determinants of patient satisfaction in a global setting. We perform an analysis to evaluate the collected literature and to fulfill the literature gap of bibliometric analysis within this theme. This review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) approach. We conducted our database search in Scopus, Web of Science, and PubMed in June 2022. Studies from 2000-2021 that followed the inclusion and exclusion criteria and that were written in English were included in the sample. We ended up with 157 articles to review. A co-citation and bibliographic coupling analysis were employed to find the most relevant sources, authors, and documents. We divided the factors influencing patient satisfaction into criteria and explanatory variables. Medical care, communication with the patient, and patient's age are among the most critical factors for researchers. The bibliometric analysis revealed the countries, institutions, documents, authors, and sources most productive and significant in patient satisfaction.

High-touch surfaces disinfection compliance in a COVID-19 intensive care unit.

Environmental cleaning and disinfection are fundamental health care-associated infection prevention measures. This study aimed to evaluate the disinfection compliance of high-touch surfaces in a COVID-19-only intensive care unit, using a fluorescent marker. It was divided into 3 phases, baseline assessment, educational feedback, and post feedback. Disinfection compliance improved significantly from the first to the third phase, 14.3% to 51.4% (P < .001), respectively.

Conceptions of Hegemonic Masculinity as a Mediator of Sexism Directed at Women / Concepções de Masculinidade Hegemônica como Mediadora do Sexismo Direcionado às Mulheres

ABSTRACT This article analyses the relationship between masculine identity and ambivalent sexism, considering concepts of masculinity and culture of honour as mediating variables in this relationship. For this purpose, two studies were carried out with male participants from two regions of Brazil, Midwest (119 participants) and Northeast (117 participants). The results indicated that the concepts of masculinity are mediators of the relationship between masculine identity and ambivalent sexism. In contrast, the culture of honour did not remain significant in the mediation model. We conclude that the conception of masculinity based on hegemonic precepts of gender restates masculine superiority in detriment to the feminine, naturalizes sexism and violent behaviour for maintenance of masculine identity.

RESUMO Este artigo analisa a relação entre identidade masculina e sexismo ambivalente, tomando as concepções de masculinidade e a cultura da honra como variáveis mediadoras dessa relação. Para alcançar este objetivo, foram realizados dois estudos com participantes homens de duas regiões do país, Centro-oeste (119 participantes) e Nordeste (117 participantes). Os resultados indicaram que as concepções de masculinidade são mediadoras da relação entre identidade masculina e o sexismo ambivalente. Em contraste, a cultura da honra não se manteve significativa no modelo da mediação. Conclui-se que a concepção de uma masculinidade, baseada em preceitos hegemônicos de gênero, reafirma a superioridade masculina em detrimento à feminina, e naturaliza práticas de sexismo e comportamentos violentos em defesa da manutenção da identidade masculina.

BNT162b2 against COVID-19 in Brazil using a test-negative design: Study protocol and statistical analysis plan.

INTRODUCTION: Real-world data on COVID-19 vaccine effectiveness are needed to validate evidence from randomized clinical trials. Accordingly, this study aims to evaluate, in a real-world setting in Brazil, the effectiveness of Pfizer-BioNTech BNT162b2 against symptomatic COVID-19 and COVID-19-related complications across diverse populations. MATERIALS AND METHODS: A test-negative case-control study with follow-up of cases is currently being conducted in Toledo, a city in southern Brazil, following a mass COVID-19 vaccination campaign with BNT162b2. The study is being conducted among patients aged 12 years or older seeking care in the public health system with acute respiratory symptoms and tested for SARS-CoV-2 on reverse transcription polymerase chain reaction (RT-PCR). Cases are RT-PCR positive and controls RT-PCR negative. Test-positive cases are prospectively followed through structured telephone interviews performed at 15 days post-enrollment, and at 1, 3, 6, 9 and 12 months. Baseline demographic, clinical, and vaccination data are being collected by means of structured interviews and medical registry records reviews at the time of enrollment. All RT-PCR-positive samples are screened for mutations to identify SARS-CoV-2 variants. ETHICS AND DISSEMINATION: The study protocol has been approved by the research ethics committee of all participant sites. Study findings will be disseminated through peer-reviewed publications and conference presentations. TRAIL REGISTRATION: Clinicatrials.gov: NCT05052307.

A Critical Analysis of Decentralizing the Portuguese Public Healthcare Provision Services.

The Portuguese health system has a universal, public, general National Health Service (NHS), tending towards free healthcare access. Created in 1979, this delivery model developed from the integration and complementarity between the different response levels (primary, hospital, continuing, and palliative care). However, over the last 40 years, the initially centralized system underwent a decentralization process with the creation of Regional Health Administrations in the five mainland administrative regions. Since then, the entire NHS has settled around this new organization. The most recent step started in 2018 with the decentralization of primary healthcare skills to 190 municipalities. This paper presents the various critical issues involved in the latest gradual decentralization process in health, intending to bring services closer to the citizens, and to be more focused on their needs. The article identifies and discusses the implications of this experience based on the steps foreseen in the already-published legal texts.