Exploring the Antioxidant and Anti-Inflammatory Potential of Saffron (Crocus sativus) Tepals Extract within the Circular Bioeconomy.

Repurposing saffron (Crocus sativus) waste presents a sustainable strategy for generating high-value products within the bioeconomy framework. Typically, flower components are discarded after stigma harvest, resulting in significant waste-350 kg of tepals per kilogram of stigmas. This research employed a comprehensive approach, integrating bioactivity studies (in vitro and in silico) with Life Cycle Assessment (LCA) evaluations, to extract and assess bioactive compounds from C. sativus tepals sourced in Tuscany, Italy. Phytochemical characterization using UPLC-MS/MS revealed a high abundance and variety of flavonoids in the hydro-ethanolic extract (CST). The antioxidant capacity was validated through various assays, and the ability to mitigate H2O2-induced oxidative stress and enhance fermentation was demonstrated in Saccharomyces cerevisiae. This study reports that C. sativus tepals extract reduces oxidative stress and boosts ethanol fermentation in yeast, paving the way for applications in the food and biofuels sectors. Further validation in RAW 264.7 macrophages confirmed CST's significant anti-inflammatory effects, indicating its potential for pharmaceutical, cosmeceutical, and nutraceutical applications. In silico studies identified potential targets involved in antioxidant and anti-inflammatory processes, shedding light on possible interaction mechanisms with Kaempferol 3-O-sophoroside (KOS-3), the predominant compound in the extract. The integration of LCA studies highlighted the environmental benefits of this approach. Overall, this research underscores the value of using waste-derived extracts through "green" methodologies, offering a model that may provide significant advantages for further evaluations compared to traditional methodologies and supporting the circular bioeconomy.

The Interplay of TLR-NFκB Signalling Pathway and Functional Immune-Related Enzymes in the Inflammatory Response of Ciona robusta.

The close phylogenetic relationship between ascidians (Tunicata) and vertebrates makes them a powerful model for studying the innate immune system. To better understand the nature and dynamics of immune responses and the mechanisms through which bacterial infections are detected and translated into inflammation in Ciona robusta, we applied an approach combining in vivo lipopolysaccharide (LPS) stimulation, immune-labelling techniques and functional enzymatic analyses. The immunohistochemistry showed that Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NFκB) were expressed during the inflammatory pharynx response 4 h post-LPS, with the formation of nodules in pharynx vessel lumen. Also, the endothelium vessels were involved in the inflammatory response. Observations of histological sections from naive and buffer-inoculated ascidians confirmed an immuno-positive response. Enzyme immune parameters-which included the activity of phenoloxidase, glutathione peroxidase, lysozyme, alkaline phosphatase and esterase-showed up-modulation 4 h after LPS injection, confirming their participation during ascidian inflammatory response. These findings provide new insights into the mechanisms underlying the LPS-induced C. robusta response and suggest that a broad innate immune mechanism, as in vertebrates, is involved in the regulation of inflammatory responses. Further findings in this direction are needed to cover knowledge gaps regarding the organized set of molecular and cellular networks involved in universal immune interactions with pathogens.

Cardiac Autonomic Neuropathy in Graves' Disease: Smoking and Age as Predictive Factors.

OBJECTIVES: Hypermetabolic state in Graves' disease (GD) has a great impact on heart homeostasis, acting directly on the heart muscle and modulating the autonomic nervous system. To characterize cardiac autonomic neuropathy (CAN) as a possible complication in patients with GD. METHODS: We evaluated euthyroid GD patients and a control group of healthy euthyroid people. CAN was assessed using autonomic tests of cardiovascular reflex and heart rate variability: respiratory, Valsalva, orthostatic and orthostatic hypotension tests, high frequency, low frequency, and very low-frequency bands. Transthoracic echocardiography was performed in GD patients. RESULTS: Sixty GD patients and 50 people in control group were assessed. CAN was diagnosed in 20% of GD and 14% in the control group. Among GD, 13.3% presented incipient, and 6.7% established CAN, while in the control group, it was verified incipient in 8% and established in 6% (P = .7479). All GD patients with CAN presented an alteration in the deep breathing test. Age and smoking were evidenced as factors associated with the presence of CAN, while higher TRAb values at diagnosis decreased the chance of CAN. CONCLUSIONS: The prevalence of CAN in euthyroid GD patients was 20%. Changes in the cardiac autonomic nervous system were identified, pointing to the importance of evaluating this complication in these patients. Smoking was a predictive factor for CAN, increasing its relationship with conditions that aggravate GD.

Impact of age on tumor characteristics and treatment outcomes in pediatric Differentiated Thyroid Carcinoma.

PURPOSE: There is a tendency to use data generated for adults in the management of pediatric Differentiated Thyroid Carcinoma, neglecting the clinical peculiarities of this condition in childhood. This study aimed to assess and compare the clinical-epidemiological characteristics and their significance in the evolution of thyroid carcinoma diagnosed in childhood across different age groups. METHODS: Seventy-seven patients diagnosed with Differentiated Thyroid Carcinoma (DTC) up to 21 years old were selected and divided into different age groups: up to 10 years, 11 to 18 years, and 19 to 21 years old. Clinical-epidemiological data and their influence in the disease progression were analyzed and compared across age groups. RESULTS: Patients diagnosed below 10 years of age were associated with tumors showing extrathyroidal extension, metastasis in regional lymph nodes, higher levels of stimulated thyroglobulin in the diagnostic iodine-131 whole-body scan (WBS), and under TSH suppression in the last assessment. Additionally, pulmonary metastasis were associated in both diagnostic and post-radioiodine dose WBSs in these younger patients. Analysis of findings in the post-radioiodine therapy WBS revealed significant differences between all age groups (p = 0.0029). The time of diagnosis was identified as a factor associated with an excellent response in subgroups up to 18 years and up to 21 years. No factors associated with dynamic responses over the 1st, 3rd and 5th years of follow-up and the persistence/recurrence of the disease were identified in the subgroup up to 18 years. In the subgroup up to 21 years, having an incomplete structural response in the 3rd year of follow-up increased the chances of recurrent or persistent response by 5.5 times, and by 32.6 times if found in the 5th year of follow-up. CONCLUSIONS: Younger patients exhibited more aggressive tumor characteristics and underwent more rigorous treatment. However, treatment response and disease status in the last assessment, whether free or recurrent/persistence, were similar when comparing the age groups of 11 to 18 and 19 to 21 years. Nonetheless, responses obtained in the 3rd and 5th years post-treatment emerged as factors associated with the persistence/recurrence of the disease in the last assessment in the age group up to 21 years but not in patients diagnosed up to 18 years, a relevant distinction considering the tumor behavior in defining the pediatric age range in thyroid cancer.

Equinins as Novel Broad-Spectrum Antimicrobial Peptides Isolated from the Cnidarian Actinia equina (Linnaeus, 1758).

Sea anemones are valuable for therapeutic research as a diversified source of bioactive molecules, due to their diverse bioactive molecules linked to predation and defence mechanisms involving toxins and antimicrobial peptides. Acid extracts from Actinia equina tentacles and body were examined for antibacterial activity against Gram-positive, Gram-negative bacteria, and fungi. The peptide fractions showed interesting minimum inhibitory concentration (MIC) values (up to 0.125 µg/mL) against the tested pathogens. Further investigation and characterization of tentacle acid extracts with significant antimicrobial activity led to the purification of peptides through reverse phase chromatography on solid phase and HPLC. Broad-spectrum antimicrobial peptide activity was found in 40% acetonitrile fractions. The resulting peptides had a molecular mass of 2612.91 and 3934.827 Da and MIC ranging from 0.06 to 0.20 mg/mL. Sequencing revealed similarities to AMPs found in amphibians, fish, and Cnidaria, with anti-Gram+, Gram-, antifungal, candidacidal, anti-methicillin-resistant Staphylococcus aureus, carbapenemase-producing, vancomycin-resistant bacteria, and multi-drug resistant activity. Peptides 6.2 and 7.3, named Equinin A and B, respectively, were synthesized and evaluated in vitro towards the above-mentioned bacterial pathogens. Equinin B exerted interesting antibacterial activity (MIC and bactericidal concentrations of 1 mg/mL and 0.25 mg/mL, respectively) and gene organization supporting its potential in applied research.

Unraveling the interplay between environmental microplastics and salinity stress on Mytilus galloprovincialis larval development: A holistic exploration.

The rise of plastic production has triggered a surge in plastic waste, overwhelming marine ecosystems with microplastics. The effects of climate change, notably changing salinity, have shaped the dynamics of coastal lagoons. Thus, understanding the combined impact of these phenomena on marine organisms becomes increasingly crucial. To address these knowledge gaps, we investigated for the first time the interactive effects of environmental microplastics (EMPs) and increased salinity on the early development of Mytilus galloprovincialis larvae. Morphological assessments using the larval embryotoxicity test revealed larval anomalies and developmental arrests induced by EMPs and increased salinity. Transcriptomic analyses targeting 12 genes involved in oxidative stress, apoptosis, DNA repair, shell formation, and stress proteins were conducted on D-larvae uncovered the potential effects of EMPs on shell biomineralization, highlighting the role of Histidine Rich Glycoproteine (HRG) and tubulin as crucial adaptive mechanisms in Mytilus sp. in response to environmental shifts. Furthermore, we explored oxidative stress and neurotoxicity using biochemical assays. Our findings revealed a potential interaction between EMPs and increased salinity, impacting multiple physiological processes in mussel larvae. Our data contribute to understanding the cumulative effects of emerging anthropogenic pollutants and environmental stressors, emphasizing the need for a holistic approach to assessing their impact on marine ecosystems.

Scolopax rusticola Carrying Enterobacterales Harboring Antibiotic Resistance Genes.

The Eurasian woodcock (Scolopax rusticola) belongs to those bird species that make systematic migratory flights in spring and autumn in search of favorable breeding and wintering areas. These specimens arrive in the Mediterranean Area from northeastern European countries during the autumn season. The purpose of this study was to assess whether woodcocks can carry antibiotic resistance genes (ARGs) along their migratory routes. Although the role of migratory birds in the spread of some zoonotic diseases (of viral and bacterial etiology) has been elucidated, the role of these animals in the spread of antibiotic resistance has not yet been clarified. In this study, we analyzed the presence of beta-lactam antibiotic resistance genes. The study was conducted on 69 strains from 60 cloacal swabs belonging to an equal number of animals shot during the 2022-2023 hunting season in Sicily, Italy. An antibiogram was performed on all strains using the microdilution method (MIC) and beta-lactam resistance genes were investigated. The strains tested showed no phenotypic resistance to any of the 13 antibiotics tested; however, four isolates of Enterobacter cloacae and three of Klebsiella oxytoca were found to carry the blaIMP-70, blaVIM-35, blaNDM-5 and blaOXA-1 genes. Our results confirm the importance of monitoring antimicrobial resistance among migratory animals capable of long-distance bacteria spread.

Effects of Proprioceptive Insoles and Specific Core Training on Postural Stability for Preventing Injuries in Tennis.

Tennis is a complex sport based on unpredictability that requires adequate physical and psychological preparation to prevent injuries. The aim of this study was to investigate the effects of 8-week specific core stability training on postural stability in competitive adolescent tennis players, aged between 14 and 19 years old. Sixty-one participants were randomly allocated into two groups: experimental (n = 32) and control (n = 29) groups. The first group wore proprioceptive insoles 8 h a day and performed a detailed training 3 times a week for 8 weeks; the second group only received proprioceptive insoles to wear 8 h a day for 8 weeks. The postural stability parameters (center-of-pressure length, center-of-pressure velocity, and 95% confidence ellipse sway area) included three assessment times: baseline (T0), intermediate test (T1), post-test (T2), and retention test (T3). Data analysis showed a significant improvement in the experimental group compared with the control group, indicating a large effect size in center-of-pressure length, ellipse sway area, and center-of-pressure velocity at T2 and T3 (p < 0.05). In conclusion, our results suggest that a specific and detailed core stability training plays a significant role in improving balance and postural stability in young tennis players, especially in terms of preventing the risk of injury.

Experimental study on physical exercise in diabetes: pathophysiology and therapeutic effects.

Diabetes is a chronic disease. Some complications can be prevented, their effects can be slowed down.  Sedentary lifestyle increases the risk of obesity and consequently the predisposition to diabetes II. The article aimed to demonstrate the positive and negative effects of exercise on active and sedentary diabetics and on pathophysiology, evaluating the effects after 3 and 6 months. The study involved 90 participants, both male and female, with type II diabetes, aged 45, divided into two groups: Group A (n=50, sedentary) and Group B (n=40, active). We evaluated anthropometric parameters, blood chemistry values, which are fundamental for the transversal evaluation of the results. In group A improvements were less noticeable than group B. The most improved parameter is blood sugar, Glycemic values and BMI. Cholesterol and Hb1Ac decreased but more slowly than previous parameters. The expectations of the study were, not only in recognizing the therapeutic and preventive powers of exercise, but above all in choosing to program a motor protocol after a team work between diabetologist, sports doctor and kinesiologist and/ or personal trainer. Physical activity is an additional therapy to insulin.

Ecosystem degradation and the spread of Covid-19.

The linkages between the emergence of zoonotic diseases and ecosystem degradation have been widely acknowledged by the scientific community and policy makers. In this paper we investigate the relationship between human overexploitation of natural resources, represented by the Human Appropriation of Net Primary Production Index (HANPP) and the spread of Covid-19 cases during the first pandemic wave in 730 regions of 63 countries worldwide. Using a Bayesian estimation technique, we highlight the significant role of HANPP as a driver of Covid-19 diffusion, besides confirming the well-known impact of population size and the effects of other socio-economic variables. We believe that these findings could be relevant for policy makers in their effort towards a more sustainable intensive agriculture and responsible urbanisation.

Step-by-Step Regeneration of Tentacles after Injury in Anemonia viridis-Morphological and Structural Cell Analyses.

Benthic marine invertebrates, such as corals, are often subjected to injury caused by several sources. Here, the differences and characteristics in injured and health tissues in terms of cellular components are shown through a histological investigation of the soft coral Anemonia viridis at 0 h, 6 h, 24 h, and 7 days after injury caused by tentacle amputation. In addition, a new tool was used for the first time in invertebrates, positron emission tomography, in order to investigate the events that occur during regeneration within a longer time period (0 h, 24 h, and 14 days after the tentacles were cut). Higher integrated density values were measured through a densitometric analysis in sections stained with Fontana-Masson at 24 h after the tentacles were cut. This suggests an increase in melanin-like containing cells and a subsequent increase in fibroblast-like cells differentiated by amoebocytes that converge to the lesion site in the early stages of inflammation and regeneration. This work provides, for the first time, an elucidation of the events that occur during wound-healing and regeneration in basal metazoan, focusing on the characterisation of immune cells and their role. Our results indicate that Mediterranean anthozoan proves to be a valuable model for studying regeneration. Many events highlighted in this research occur in different phyla, suggesting that they are highly conserved.

Short-Term Effects of Specific Sensorimotor Training on Postural Assessment in Healthy Individuals: A Pilot Study with a Randomized Placebo-Controlled Trial.

It is well-known that sensorimotor training aims to increase the performance of the sensorimotor system to maintain an upright position. Through the use of a randomized placebo-controlled trial, the specific aim of this study was to investigate the short-term effects of a specific session of sensorimotor training on postural balance, stability and coordination in healthy, recreationally active participants. Ninety subjects were randomly allocated into three groups: experimental (n = 30), placebo (n = 32) and control (n = 28). The experimental group performed a 5 min warm-up, with the sensorimotor training consisting of 60-min specific sensorimotor exercises; the control group was not allowed to perform any sensorimotor training; the placebo group observed a video clip of an individual belonging to the experimental group performing the sensorimotor training accurately. All participants were seen three times per week for 4 weeks. Before and after the entire training, all groups of participants undertook stabilometric parameter assessment. The intervention-mediated sensorimotor training confirmed significant enhancement in the proprioceptive system. Significant improvement in the motor and/or sensory function was observed in the experimental and placebo groups. In conclusion, our findings suggest that specific sensorimotor training performed 3 days per week for 4 weeks could improve postural balance, stability and coordination in healthy individuals.

Response of Sabella spallanzanii to multiple stressors. The combined effect of infection and copper sulphate.

The aim of this work is to study the immune responses of the polychaete Sabella spallanzanii after exposure to copper sulphate, an immunomodulating agent in marine organisms, and the multiple stresses caused by Escherichia coli infection, to validate the species as a model organism in marine-coastal biomonitoring programmes. Polychaetes were housed in laboratory and divided into five experimental groups: 1. Control (no microinjected), 2. filtered seawater + TBS injection (control of point 3), 3. filtered seawater + E. coli injection (control of point 4), 4. CuSO4 + TBS injection (control of point 5), and 5. CuSO4 + E. coli injection. The immune variables, esterase and alkaline phosphatase activity, cytotoxicity and detoxifying/antioxidant enzymes such as glutathione peroxidase were evaluated in total body extracts of the animals. Moreover, toll-like receptor, allograft inflammatory factor-1, lysozyme and haemagglutinating activity were investigated to highlight possible interactions. Indeed, the results of this work demonstrate the immunomodulating effect of copper sulphate on S. spallanzanii total body extracts related to oxidative stress and inflammatory markers.

Semblance of a physician: medical students' experiences during Type 2 Diabetes Mellitus clinic simulation / Com o semblante de médico: experiências de estudantes de medicina durante a simulação clínica em Diabetes tipo 2 / Con el semblante de médico: experiencias de estudiantes de medicina durante la simulación clínica en Diabetes tipo 2

This qualitative research aimed to understand the experiences of a fourth-year medical class during two clinical simulation scenarios of type 2 diabetes management in Primary Care. Ten simulated students were interviewed. The results describe the symbolic conception of diabetes and its treatment as anguish, damage, condemnation and an unpleasant sentence that affects the appropriation and performance of the medical role by students. Considering Medical Psychology, we suggest that such subjective factors should be addressed in teaching-learning in addition to commonly cognitive aspects mentioned in the medical education literature in order for students to develop the work profile to cope with diabetes in Primary Care. Clinical simulation facilitates the subjective approach through its group support component to promote reflection, insights and self-awareness.(AU)

Esta pesquisa qualitativa objetivou compreender as experiências de estudantes de um quarto ano médico durante dois cenários de simulação clínica do manejo do diabetes tipo 2 na Atenção Primária. Dez estudantes simulados foram entrevistados. Os resultados descrevem a concepção simbólica do diabetes e seu tratamento como angústia, dano, condenação e uma sentença desagradável que afeta a apropriação e o desempenho do papel médico pelos estudantes. Considerando a Psicologia Médica, sugerimos que os fatores subjetivos apontados devem ser abordados no ensino-aprendizado além de aspectos cognitivos mais comuns na literatura de educação médica para que os estudantes desenvolvam o perfil de trabalho ao enfrentamento do diabetes na Atenção Primária. A simulação clínica permite esta abordagem subjetiva por seu componente de suporte grupal promovendo reflexão, insights e autoconsciência.(AU)

Esta encuesta cualitativa tuvo el objetivo de comprender las experiencias de estudiantes del cuarto año de medicina durante dos escenarios de simulación clínica del manejo de la diabetes tipo 2 en la Atención Primaria. Fueron entrevistados 10 estudiantes simulados. Los resultados describen la concepción simbólica de la diabetes y su tratamiento, tales como angustia, daño, condenación y una sentencia desagradable que afecta la apropiación y el desempeño del papel médico por parte de los estudiantes. Considerando la Psicología Médica, sugerimos que los factores subjetivos señalados deben abordarse en la enseñanza-aprendizaje, más allá de aspectos cognitivos más comunes en la literatura de educación médica para que los estudiantes desarrollen el perfil de trabajo para el enfrentamiento de la diabetes en la Atención Primaria. La simulación clínica permite este abordaje subjetivo por su componente de soporte grupal promoviendo reflexión, insights y autoconciencia.(AU)

Early Diagnosis and Linkage to Care: an Experience over 14 years of Point of Care Rapid HIV Testing.

Point-of-care rapid testing is one of the strategies to increase HIV screening. We present data on over 14 years of the "EASY Test Program", an ongoing cross-sectional collaborative project that provides free and anonymous rapid HIV testing in the metropolitan city of Milan, Italy. Overall, 22,186 HIV tests were performed, with a 0.52% prevalence of HIV infection; 100% of those diagnosed with HIV were linked to care. The "EASY Test Program" is an appropriate test-and-treat strategy, allowing a fast HIV assessment (24 hours). Motivated clinicians, in partnership with community associations, can perform an easy HIV screening out of hospitals in alternative settings, among individuals who in the majority of cases had never tested for HIV, ultimately providing an effective linkage to care.

Higher systemic inflammatory status and cardiovascular risk associated with Charcot arthropathy unrelated to infection or extremity amputation.

Purpose: People with diabetes and Charcot arthropathy have higher mortality than people with diabetes without this complication. Are the causes of this higher mortality exclusively infectious or of a cardiovascular origin? We aimed to study aspects related to cardiovascular risk and inflammation in a population of people with type 2 diabetes with and without Charcot arthropathy. Methods: A cross-sectional study was performed in people with diabetes and Charcot Eickenholtz III arthropathy, matched for sex and age, with two groups of people with diabetes without Charcot arthropathy with and without peripheral sensory-motor neuropathy, in the absence of active infection. All participants underwent clinical and laboratory evaluation at the time of the interview, and their cardiovascular risk was calculated according to the United Kingdom Prospective Diabetes Study (UKPDS) calculator. Results: We evaluated 69 people with type 2 diabetes (21 with Charcot arthropathy, 24 with diabetic peripheral neuropathy and 24 without this neuropathy), with a mean age between 57 and 61 years and with a diabetes duration of more than 10 years. Patients with Charcot arthropathy had a longer duration of diabetes; more frequency of dyslipidemia; and higher levels of uric acid, microalbuminuria and C-reactive protein than the other two groups. A total of 73.9% of the people evaluated had high or very high cardiovascular risk according to the UKPDS score. Conclusion: The people with type 2 diabetes presenting Charcot arthropathy had greater values of systemic inflammatory parameters, even in the chronic phase of the complication, with a higher risk of mortality unrelated to infections.

Full Diabetic Foot Ulcer Healing and Pain Relief Based on Platelet-Rich-Plasma gel Formulation Treatment and the Involved Pathways.

Diabetic foot ulcer is a severe Diabetic Mellitus-associated complication. It is induced by poor glycemic control, which leads to peripheral neuropathy and vascular diseases. Platelet-rich plasma could be beneficial for healing processes due to its active biomolecules that promotes immunomodulation, angiogenesis, cell proliferation and analgesia.

Invertebrate Models in Innate Immunity and Tissue Remodeling Research.

The aim of this Special Issue is to highlight the close functional and highly conserved link between innate immunity, homeostasis maintenance, inflammation, tissue remodeling and regeneration [...].

Non-Alcoholic Fatty Liver Disease in Long-Term Type 2 Diabetes: Role of rs738409 PNPLA3 and rs499765 FGF21 Polymorphisms and Serum Biomarkers.

Fibroblast growth factor 21 (FGF21) signaling and genetic factors are involved in non-alcoholic fatty liver disease (NAFLD) pathogenesis. However, these factors have rarely been studied in type 2 diabetes mellitus (T2D) patients from admixed populations such as in those of Brazil. Therefore, we aimed to evaluate rs738409 patanin-like phospholipase domain-containing protein (PNPLA3) and rs499765 FGF21 polymorphisms in T2D, and their association with NAFLD, liver fibrosis, and serum biomarkers (FGF21 and cytokeratin 18 levels). A total of 158 patients were included, and the frequency of NAFLD was 88.6%, which was independently associated with elevated body mass index. Significant liver fibrosis (≥F2) was detected by transient elastography (TE) in 26.8% of NAFLD patients, and was independently associated with obesity, low density lipoprotein, and gamma-glutamyl transferase (GGT). PNPLA3 GG genotype and GGT were independently associated with cirrhosis. PNPLA3 GG genotype patients had higher GGT and AST levels; PNPLA3 GG carriers had higher TE values than CG patients, and FGF21 CG genotype patients showed lower gamma-GT values than CC patients. No differences were found in serum values of FGF21 and CK18 in relation to the presence of NAFLD or liver fibrosis. The proportion of NAFLD patients with liver fibrosis was relevant in the present admixed T2D population, and was associated with PNPLA3 polymorphisms.

Cardiovascular Autonomic Reflex Tests and 7 Heart Rate Variability Indices for Early Diagnosis of Cardiovascular Autonomic Neuropathy in Type 2 Diabetes Individuals.

BACKGROUND: Cardiovascular autonomic neuropathy (CAN) is a common complication of type 2 Diabetes mellitus (T2D), and prevalence varies according to the methodology used. CAN should be diagnosed in the subclinical stage when an intensive treatment of T2D could avoid the progression to irreversible phases. OBJECTIVE: Determine the prevalence of early involvement (EI) of CAN in T2D individuals comparing two methodologies. METHODS: This was a cross-sectional study that included 183 T2D individuals who were monitored in a Tertiary centre. The diagnosis of CAN was based on the results of four cardiovascular autonomic reflex tests (CARTs: expiration-inspiration index, Valsalva maneuver, orthostatic test, and changes in blood pressure after standing) and of seven heart rate variability (7HRV) indices (CARTs plus the spectral analysis). The findings were validated in an independent cohort comprised of 562 T2D individuals followed in a Primary care setting. RESULTS: With the use of 7HRV, 30.6% and 77.8% of individuals in the Tertiary and in the Primary centers, respectively, were classified as without CAN; 25.1% and 15.3% as EI and 44.3% and 6.9% as definitive CAN, respectively. The use of CARTs decreased the proportion of individuals without CAN in both centers (7.1% and 47%) and increased the frequency of EI (30.6% and 36.6%) and definitive CAN (62.3% and 16.4%), respectively. The concordance between both evaluated methodologies was weak. CONCLUSION: Higher proportions of T2D individuals were diagnosed with EI and with definitive CAN with the use of CARTs.