Search

Background: The combination of highly localized impedance (LI) and contact force (CF) may improve tissue characterization and lesion prediction during radiofrequency (RF) pulmonary vein isolation (PVI) in patients with atrial fibrillation (AF)., Objective: We report the outcomes of our acute and long-term clinical evaluation of CF-LI-guided PVI in consecutive AF ablation cases from an international multicenter clinical setting., Methods: Three hundred twenty-four consecutive patients from 20 European centers undergoing RF catheter ablation with the Stablepoint™ catheter were enrolled in the CHARISMA registry. Of these, 275 had a minimum follow-up of 1 year and were included in the primary analysis., Results: The mean procedure duration was 115 ± 47 min, and the mean fluoroscopy time was 9.9 ± 6 min. At the end of the procedures, all PVs had been successfully isolated in all study patients. Minor complications were reported in 12 patients (4.4%). At 1 year, 36 (13.1%) patients had had an AF recurrence, and freedom from antiarrhythmic drugs and AF recurrence was achieved in 228 (82.9%) patients. The recurrence rate was higher in patients with persistent AF (21/116, 18.1%) than in those with paroxysmal AF (15/159, 9.4%; p = 0.0459). On multivariate logistic analysis adjusted for baseline confounders, only time > 6 months from first diagnosis of AF to ablation (HR = 2.93, 95%CI 1.03 to 8.36, p = 0.0459) was independently associated with recurrences., Conclusion: An ablation strategy for PVI guided by CF-LI technology proved safe and effective and resulted in a low recurrence rate of AF over 1-year follow-up, irrespective of the underlying AF type., Clinical Trial Registration: Catheter Ablation of Arrhythmias with a High-Density Mapping System in Real-World Practice. (CHARISMA). URL: http://clinicaltrials.gov/ Identifier: NCT03793998., Competing Interests: Declarations. Ethics approval: This study was performed in line with the principles of the Declaration of Helsinki. The locally appointed Ethics Committee approved the research protocol. Consent to participate: Informed consent was obtained from all individual participants included in the study. Consent for publication: Patients signed informed consent regarding publishing their data. Conflict of interest: M. Malacrida and R. Ricci Maga are employees of Boston Scientific. The other authors have no conflicts of interest to declare that are relevant to the content of this article., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Recent data on pulsed field ablation (PFA) for atrial fibrillation (AF) ablation suggest a progressive reduction in procedural times. Real-world data regarding the relationship between the learning curve of PFA and clinical outcomes are scarce. The objective was to evaluate the PFA learning curve and its impact on acute outcomes., Methods: Consecutive patients undergoing AF ablation with the FARAPULSE™ PFA system were included in a prospective, non-randomized multicenter study. Procedural times were stratified on the operators' learning curve. Comparative analysis of skin-to-skin time was conducted with radiofrequency (RF) and cryoablation (CB) pulmonary vein isolation (PVI) procedures performed by the same operators in the previous year., Results: Among 752 patients, 35.1% were females, and 66.9% had paroxysmal AF; mean age was 62.2 ± 10 years. A total of 62.5% of procedures were performed by operators with > 20 PFA procedures. Both time to PVI (25.6 ± 10 min vs 16.5 ± 8, p < 0.0001) and fluoroscopy time (19.8 ± 8 min vs 15.9 ± 8 min, p = 0.0045) significantly improved after 10 associated with consistent linear trend towards procedural time reduction (R 2 0.92-0.68 across various procedural metrics). Current PFA skin-to-skin time was lower than the historical skin-to-skin one in 217 (62.4%) procedures; it was similar in 112 (32.2%) cases and higher than the historical skin-to-skin one in 19 (5.5%). No major complications were reported., Conclusions: In this nationwide multicentric experience, the novel PFA system proved to be fast, safe, and acutely effective in both paroxysmal and persistent AF patients. The learning curve appears to be rapid, as improvements in procedural parameters were observed after only a few procedures., Clinical Trial Registration: Advanced TecHnologies For SuccEssful AblatioN of AF in Clinical Practice (ATHENA). URL: http://clinicaltrials.gov/ Identifier: NCT05617456., Competing Interests: Declarations. Ethics approval: This study was performed in line with the principles of the Declaration of Helsinki. The locally appointed Ethics Committee approved the research protocol. Consent to participate: Informed consent was obtained from all individual participants included in the study. Consent for publication: Patients signed informed consent regarding publishing their data. Competing interests: M.M and A.V.P. are employees of Boston Scientific. S.B. and P.R. received teaching and proctor’s fees and honoraria from Boston Scientific and Medtronic. A.B. and M.P. received teaching and proctor’s fees and honoraria from Boston Scientific. G.F. is a clinical proctor for Boston Scientific. C.T. received speaker’s and proctor’s fees from Boston Scientific, Medtronic, Abbott, and Biosense Webster. He serves in the Advisory Board of Medtronic, Boston Scientific. S.B. and P.R. received speaker’s and proctor’s fees from Boston Scientific and Medtronic. The other authors have no conflicts of interest to declare. The other authors have no conflicts of interest to declare., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Food selectivity is common in children with Autism Spectrum Disorder (ASD). The treatment used can be invasive and difficult to implement, necessitating the exploration of multicomponent approaches. This study presents the case of a 9-year-old autistic girl with severe food selectivity, who ate exclusively liquid and semi-solid foods. A multicomponent intervention protocol was developed, including stimulus fading and positive reinforcement techniques, to increase acceptance of new textures and foods. Treatment sessions showed significant improvement in acceptance of semi-solid and novel foods, with a reduction in problem behaviors associated with mealtime. This study suggests that a multicomponent intervention can significantly improve food acceptance and reduce mealtime distress, proving to be a practical and effective treatment strategy in an autistic child. The intervention led to an increase in food acceptance and a reduction in mealtime-related distress, potentially improving the child and family's quality of life., Competing Interests: Authors RM, LT, CP were employed by Progetto Dopo di Noi Soc. Coop. Arl. Authors SS were employed by I Corrieri dell’Oasi Soc. Coop. Soc. Authors MG were employed by Cooperativa dalla Luna. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Maggio, Turriziani, Suraniti, Graziano, Patanè, Randazzo, Passantino, Di Cara, Quartarone and Cucinotta.)

Background: Worldwide, three million deaths each year are reported due to the harmful use of alcohol. To date, only a few drugs have been approved for the treatment of Alcohol Use Disorder (AUD). This systematic review and meta-analysis aim to assess the long-term efficacy and safety of sodium oxybate (SMO) treatment in patients with AUD., Methods: We followed the PRISMA statement guidelines and searched PubMed and ISI Web of Science to retrieve the studies of interest. In total, 13 studies on long-term (>12 weeks) SMO administra- tion in patients with AUD were included in this systematic review, and 7 were included in the meta- analysis., Results: Overall, the abstinence rate after 12 weeks of treatment was similar in the SMO and placebo groups, while it was significantly in favour of SMO compared to Naltrexone (NTX). The completion rate was similar in all three conditions. Mean corpuscular volume (MCV) levels favoured SMO over NTX, while Alcohol Craving Scale (ACS) scores did not favour SMO. The incidence of adverse reactions varied widely between studies., Conclusion: SMO in the chronic treatment of patients with AUD showed no superiority to placebo in our analysis of published RCTs, although many observational studies reported its beneficial effect in the long term. On the contrary, SMO was superior to NTX treatment on abstinence. The rate of study completion was similar in the three groups. Safety was not an issue in any of the studies included. Further studies are needed to better assess SMO efficacy and safety in the long term.

Background: No data have been reported on cooling characteristics and the impact of variant pulmonary vein (PV) anatomy on atrial fibrillation (AF) recurrences after POLARx cryoballoon (CB) ablation., Objective: The purpose of this study was to analyze the impact of PV anatomy variants and cooling characteristics after CB ablation from a large multicenter prospective registry., Methods: The primary end point was defined as 1-year absence of any atrial tachyarrhythmias (ATAs: AF/atrial flutter/atrial tachycardia). Correlation between ATA recurrences and anatomy variants/cooling characteristics were evaluated. The secondary outcome was the rate of major periprocedural complications., Results: A total of 429 consecutive patients diagnosed with paroxysmal AF (83.4%) or persistent AF (peAF; 16.6%) were enrolled. Twenty-eight patients (6.6%) exhibited an anatomical variant (common ostium: 4.0%; adjunctive PV: 2.6%). Nadir temperature, thaw time, and total deflation time were different between standard PVs and PV variants. After the blanking period, over a mean of 431 ± 99 days of follow-up, 63 patients (14.7%) suffered an ATA recurrence. Patients with recurrences had both a shorter thaw time (18.5 ± 7 seconds vs 19.8 ± 7 seconds; P = .0012) and a shorter total deflation time, whereas time to isolation was longer (57.4 ± 42 seconds vs 49.1 ± 33 seconds; P = .04). Patients with anatomy variants showed a similar ATA recurrence rate (5 of 28 [17.9%]) to the standard PV anatomy group (58 of 401 [14.5%]) (P = .584), with a hazard ratio (HR) of 1.43 (95% confidence interval [CI] 0.49-4.13; log-rank, P = .4384). After adjusting for confounders, heart failure (HR 4.12; 95% CI 1.75-9.73; P = .0013) and peAF (HR 1.81; 95% CI 1.03-3.18; P = .0433) remained associated with ATA recurrence during follow-up., Conclusion: The POLARx CB system demonstrated long-term efficacy, along with a safe profile, in both patients with paroxysmal AF and those with peAF, regardless of the presence PV variants. Time to isolation was longer in patients with ATA recurrences during follow-up., Clinical Trial Registration: Catheter Ablation of Arrhythmias with a High-Density Mapping System in Real-World Practice (CHARISMA)., Clinicaltrials: gov identifier: NCT03793998. Registration date: January 4, 2019., Competing Interests: Disclosures Mr Malacrida is a salaried employee of Boston Scientific. Dr Tondo is a member of the Boston Scientific advisory board. The other authors have no conflicts of interest to declare., (Copyright © 2024 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: Intracardiac echocardiography (ICE) is increasingly employed in atrial fibrillation (AF) ablation procedures, with the potential to enhance procedural efficacy. Nevertheless, there is currently a lack of evidence assessing the impact of ICE on the efficiency, effectiveness, and safety outcomes in the context of novel pulsed-field ablation (PFA) for AF., Purpose: We aimed to assess whether the use of ICE could improve procedural parameters in a large population undergoing AF ablation with FARAPULSE™ catheter., Methods: Consecutive patients who had undergone PFA of AF from nine Italian centers were included. In procedures where the ICE catheter was employed for guidance (ICE-guided group), it was used to maneuver the PFA catheter within the left atrium to achieve optimal contact with atrial structures., Results: We analyzed 556 patients: 357 (66%) with paroxysmal AF, 499 (89.7%) de novo PVI. ICE-guided procedures (n = 138) were propensity matched with patients with a standard approach (n = 138), and their outcomes were compared. During ICE-guided procedures, no improvement in procedural metrics was recorded (ICE vs Standard, 23 ± 6 min vs 18.5 ± 9 min for time to PVI, p < 0.0001; 38.8 ± 7 vs 32.5 ± 5 number of PFA deliveries to achieve PVI, p < 0.0001; 68.8 ± 19 min vs 71.8 ± 29 min for primary operator time, p = 0.5301; 16.1 ± 8 min vs 18.2 ± 10 min for fluoroscopy time, p = 0.5476) except for support time (76.8 ± 26 min vs 91.4 ± 37 min, p = 0.0046). No major procedure-related adverse events were reported., Conclusion: Our findings confirmed that PFA could be consistently performed in a rapid, safe, and efficacious manner. The use of ICE to guide PFA was not associated with an improvement in procedural metrics., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Cushing's syndrome due to ectopic ACTH secretion is a rare clinical condition resulting from a dysregulated ACTH secretion by neuroendocrine tumors. The overall incidence of endogenous Cushing's syndrome is 0.7-2.4 per million people annually, with children accounting for only 10% of new cases each year., Case Report: When the patient first presented clinically, she was a 17-year-old girl who displayed psychiatric symptoms. Blood tests showed diabetes mellitus and hypokalemia. She had hypertension and vertebral collapses. The hormone assay showed hypercortisolism, and because of this, she began taking metyrapone. The results of the 68Ga-PET/DOTATOC and 18FDG-PET scans were negative. The clinical course was intermittent. After one year, a 68Ga-PET/DOTATOC showed a nodule in the thymic lodge. Despite the thymectomy, the hypercortisolism persisted. A subsequent 68Ga-PET/DOTATOC repeated three months after surgery revealed an uptake corresponding to the thymic lodge. She underwent another surgery, which is finally being resolved. ACTH levels were monitored before, during, and post-surgery. The laboratory provided the ACTH results promptly, and thoracic surgeons waited for hormonal results before concluding the procedure. The adopted strategy permitted us to monitor the surgery outcome., Conclusion: The heterogeneity of ectopic Cushing's syndrome makes diagnosis difficult. 68Ga-PET/DOTATOC proves to be a tracer with good sensitivity and specificity for the identification of ACTH-secreting neuroendocrine lesions. The short half-life of ACTH is found to be a strategy to monitor the complete surgical resection of the neuroendocrine lesion.

Introduction: Limited data exist on pulsed-field ablation (PFA) in patients with persistent atrial fibrillation (PeAF) undergoing left atrial posterior wall isolation (LAPWI)., Methods: The Advanced TecHnologies For SuccEssful AblatioN of AF in Clinical Practice (ATHENA) prospective registry included consecutive patients referred for PeAF catheter ablation at 9 Italian centers, treated with the FARAPULSE TM -PFA system. The primary efficacy and safety study endpoints were the acute LAPWI rate, freedom from arrhythmic recurrences and the incidence of major periprocedural complications. Patients undergoing pulmonary vein isolation (PVI) alone, PWI + LAPWI and redo procedures were compared., Results: Among 249 patients, 21.7% had long-standing PeAF, 79.5% were male; mean age was 63 ± 9 years. LAPWI was performed in 57.6% of cases, with 15.3% being redo procedures. Median skin-to-skin times (PVI-only 68 [60-90] vs. PVI + LAPWI 70 [59-88] mins) did not differ between groups. 45.8% LAPWI cases were approached with a 3D-mapping system, and 37.3% with intracardiac echocardiography. LAPWI was achieved in all patients by means of PFA alone, in 88.8% cases at first pass. LAPWI was validated either by an Ultrahigh-density mapping system or by recording electrical activity + pacing maneuvers. No major complications occurred, while 2.4% minor complications were detected. During a median follow-up of 273 [191-379] days, 41 patients (16.5%) experienced an arrhythmic recurrence after the 90-day blanking period, with a mean time to recurrence of 223 ± 100 days and no differences among ablation strategies., Conclusion: LAPWI with PFA demonstrates feasibility, rapidity, and safety in real-world practice, offering a viable alternative for PeAF patients. LAPWI is achievable even with a fluoroscopy-only method and does not significantly extend overall procedural times., (© 2024 The Authors. Journal of Cardiovascular Electrophysiology published by Wiley Periodicals LLC.)

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fasciani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Our recent study revealed that fluorescent lamp light can penetrate deep into the brain of mice and rats leading to the development of typical histological characteristics associated with Parkinson's disease such as the loss of dopamine neurons in the substantia nigra. Monochromatic LED lights were thus used in this work to deepen our knowledge on the effects of the major wavelength peaks of fluorescent light on mouse and human dopaminergic cells. In particular, we exposed immortalized dopaminergic MN9D neuronal cells, primary cultures of mouse mesencephalic dopaminergic cells and human dopaminergic neurons differentiated from induced pluripotent stem cells (hiPSC) to different LED light wavelengths. We found that chronic exposure to LED light reduced overall undifferentiated MN9D cell number, with the most significant effects observed at wavelengths of 485 nm and 610 nm. Moreover, LED light especially at 610 nm was able to negatively impact on the survival of mouse mesencephalic dopaminergic cells and of human dopaminergic neurons derived from hiPSC. Notably, differentiated MN9D dopaminergic cells, which closely resemble mature dopamine neuronal phenotype, acutely exposed for 3 h at 610 nm, showed a clear increase in ROS production and cytotoxicity compared to controls undifferentiated MN9D cells. These increases were even more pronounced by the co-treatment with the oxidative agent H 2 O 2 . Collectively, these findings suggest that specific wavelengths, particularly those capable of penetrating deep into the brain, could potentially pose an environmental hazard in relation to Parkinson's disease., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Adrenal hemorrhage is a rare, but important, diagnosis to recognize, in particular when there is involvement of both adrenal glands. Bilateral adrenal hemorrhage can in fact lead to adrenal insufficiency, with dramatic consequences if not promptly recognized and treated. It is normally caused by systemic conditions that lead to the vasoconstriction and thrombosis of the adrenal vein. Oftentimes, the clinical diagnosis of this condition can be very challenging, as its signs and symptoms are generalized and nonspecific (abdominal pain, nausea, and fatigue). Here, we present the cases of two patients admitted to the Emergency Department in 2016 and 2022 with acute abdominal pain, having recently undergone surgery and subsequently prescribed low-molecular-weight heparin. In both cases, laboratory results revealed neutrophilic leukocytosis and an unexplained anemia. Due to the persistence of abdominal pain despite medication, a CT scan was performed, showing an enlargement of both adrenal glands suggestive of bilateral adrenal hemorrhage. Adrenal function was tested that correlated with a diagnosis of adrenal insufficiency, and both patients were promptly treated with parenteral hydrocortisone as a result. On 5 years' follow-up from the acute event, the second patient's adrenal function had returned to normal, and he has not needed further adrenal replacement therapy; the first patient however demonstrated persistence of adrenal failure requiring replacement therapy. In this paper, through our experience and a literature analysis, we will aim to outline some clues to identify patients at potential risk of bilateral adrenal hemorrhage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Aloini, Manella, Biondo, Maggio, Roberto, Ricci, Lardo, Addario Chieco and Stigliano.)

Introduction: Autism spectrum disorder is a lifelong neurodevelopmental disorder. The profile of functioning in autistic people is very heterogeneous, and it is necessary to take into account individual characteristics to better support integration in the workplace. However, unemployment rates are higher for autistic people than for other types of disabilities. We present a prospective case series to explore the feasibility and efficacy of an individual-supported program to enhance placement in a sheltered work environment delivered by an Italian community day care center., Methods: Autistic subjects, aged from 12 to 31 years, participated in an individual-supported program regarding employment in sheltered art workshops, integrated into the regular activity of a semi-residential center three times a week for 1 year. Their feasibility retention rate and time worked per session were registered; moreover, working methods efficacy and self-organization improvement were tracked by the Likert-based rating system. Secondary outcome measures span functional levels, challenge behaviors, and sensory problems., Results: All the individuals presented a good adaptation to the environment, with a significant increase in time worked per session. After 1 year, the intervention allowed an increase in tasks completed in an assigned complex job and an improvement in self-organization within the work schedule in a group of subjects consisting mainly of severe-to-moderate levels of autism severity (86.6%). Finally, we observed a significant increase in independent functioning areas of the TEACCH transitional assessment profile. Challenge behaviors and sensory problems were also recorded., Conclusion: This case series supports the idea that individual-supported programs for placement in sheltered job environments delivered by community day care centers could be feasible and effective for ASD with higher levels of severity and co-occurring intellectual disability. Further targeted studies based on community models and accessible methods need to be planned to define the effectiveness of the intervention and promote improved practice at the community level with a better social impact., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Maggio, Turriziani, Campestre, Di Cara, Tripodi, Impallomeni, Quartarone, Passantino and Cucinotta.)

Unstructured regions in functional proteins have gained attention in recent years due to advancements in informatics tools and biophysical methods. G protein-coupled receptors (GPCRs), a large family of cell surface receptors, contain unstructured regions in the form of the i3 loop and C-terminus. This review provides an overview of the functional significance of these regions in GPCRs. GPCRs transmit signals from the extracellular environment to the cell interior, regulating various physiological processes. The i3 loop, located between the fifth and sixth transmembrane helices, and the C-terminus, connected to the seventh transmembrane helix, are determinant of interactions with G proteins and with other intracellular partners such as arrestins. Recent studies demonstrate that the i3 loop and C-terminus play critical roles in allosterically regulating GPCR activation. They can act as autoregulators, adopting conformations that, by restricting G protein access, modulate receptor coupling specificity. The length and unstructured nature of the i3 loop and C-terminus provide unique advantages in GPCR interactions with intracellular protein partners. They act as "fishing lines", expanding the radius of interaction and enabling GPCRs to tether scaffolding proteins, thus facilitating receptor stability during cell membrane movements. Additionally, the i3 loop may be involved in domain swapping between GPCRs, generating novel receptor dimers with distinct binding and coupling characteristics. Overall, the i3 loop and C-terminus are now widely recognized as crucial elements in GPCR function and regulation. Understanding their functional roles enhances our comprehension of GPCR structure and signaling complexity and holds promise for advancements in receptor pharmacology and drug development.

The development of formulations adapted to the patient's age is a challenge in the pharmaceutical industry. Pediatric and geriatric patients may have difficulties in swallowing oral medications when an adequate formulation is not available. Carvedilol is a poorly water-soluble drug used to treat cardiovascular problems; it is commercialized in several countries only as solid oral formulations, which are often manipulated at the point of administration to treat pediatric or geriatric patients. The purpose of this work was to obtain a new dosage form of Carvedilol using safe excipients, suitable for administration to pediatric and geriatric patients. To improve the solubility of Carvedilol, the effect of several factors was analyzed and optimized. Subsequently, to improve the physical stability of the formulations, two preparation methods were analyzed by adding HPMC. In "method 1," HPMC was dissolved in buffer and incorporated into a mixture of Carvedilol-PEG 400, while in "method 2," Carvedilol was solubilized in buffer containing PEG 400, and then, HPMC was added. Finally, microbiological tests were performed to the stable formulations. The factors "pH value" and "concentration of PEG" affected the solubility of Carvedilol. A formulation containing Carvedilol (3 mg/mL), pH=3, PEG 400 (15% v/v), and HPMC (0.25% w/v) prepared by method 2 was stable for 180 days at 4 °C while those containing Carvedilol (5 mg/mL), pH=3, PEG 400 (27% v/v), and HPMC (0.5% w/v), prepared by method 2, were stable for 180 days at 4 and 25°C. These oral liquid formulations were physicochemical and microbiologically stable for 6 months., (© 2023. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Background: Highly localized impedance (LI) measurements during atrial fibrillation (AF) ablation have the potential to act as a reliable predictor of the durability of the lesions created., Objective: We aimed to collect data on the procedural parameters affecting LI-guided ablation in a large multicenter registry., Methods: A total of 212 consecutive patients enrolled in the CHARISMA registry and undergoing their first pulmonary vein (PV) isolation for paroxysmal and persistent AF were included., Results: In all, 13,891 radiofrequency (RF) applications of ≥3 s duration were assessed. The first-pass PV isolation rate was 93.3%. A total of 80 PV gaps were detected. At successful ablation spots, baseline LI and absolute LI drop were larger than at PV gap spots (161.4 ± 19 Ω vs. 153.0 ± 13 Ω, p  < 0.0001 for baseline LI; 22.1 ± 9 Ω vs. 14.4 ± 5 Ω, p  < 0.0001 for LI drop). On the basis of Receiver operating characteristic curve analysis, the ideal LI drop, which predicted successful ablation, was >21 Ω at anterior sites and >18 Ω at posterior sites. There was a non-linear association between the magnitude of LI drop and contact-force (CF) ( r  = 0.14, 95% CI: 0.13-0.16, p  < 0.0001) whereas both CF and LI drop were inversely related with delivery time (DT) (-0.22, -0.23 to -0.20, p  < 0.0001 for CF; -0.27, -0.29 to -0.26, p  < 0.0001 for LI drop)., Conclusion: An LI drop >21 Ω at anterior sites and >18 Ω at posterior sites predicts successful ablation. A higher CF was associated with an increased likelihood of ideal LI drop. The combination of good CF and adequate LI drop allows a significant reduction in RF DT., Clinical Trial Registration: http://clinicaltrials.gov/, identifier: NCT03793998., Competing Interests: MM and GC are employees of Boston Scientific. MA is a consultant for Boston Scientific and Biosense Webster and a clinical proctor for Medtronic, and has received educational fees from Abbott. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor TS declared a past co-authorship with the author FS., (© 2023 Lepillier, Maggio, De Sanctis, Malacrida, Stabile, Zakine, Champ-Rigot, Anselmino, Segreti, Dell'Era, Garnier, Mascia, Pandozi, Dello Russo, Scaglione, Cosaro, Ferraro, Paziaud, Maglia and Solimene.)

Background: Clinical evidence has shown frequent hypogonadism following mitotane (MTT) treatment in male patients with adrenocortical carcinoma. This study aimed to evaluate the impact of MTT on male gonadal function., Methods: Morphological analysis of testes and testosterone assays were performed on adult CD1 MTT-treated and untreated mice. The expression of key genes involved in interstitial and tubular compartments was studied by real-time PCR. Moreover, quantitative and qualitative analysis of spermatozoa was performed., Results: Several degrees of damage to the testes and a significant testosterone reduction in MTT-treated mice were observed. A significant decline in 3βHsd1 and Insl3 mRNA expression in the interstitial compartment confirmed an impairment of androgen production. Fsh-R mRNA expression was unaffected by MTT, proving that Sertoli cells are not the drug's primary target. Sperm concentrations were significantly lower in MTT-treated animals. Moreover, the drug caused a significant increase in the percentage of spermatozoa with abnormal chromatin structures., Conclusion: MTT negatively affects the male reproductive system, including changes in the morphology of testicular tissue and reductions in sperm concentration and quality.

Pancreatic islet transplantation is a therapeutic option for achieving physiologic regulation of plasma glucose in Type 1 diabetic patients. At the same time, mesenchymal stem cells (MSCs) have demonstrated their potential in controlling graft rejection, the most fearsome complication in organ/tissue transplantation. MSCs can interact with innate and adaptive immune system cells either through direct cell-cell contact or through their secretome including exosomes. In this review, we discuss current findings regarding the graft microenvironment of pancreatic islet recipient patients and the crucial role of MSCs operation as cell managers able to control the immune system to prevent rejection and promote endogenous repair. We also discuss how challenging stressors, such as oxidative stress and impaired vasculogenesis, may jeopardize graft outcomes. In order to face these adverse conditions, we consider either hypoxia-exposure preconditioning of MSCs or human stem cells with angiogenic potential in organoids to overcome islets' lack of vasculature. Along with the shepherding of carbon nanotubes-loaded MSCs to the transplantation site by a magnetic field, these studies look forward to exploiting MSCs stemness and their immunomodulatory properties in pancreatic islet transplantation.

Bipolar disorders (BDs) are a heterogeneous group of severe affective disorders generally described by the alternation of (hypo)manic, depressive, and mixed phases, with euthymic intervals of variable duration. BDs are burdened with high psychiatric and physical comorbidity, increased suicide risk and reduced life expectancy. In addition, BDs can progress into complicated forms (e.g., mixed states, rapid/irregular cycling), which are more difficult to treat and often require personalized pharmacological combinations. Mood stabilizers, particularly Lithium and Valproic acid (VPA), still represent the cornerstones of both acute and chronic pharmacotherapies of BDs. Lithium is the gold standard in BD-I and BDII with typical features, while VPA seems more effective for atypical forms (e.g., mixed-prevalence and rapid-cycling). However, despite appropriate mood stabilization, many patients show residual symptoms, and more than a half recur within 1-2 years, highlighting the need of additional strategies. Among these, the association of atypical antipsychotics (AAPs) with mood stabilizers is recurrent in the treatment of acute phases, but it is also being growingly explored in the maintenance pharmacotherapy. These combinations are clinically more aggressive and often needed in the acute phases, whereas simplifying pharmacotherapies to mood stabilizers only is preferable in the long-term, whenever possible. When mood stabilizers are not enough for maintenance treatment, Quetiapine and, less consistently, Aripiprazole have been proposed as the most advisable adjunctive strategies, for their safety and tolerability profiles. However, in view of the increased risk of serious adverse effects, a careful patient-centered balance between costs and benefits is mandatory., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The use of a higher dose per fraction to overcome the high radioresistance of prostate cancer cells has been unsuccessfully proposed. Herein, we present PC3 and DU-145, castration-resistant prostate cancer cell lines that survived a clinically used ultra-higher dose per fraction, namely, radioresistant PC3 and DU-145 cells (PC3RR and DU-145RR). Compared to PC3, PC3RR showed a higher level of aggressive behaviour, with enhanced clonogenic potential, DNA damage repair, migration ability and cancer stem cell features. Furthermore, compared to PC3, PC3RR more efficiently survived further radiation by increasing proliferation and down-regulating pro-apoptotic proteins. No significant changes of the above parameters were described in DU-145RR, suggesting that different prostate cancer cell lines that survive ultra-higher dose per fraction do not display the same grade of aggressive phenotype. Furthermore, both PC3RR and DU-145RR increased antioxidant enzymes and mesenchymal markers. Our data suggest that different molecular mechanisms could be potential targets for future treatments plans based on sequential strategies and synergistic effects of different modalities, possibly in a patient-tailored fashion. Moreover, PC3RR cells displayed an increase in specific markers involved in bone remodeling, indicating that radiotherapy selects a PC3 population capable of migrating to secondary metastatic sites. Finally, PC3RR cells showed a better sensitivity to Docetaxel as compared to native PC3 cells. This suggests that a subset of patients with castration-resistant metastatic disease could benefit from upfront Docetaxel treatment after the failure of radiotherapy.

Introduction: Unrecognized incomplete pulmonary vein (PV) isolation during the index procedure, can be a major cause of clinical recurrences of atrial fibrillation (AF) after cryoballoon (CB) ablation. We aimed to characterize the extension of the lesions produced by CB ablation and to assess the value of using an ultra-high resolution electroanatomic mapping (UHDM) system to detect incomplete CB lesions., Materials and Methods: Twenty-nine consecutive patients from the CHARISMA registry undergoing AF ablation at four Italian centers were prospectively evaluated. The Rhythmia™ mapping system and the Orion™ (Boston Scientific) mapping catheter were used to systematically map the left atrium and PVs before and after cryoablation., Results: A total of 116 PVs were targeted and isolated. Quantitative assessment of the lesions revealed a significant reduction of the antral surface area of the PV, resulting in an ablated area of 5.7 ± 0.7 cm 2 and 5.1 ± 0.8 cm 2 for the left PV pair and right PV pair, respectively ( p = 0.0068). The mean posterior wall (PW) area was 22.9 ± 2 cm 2 and, following PV isolation, 44.8 ± 6% of the PW area was ablated. After CB ablation, complete isolation of each PV was documented by the POLARMap™ catheter in all patients. By contrast, confirmatory UHDM and the Lumipoint™ tool unveiled PV signals in 1 out of 114 of the PVs (0.9%). Over 30-day follow-up, no major procedure-related adverse events were reported. After a mean follow-up of 333 days, 89.7% of patients were free from arrhythmia recurrence., Conclusion: The lesion extension achieved by the new CB ablation system involved the PV antrum, with less than 50% of the PW remaining untouched. The new system, with short tip and circular mapping catheter, failed to achieve PV isolation in only 0.9% of all PVs treated., Clinical Trial Registration: [http://clinicaltrials.gov/], identifier [NCT03793998]., Competing Interests: Authors MM and FPi were salaried Boston Scientific employees. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Spera, Narducci, Bencardino, Perna, Bisignani, Pinnacchio, Tondo, Maggio, Stabile, Iacopino, Tundo, Ferraro, De Simone, Malacrida, Pintus, Crea and Pelargonio.)

The architecture of eukaryotic cells is defined by extensive membrane-delimited compartments, which entails separate metabolic processes that would otherwise interfere with each other, leading to functional differences between cells. G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors, and their signal transduction is traditionally viewed as a chain of events initiated from the plasma membrane. Furthermore, their intracellular trafficking, internalization, and recycling were considered only to regulate receptor desensitization and cell surface expression. On the contrary, accumulating data strongly suggest that GPCRs also signal from intracellular compartments. GPCRs localize in the membranes of endosomes, nucleus, Golgi and endoplasmic reticulum apparatuses, mitochondria, and cell division compartments. Importantly, from these sites they have shown to orchestrate multiple signals that regulate different cell pathways. In this review, we summarize the current knowledge of this fascinating phenomenon, explaining how GPCRs reach the intracellular sites, are stimulated by the endogenous ligands, and their potential physiological/pathophysiological roles. Finally, we illustrate several mechanisms involved in the modulation of the compartmentalized GPCR signaling by drugs and endogenous ligands. Understanding how GPCR signaling compartmentalization is regulated will provide a unique opportunity to develop novel pharmaceutical approaches to target GPCRs and potentially lead the way towards new therapeutic approaches.

Autism spectrum disorder is a neurodevelopmental disorder with a rising prevalence disorder. This high-cost/high-burden condition needs evidence-based behavioral treatments that are able to reduce the impact of symptoms on children’s functioning. This retrospective chart review study compared the impact of different types of early interventions on toddlers diagnosed with an autism spectrum disorder developmental profile. Analyses were conducted on 90 subjects (mean = 27.76 months, range 18−44 months; M:F = 4.29:1), of which 36 children underwent the usual treatment, 13 children underwent an intervention based on early intensive behavioral intervention (EIBI) and 41 children received the Early Start Denver Model, for one year, with the same weekly frequency of about 6 h a week. A significant decrease in the severity of autism symptoms was observed for all children when looking at the Ados-2 severity score (average difference = 3.05, SD = 0.71, p = < 0.001) and the Ados-2 social subscale (average difference = 2.87, SD = 0.59, p < 0.001). Otherwise, for most of the Griffiths subscales, we found a significant improvement only for those children who underwent the Early Start Denver Model intervention (General Quotient average difference = 14.47, SD = 3.22, corrected p < 0.001). Analyzing the influence of age on the investigated scores, we found a significant association with the Eye−hand Coordination Quotient (p = 0.003), Performance Quotient (p = 0.042) and General Quotient (p = 0.006). In all these domains, a mild negative correlation with age was observed, as measured by the Pearson’s correlation coefficient (r = −0.32, p = 0.002; r = −0.21, p = 0.044; r = −0.25, p = 0.019, respectively), suggesting less severe developmental skills at the start of treatment for older children. Our results are consistent with the literature that underlines the importance of early intervention, since prompt diagnosis can reduce the severity of autism symptoms; nevertheless, in toddlers, our study demonstrated that an intervention model based on naturalistic developmental behavioral principles such as the Early Start Denver Model is more effective on children’s developmental profile. Further studies are required to assess the extent of effectiveness of different early intervention models in community settings.

Background: The association between cortisol secretion and mortality in patients with adrenal incidentalomas is controversial. We aimed to assess all-cause mortality, prevalence of comorbidities, and occurrence of cardiovascular events in uniformly stratified patients with adrenal incidentalomas and cortisol autonomy (defined as non-suppressible serum cortisol on dexamethasone suppression testing)., Methods: We conducted an international, retrospective, cohort study (NAPACA Outcome) at 30 centres in 16 countries. Eligible patients were aged 18 years or older with an adrenal incidentaloma (diameter ≥1 cm) detected between Jan 1, 1996, and Dec 31, 2015, and availability of a 1 mg dexamethasone suppression test result from the time of the initial diagnosis. Patients with clinically apparent hormone excess, active malignancy, or follow-up of less than 36 months were excluded. Patients were stratified according to the 0800-0900 h serum cortisol values after an overnight 1 mg dexamethasone suppression test; less than 50 nmol/L was classed as non-functioning adenoma, 50-138 nmol/L as possible autonomous cortisol secretion, and greater than 138 nmol/L as autonomous cortisol secretion. The primary endpoint was all-cause mortality. Secondary endpoints were the prevalence of cardiometabolic comorbidities, cardiovascular events, and cause-specific mortality. The primary and secondary endpoints were assessed in all study participants., Findings: Of 4374 potentially eligible patients, 3656 (2089 [57·1%] with non-functioning adenoma, 1320 [36·1%] with possible autonomous cortisol secretion, and 247 [6·8%] with autonomous cortisol secretion) were included in the study cohort for mortality analysis (2350 [64·3%] women and 1306 [35·7%] men; median age 61 years [IQR 53-68]; median follow-up 7·0 years [IQR 4·7-10·2]). During follow-up, 352 (9·6%) patients died. All-cause mortality (adjusted for age, sex, comorbidities, and previous cardiovascular events) was significantly increased in patients with possible autonomous cortisol secretion (HR 1·52, 95% CI 1·19-1·94) and autonomous cortisol secretion (1·77, 1·20-2·62) compared with patients with non-functioning adenoma. In women younger than 65 years, autonomous cortisol secretion was associated with higher all-cause mortality than non-functioning adenoma (HR 4·39, 95% CI 1·93-9·96), although this was not observed in men. Cardiometabolic comorbidities were significantly less frequent with non-functioning adenoma than with possible autonomous cortisol secretion and autonomous cortisol secretion (hypertension occurred in 1186 [58·6%] of 2024 patients with non-functioning adenoma, 944 [74·0%] of 1275 with possible autonomous cortisol secretion, and 179 [75·2%] of 238 with autonomous cortisol secretion; dyslipidaemia occurred in 724 [36·2%] of 1999 patients, 547 [43·8%] of 1250, and 123 [51·9%] of 237; and any diabetes occurred in 365 [18·2%] of 2002, 288 [23·0%] of 1250, and 62 [26·7%] of 232; all p values <0·001)., Interpretation: Cortisol autonomy is associated with increased all-cause mortality, particularly in women younger than 65 years. However, until results from randomised interventional trials are available, a conservative therapeutic approach seems to be justified in most patients with adrenal incidentaloma., Funding: Deutsche Forschungsgemeinschaft, Associazione Italiana per la Ricerca sul Cancro, Università di Torino., Competing Interests: Declaration of interests IB has served as a consultant for Corcept Therapeutics, Sparrow Pharmaceutics, and Spruce Biosciences; was as a member of advisory or data safety monitoring boards for Adrenas Therapeutics, Recordati, and Strongbridge Biopharma (in all cases, institution fees were provided); and reports personal honoraria from Elsevier ClinicalKey. IC reports consulting fees and honoraria from HRA Pharma Rare Diseases and Recordati; was a member of advisory or data safety monitoring boards for HRA Pharma Rare Diseases and Recordati; and has participated in clinical studies from Corcept Therapeutics. ACh reports personal support for attending meetings or travel from Sanofi; personal honoraria from Ipsen; and was a member of advisory or data safety monitoring boards for Ipsen. TDe reports personal consulting fees for being a member of advisory or data safety monitoring boards for HRA Pharma Rare Diseases and Recordati; personal honoraria from Novartis; and has participated in clinical studies from Corcept Therapeutics and HRA Pharma Rare Diseases (for these, institution fees were provided). MF has participated in clinical studies from Corcept Therapeutics and HRA Pharma Rare Diseases (for these, institution fees were provided). LM was a member of the expert panel “Focus Area Adrenal and Cardiovascular Endocrinology” from the European Society of Endocrinology, and led the working group 5 of the project “CA20122—Harmonizing clinical care and research on adrenal tumours in European countries” from the European Cooperation in Science in Technology. JN-P reports grants from Diurnal Group; and has served as a consultant for and received honoraria from HRA Pharma Rare Diseases, Crinetics Pharmaceuticals, and Recordati (in all cases, institution fees were provided). CS reports consulting fees and honoraria from HRA Pharma Rare Diseases and Recordati; was a member of advisory or data safety monitoring boards for HRA Pharma Rare Diseases and Recordati; and has served as coordinator of the Pituitary Club of the Italian Society of Endocrinology. MT reports personal consulting fees (for being a member of advisory or data safety monitoring boards for Corcept Therapeutics and HRA Pharma Rare Diseases); and has participated in clinical studies from HRA Pharma Rare Diseases (for which institution fees were provided). ST reports personal support for attending meetings or travel from Ipsen, Pfizer, and Recordati; personal honoraria from Recordati; and has participated in clinical studies from Crinetics Pharmaceuticals, Novartis, and Strongbridge Biopharma. All other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

New radioimaging techniques, exploiting the quantitative variables of imaging, permit to identify an hypothetical pathological tissue. We have applied this potential in a series of 72 adrenal incidentalomas (AIs) followed at our center, subdivided in functioning and non-functioning using laboratory findings. Each AI was studied in the preliminary non-contrast phase with a specific software (Mazda), surrounding a region of interest within each lesion. A total of 314 features were extrapolated. Mean and standard deviations of features were obtained and the difference in means between the two groups was statistically analyzed. Receiver Operating Characteristic (ROC) curves were used to identify an optimal cutoff for each variable and a prediction model was constructed via multivariate logistic regression with backward and stepwise selection. A 11-variable prediction model was constructed, and a ROC curve was used to differentiate patients with high probability of functioning AI. Using a threshold value of >-275.147, we obtained a sensitivity of 93.75% and a specificity of 100% in diagnosing functioning AI. On the basis of these results, computed tomography (CT) texture analysis appears a promising tool in the diagnostic definition of AIs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Maggio, Messina, D’Arrigo, Maccagno, Lardo, Palmisano, Poggi, Monti, Matarazzo, Laghi, Pugliese and Stigliano.)

(+)-4-Propyl-9-hydroxynaphthoxazine ((+)PHNO) is a high affinity, preferential dopamine D 3 versus D 2 agonist employed in view of its high specificity and excellent signal-to-noise ratio as a radiotracer for positron emission tomography (PET) imaging. Surprisingly, its profile at other classes of monoamine receptor remains undocumented. In addition to hD 3 and hD 2L receptors, (+)PHNO revealed high affinity at hD 4.4 but not hD 1 or hD 5 receptors. It also revealed significant affinity for several other G protein-coupled monoaminergic receptors, in particular h5-HT 1A and h5-HT 7 . (+)PHNO behaved as a full agonist at hD 4.4 and h5-HT 1A receptors with potencies comparable to its actions at hD 3 and hD 2L receptors, and with less potency at 5-HT 7 receptors. In binding assays with membranes derived from cells co-expressing hD 3 and hD 2L receptors and labeled with [ 3 H]Nemonapride or [ 3 H]Spiperone, the proportion of high affinity binding sites recognized by (+)PHNO was higher than an equivalent mixture of membranes from cells expressing hD 3 or hD 2L receptors, suggesting that (+)PHNO promotes formation of hD 3 -hD 2L heterodimers. Further, in cells co-expressing hD 3 and hD 2L receptors, (+)PHNO showed higher efficacy for inhibiting forskolin stimulated adenylyl cyclase and inducing adenylyl cyclase super-sensitization than in cells transfected with only hD 2L receptors. In conclusion, (+)PHNO is a potent agonist at hD 4 . 4 , h5-HT 1A and h5-HT 7 as well as hD 3 and hD 2L receptors, and it potently activates dopamine hD 3 -hD 2L heterodimers. These interactions should be considered when interpreting PET studies with [ 11 C](+)PHNO and may be relevant to its functional and potential clinical properties in Parkinson's disease and other disorders., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: The antral region of pulmonary veins (PV)s seems to play a key role in a strategy aimed at preventing atrial fibrillation (AF) recurrence. Particularly, low-voltage activity in tissue such as the PV antra and residual potential within the antral scar likely represent vulnerabilities in antral lesion sets, and ablation of these targets seems to improve freedom from AF. The aim of this study is to validate a structured application of an approach that includes the complete abolition of any antral potential achieving electrical quiescence in antral regions., Methods: The improveD procEdural workfLow for cathETEr ablation of paroxysmal AF with high density mapping system and advanced technology (DELETE AF) study is a prospective, single-arm, international post-market cohort study designed to demonstrate a low rate of clinical atrial arrhythmias recurrence with an improved procedural workflow for catheter ablation of paroxysmal AF, using the most advanced point-by-point RF ablation technology in a multicenter setting. About 300 consecutive patients with standard indications for AF ablation will be enrolled in this study. Post-ablation, all patients will be monitored with ambulatory event monitoring, starting within 30 days post-ablation to proactively detect and manage any recurrences within the 90-day blanking period, as well as Holter monitoring at 3, 6, 9, and 12 months post-ablation. Healthcare resource utilization, clinical data, complications, patients' medical complaints related to the ablation procedure and patient's reported outcome measures will be prospectively traced and evaluated., Discussion: The DELETE AF trial will provide additional knowledge on long-term outcome following a structured ablation workflow, with high density mapping, advanced algorithms and local impedance technology, in an international multicentric fashion. DELETE AF is registered at ClinicalTrials.gov (NCT05005143)., (© 2022 The Authors. Clinical Cardiology published by Wiley Periodicals LLC.)

Purpose: Highly localized impedance (LI) measurements during atrial fibrillation (AF) ablation have emerged as a viable real-time indicator of tissue characteristics and the consequent durability of the lesions created. We investigated the impact of catheter-tissue contact force (CF) on LI behavior during pulmonary vein isolation (PVI)., Methods: Forty-five consecutive patients of the CHARISMA registry undergoing de novo AF radiofrequency (RF) catheter ablation with a novel open-irrigated-tip catheter endowed with CF and LI measurement capabilities (Stablepoint™ catheter, Boston Scientific) were included., Results: A total of 2895 point-by-point RF applications were analyzed (RF delivery time (DT) = 8.7±4s, CF = 13 ±±8 g, LI drop = 23 ±±7 Ω). All PVs were successfully isolated in an overall procedure time of 118 ±±34 min (fluoroscopy time = 13 ±±8 min). The magnitude of LI drop weakly correlated with CF (r = 0.13, 95% confidence interval (CI): 0.09 to 0.16, p < 0.0001), whereas both CF and LI drop inversely correlated with DT (r = -0.26, 95%CI: -0.29 to -0.22, p < 0.0001 for CF; r = -0.36, 95%CI: -0.39 to -0.33, p < 0.0001 for LI). For each 10 g of CF, LI drop markedly increased from 22.4 ± 7 Ω to 24.0 ± 8 Ω at 5 to 25 g CF intervals (5-14 g of CF vs 15-24 g of CF, p < 0.0001), whereas it showed smooth transition over 25 g (24.8 ± 7Ω at ≥ 25 g CF intervals, p = 0.0606 vs 15-24 g of CF). No major complications occurred during the procedures or within 30 days., Conclusions: CF significantly affects LI drop and probable consequent lesion formation during RF PVI. The benefit of higher contact (> 25 g) between the catheter and the tissue appears to have less impact on LI drop., Trial Registration: Catheter Ablation of Arrhythmias With High Density Mapping System in the Real World Practice (CHARISMA). URL: http://clinicaltrials.gov/ Identifier: NCT03793998., (© 2022. The Author(s).)

Therapeutic drug monitoring (TDM) is an effective tool used to improve the pharmacological treatment in clinical practice, especially to detect subtherapeutic drug plasma concentration (Cp) in order to consider a change of dosage during treatment and reach its putative therapeutic range. In this study, we report the Cp values of lithium and valproic acid (VPA), alone and in combination, mostly in bipolar patients admitted to an Italian clinical center of the University of Pisa during the years 2016-2020, which include 12,294 samples of VPA, 7449 of lithium and 1118 of both in combination. Lithium and VPA are the most utilized drugs in treating bipolar disorders, and their TDM is strongly recommended by recent guidelines. In relation to lithium Cp monitoring, several studies have underlined that 0.5-0.8 mmol/L is the optimal range for chronic treatment, and below 0.4 mmol/L, it is unlikely to produce a clinical response. For VPA, the therapeutic range is 50-100 μg/mL and a linear correlation between Cp and clinical efficacy has been proposed, where below 50 μg/mL, the clinical efficacy of VPA has not been proven thus far. Toxic levels of both drugs were rarely found in our study, while a high percentage of patients, about one-third, had sub-therapeutic Cp during their treatments. In addition, in several cases of patients receiving multiple blood sampling, the initial subtherapeutic Cp changed only partially without reaching its therapeutic window. In relation to age, we found a higher percentage of lithium and VPA Cp values in range in the adolescents than in the adults and elderly groups. No differences were reported when analyzing the distribution of Cp values in males and females. In conclusion, this present study suggests that TDM is widely used by many specialists, but there is still a window of improvement for optimizing pharmacological treatments in clinical practice.

Information flow from a source to a receiver becomes informative when the recipient can process the signal into a meaningful form. Information exchange and interpretation is essential in biology and understanding how cells integrate signals from a variety of information-coding molecules into complex orchestrated responses is a major challenge for modern cell biology. In complex organisms, cell to cell communication occurs mostly through neurotransmitters and hormones, and receptors are responsible for signal recognition at the membrane level and information transduction inside the cell. The G protein-coupled receptors (GPCRs) are the largest family of membrane receptors, with nearly 800 genes coding for these proteins. The recognition that GPCRs may physically interact with each other has led to the hypothesis that their dimeric state can provide the framework for temporal coincidence in signaling pathways. Furthermore, the formation of GPCRs higher order oligomers provides the structural basis for organizing distinct cell compartments along the plasma membrane where confined increases in second messengers may be perceived and discriminated. Here, we summarize evidence that supports these conjectures, fostering new ideas about the physiological role played by receptor homo- and hetero-oligomerization in cell biology.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. About 25% of RMS expresses fusion oncoproteins such as PAX3/PAX7-FOXO1 (fusion-positive, FP) while fusion-negative (FN)-RMS harbors RAS mutations. Radiotherapy (RT) plays a crucial role in local control but metastatic RMS is often radio-resistant. HDAC inhibitors (HDACi) radio-sensitize different cancer cells types. Thus, we evaluated MS-275 (Entinostat), a Class I and IV HDACi, in combination with RT on RMS cells in vitro and in vivo. MS-275 reversibly hampered cell survival in vitro in FN-RMS RD (RASmut) and irreversibly in FP-RMS RH30 cell lines down-regulating cyclin A, B, and D1, up-regulating p21 and p27 and reducing ERKs activity, and c-Myc expression in RD and PI3K/Akt/mTOR activity and N-Myc expression in RH30 cells. Further, MS-275 and RT combination reduced colony formation ability of RH30 cells. In both cell lines, co-treatment increased DNA damage repair inhibition and reactive oxygen species formation, down-regulated NRF2 , SOD , CAT and GPx4 anti-oxidant genes and improved RT ability to induce G2 growth arrest. MS-275 inhibited in vivo growth of RH30 cells and completely prevented the growth of RT-unresponsive RH30 xenografts when combined with radiation. Thus, MS-275 could be considered as a radio-sensitizing agent for the treatment of intrinsically radio-resistant PAX3-FOXO1 RMS.

Neurotoxins such as rotenone, 1-methyl-4-phenylpyridinium (MPP + ) and 6-hydroxydopamine (6-OHDA) are well known for their high toxicity on dopaminergic neurons and are associated with Parkinson's disease (PD) in murine models and humans. In addition, PD patients often have glucose intolerance and may develop type 2 diabetes (T2D), whereas T2D patients have higher risk of PD compared to general population. Based on these premises, we evaluated the toxicity of these three toxins on pancreatic β-cell lines (INS-1 832/13 and MIN6) and we showed that rotenone is the most potent for reducing β-cells viability and altering mitochondrial structure and bioenergetics in the low nanomolar range, similar to that found in dopaminergic cell lines. MPP + and 6-OHDA show similar effects but at higher concentration. Importantly, rotenone-induced toxicity was counteracted by α-tocopherol and partially by metformin, which are endowed with strong antioxidative and cytoprotective properties. These data show similarities between dopaminergic neurons and β-cells in terms of vulnerability to toxins and pharmacological agents capable to protect both cell types.

Purpose: No data are currently available on female sexual dysfunction (FSD) in primary adrenal insufficiency (PAI) and the possible impact of replacement therapy. The aim of this study was to evaluate the prevalence of FSD and sexual distress (SD), and to evaluate the possible impact of replacement therapy on sexuality in women with PAI., Methods: Female Sexual Function Index-6 (FSFI-6) and Sexual Distress Scale (SDS) questionnaires were administered to 22 women with PAI and 23 healthy women matched for age as controls., Results: The prevalence of sexual symptoms measured by FSFI-6 (total score < 19) was significantly higher in women with PAI (15/22; 68.2%) compared to the controls (2/23; 8.7%; p = 0.001). Regarding the questionnaire items, significantly different scores were found for desire ( p < 0.001), arousal ( p = 0.0006), lubrication ( p = 0.046) and overall sexual satisfaction ( p < 0.0001) in women with PAI compared to the controls. The rate of FSD (FSFI < 19 with SDS >15) was 60% in patients with PAI. A significant inverse correlation was found between FSFI-6 total scores and SD (r = -0.65; p = 0.0011), while a significant direct correlation was found between FSFI-6 total scores and serum cortisol levels (r = 0.55; p = 0.035)., Conclusions: A higher prevalence of FSD was found in women affected by PAI compared to healthy women. Desire seems to be the most impaired aspect of sexual function. Moreover, sexual dysfunction in this population seems to be related to sexual distress and cortisol levels.

H3K9 methylation maintains cell identity orchestrating stable silencing and anchoring of alternate fate genes within the heterochromatic compartment underneath the nuclear lamina (NL). However, how cell type-specific genomic regions are specifically targeted to the NL is still elusive. Using fibro-adipogenic progenitors (FAPs) as a model, we identified Prdm16 as a nuclear envelope protein that anchors H3K9-methylated chromatin in a cell-specific manner. We show that Prdm16 mediates FAP developmental capacities by orchestrating lamina-associated domain organization and heterochromatin sequestration at the nuclear periphery. We found that Prdm16 localizes at the NL where it cooperates with the H3K9 methyltransferases G9a/GLP to mediate tethering and silencing of myogenic genes, thus repressing an alternative myogenic fate in FAPs. Genetic and pharmacological disruption of this repressive pathway confers to FAP myogenic competence, preventing fibro-adipogenic degeneration of dystrophic muscles. In summary, we reveal a druggable mechanism of heterochromatin perinuclear sequestration exploitable to reprogram FAPs in vivo., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The lack of an effective medical treatment for adrenocortical carcinoma (ACC) has prompted the search for better treatment protocols for ACC neoplasms. Sorafenib, a tyrosine kinase inhibitor has exhibited effectiveness in the treatment of different human tumors. Therefore, the aim of this study was to understand the mechanism through which sorafenib acts on ACC, especially since treatment with sorafenib alone is sometimes unable to induce a long-lasting antiproliferative effect in this tumor type. The effects of sorafenib were tested on the ACC cell line H295R by evaluating cell viability, apoptosis and VEGF receptor signaling which was assessed by analyzing VE-cadherin and β-catenin complex formation. We also tested sorafenib on an in vitro 3D cell culture model using the same cell line. Apoptosis was observed after sorafenib treatment, and coimmunoprecipitation data suggested that the drug prevents formation VEGFR-VE-cadherin and β-catenin proteins complex. These results were confirmed both by ultrastructural analysis and by a 3D model where we observed a disaggregation of spheres into single cells, which is a crucial event that represents the first step of metastasis. Our findings suggest that although sorafenib induces apoptotic cell death a small portion of cells survive the treatment and have characteristics of a malignancy. Based on our data we recommend against the use of sorafenib in patients with ACC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cerquetti, Bucci, Raffa, Amendola, Maggio, Lardo, Petrangeli, Torrisi, Toscano, Pugliese and Stigliano.)

Cancer is a heterogeneous disease and its treatment remains unsatisfactory with inconstant therapeutic responses. This variability could be related, at least in part, to different and highly personalized gut microbiota compositions. Different studies have shown an impact of microbiota on antitumor therapy. It has been demonstrated that some gut bacteria influences the development and differentiation of immune cells, suggesting that different microbiota compositions could affect the efficacy of the antitumor vaccine. Emerging data suggest that recognition of neoantigens for the generation of neoantigen cancer vaccines (NCVs) could have a key role in the activity of clinical immunotherapies. However, it is still unknown whether there is a crosstalk between microbiota and NCV. This study aimed to understand the possible mechanisms of interaction between gut microbiota and NCV delivered by DNA-electroporation (DNA-EP). We found that decreased microbiota diversity induced by prolonged antibiotic (ATB) treatment is associated with higher intratumor specific immune responses and consequently to a better antitumor effect induced by NCV delivered by DNA-EP., Competing Interests: Authors do not have a conflict of interest to be declared., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Atypical antipsychotics (AAPs) are commonly prescribed medications to treat schizophrenia, bipolar disorders and other psychotic disorders. However, they might cause metabolic syndrome (MetS) in terms of weight gain, dyslipidemia, type 2 diabetes (T2D), and high blood pressure, which are responsible for reduced life expectancy and poor adherence. Importantly, there is clear evidence that early metabolic disturbances can precede weight gain, even if the latter still remains the hallmark of AAPs use. In fact, AAPs interfere profoundly with glucose and lipid homeostasis acting mostly on hypothalamus, liver, pancreatic β-cells, adipose tissue, and skeletal muscle. Their actions on hypothalamic centers via dopamine, serotonin, acetylcholine, and histamine receptors affect neuropeptides and 5'AMP-activated protein kinase (AMPK) activity, thus producing a supraphysiological sympathetic outflow augmenting levels of glucagon and hepatic glucose production. In addition, altered insulin secretion, dyslipidemia, fat deposition in the liver and adipose tissues, and insulin resistance become aggravating factors for MetS. In clinical practice, among AAPs, olanzapine and clozapine are associated with the highest risk of MetS, whereas quetiapine, risperidone, asenapine and amisulpride cause moderate alterations. The new AAPs such as ziprasidone, lurasidone and the partial agonist aripiprazole seem more tolerable on the metabolic profile. However, these aspects must be considered together with the differences among AAPs in terms of their efficacy, where clozapine still remains the most effective. Intriguingly, there seems to be a correlation between AAP's higher clinical efficacy and increase risk of metabolic alterations. Finally, a multidisciplinary approach combining psychoeducation and therapeutic drug monitoring (TDM) is proposed as a first-line strategy to avoid the MetS. In addition, pharmacological treatments are discussed as well.

Purpose: Herein we describe the in vitro and in vivo activity of FK228 (Romidepsin), an inhibitor of class I HDACs, in counteracting and radiosensitizing embryonal (ERMS, fusion-negative) and alveolar (ARMS, fusion-positive) rhabdomyosarcoma (RMS)., Methods: RH30 (ARMS, fusion-positive) and RD (ERMS, fusion-negative) cell lines and human multipotent mesenchymal stromal cells (HMSC) were used. Flow cytometry analysis, RT-qPCR, western blotting and enzymatic assays were performed. Irradiation was delivered by using an x-6 MV photon linear accelerator. FK228 (1.2 mg/kg) in vivo activity, combined or not with radiation therapy (2 Gy), was assessed in murine xenografts., Results: Compared to HMSC, RMS expressed low levels of class I HDACs. In vitro, FK228, as single agents, reversibly downregulated class I HDACs expression and activity and induced oxidative stress, DNA damage and a concomitant growth arrest associated with PARP-1-mediated transient non-apoptotic cell death. Surviving cells upregulated the expression of cyclin A, B, D1, p27, Myc and activated PI3K/Akt/mTOR and MAPK signaling, known to be differently involved in cancer chemoresistance. Interestingly, while no radiosensitizing effects were detected, in vitro or in vivo, on RD cells, FK228 markedly radiosensitized RH30 cells by impairing antioxidant and DSBs repair pathways in vitro. Further, FK228 when combined with RT in vivo significantly reduced tumor mass in mouse RH30 xenografts., Conclusion: FK228 did not show antitumor activity as a single agent whilst its combination with RT resulted in radiosensitization of fusion-positive RMS cells, thus representing a possible strategy for the treatment of the most aggressive RMS subtype.

Adult neurogenesis consists in the generation of newborn neurons from neural stem cells taking place in the adult brain. In mammals, this process is limited to very few areas of the brain, and one of these neurogenic niches is the subgranular layer of the dentate gyrus (DG) of the hippocampus. Adult newborn neurons are generated from quiescent neural progenitors (QNPs), which differentiate through different steps into mature granule cells (GCs), to be finally integrated into the existing hippocampal circuitry. In animal models, adult hippocampal neurogenesis (AHN) is relevant for pattern discrimination, cognitive flexibility, emotional processing and resilience to stressful situations. Imaging techniques allow to visualize newborn neurons within the hippocampus through all their stages of development and differentiation. In humans, the evidence of AHN is more challenging, and, based on recent findings, it persists through adulthood, even if it declines with age. Whether this process has an important role in human brain function and how it integrates into the existing hippocampal circuitry is still a matter of exciting debate. Importantly, AHN deficiency has been proposed to be relevant in many psychiatric disorders, including mood disorders, anxiety, post-traumatic stress disorder and schizophrenia. This review aims to investigate how AHN is altered in different psychiatric conditions and how pharmacological treatments can rescue this process. In fact, many psychoactive drugs, such as antidepressants, mood stabilizers and atypical antipsychotics (AAPs), can boost AHN with different results. In addition, some non-pharmacological approaches are discussed, as well., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Some studies show that the diagnosis of Autism Spectrum Disorder could be considered reliable and stable in children aged 18 to 24 months. Nevertheless, the diagnostic stability of early ASD diagnosis has not yet been fully demonstrated. This observational study examines the one-year diagnostic stability of autism spectrum disorder diagnosis in a clinical sample of 147 children diagnosed between 18 and 48 months of age. The ADOS-2 scores were used in order to stratify children in three levels of symptom severity: Autism (AD; comparison score 5-7), Autism Spectrum Disorder (ASD; comparison score 3-4), and Sub-Threshold Symptoms; (STS; comparison score 1-2). Results: Overall, the largest part of children and toddlers diagnosed with autism spectrum disorder between 18 and 48 months continued to show autistic symptoms at one-year follow-up evaluation. Nevertheless, a significant percentage of children with higher ADOS severity scores exhibited a reduction of symptom severity and, therefore, moved towards a milder severity class one year later. Conversely, the number of subjects of the STS group meaningfully increased. Therefore, at one-year follow-up a statistically significant (χ 2 (2) = 181.46, p < 0.0001) percentage of subjects (25.2% of the total) who had received a categorical diagnosis of Autistic Disorder or Autism Spectrum Disorder in baseline no longer met the criteria for a categorical diagnosis. Furthermore, children who no longer met the criteria for autism spectrum disorder continue to show delays in one or more neurodevelopmental areas, possibly related to the emergence of other neurodevelopmental/neuropsychiatric disorders. Overall, the comprehensive results of the study account for a high sensibility but a moderate stability of ASD early diagnosis.

Schizophrenia was first described by Emil Krapelin in the 19th century as one of the major mental illnesses causing disability worldwide. Since the introduction of chlorpromazine in 1952, strategies aimed at modifying the activity of dopamine receptors have played a major role for the treatment of schizophrenia. The introduction of atypical antipsychotics with clozapine broadened the range of potential targets for the treatment of this psychiatric disease, as they also modify the activity of the serotoninergic receptors. Interestingly, all marketed drugs for schizophrenia bind to the orthosteric binding pocket of the receptor as competitive antagonists or partial agonists. In recent years, a strong effort to develop allosteric modulators as potential therapeutic agents for schizophrenia was made, mainly for the several advantages in their use. In particular, the allosteric binding sites are topographically distinct from the orthosteric pockets, and thus drugs targeting these sites have a higher degree of receptor subunit specificity. Moreover, "pure" allosteric modulators maintain the temporal and spatial fidelity of native orthosteric ligand. Furthermore, allosteric modulators have a "ceiling effect", and their modulatory effect is saturated above certain concentrations. In this review, we summarize the progresses made in the identification of allosteric drugs for dopamine and serotonin receptors, which could lead to a new generation of atypical antipsychotics with a better profile, especially in terms of reduced side effects.

Objective: Sub-motor threshold 5 Hz repetitive paired associative stimulation (5 Hz-rPAS 25ms ) produces a long-lasting increase in corticospinal excitability. Assuming a spike-timing dependent plasticity-like (STDP-like) mechanism, we hypothesized that 5 Hz-rPAS at a shorter inter-stimulus interval (ISI) of 15 ms (5 Hz-rPAS 15ms ) would exert a lasting inhibitory effect on corticospinal excitability., Methods: 20 healthy volunteers received two minutes of 5 Hz-rPAS 15ms . Transcranial magnetic stimulation (TMS) was applied over the motor hotspot of the right abductor pollicis brevis muscle at 90% active motor threshold. Sub-motor threshold peripheral electrical stimulation was given to the left median nerve 15 ms before each TMS pulse. We assessed changes in mean amplitude of the unconditioned motor evoked potential (MEP), short-latency intracortical inhibition (SICI), intracortical facilitation (ICF), short-latency afferent inhibition (SAI), long-latency afferent inhibition (LAI), and cortical silent period (CSP) before and for 60 minutes after 5-Hz rPAS 15ms ., Results: Subthreshold 5-Hz rPAS 15ms produced a 20-40% decrease in mean MEP amplitude along with an attenuation in SAI, lasting at least 60 minutes. A follow-up experiment revealed that MEP facilitation was spatially restricted to the target muscle., Conclusions: Subthreshold 5-Hz rPAS 15ms effectively suppresses corticospinal excitability. Together with the facilitatory effects of subthreshold 5-Hz rPAS 25ms (Quartarone et al., J Physiol 2006;575:657-670), the results show that sub-motor threshold 5-Hz rPAS induces STDP-like bidirectional plasticity in the motor cortex., Significance: The results of the present study provide a new short-time paradigm of long term depression (LTD) induction in human sensory-motor cortex., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Conflict of interest Hartwig R. Siebner has received honoraria as speaker and consultant from Sanofi Genzyme, Denmark and as senior editor (NeuroImage) from Elsevier Publishers, Amsterdam, The Netherlands, royalties as book editor from Springer Publishers, Stuttgart, and a research fund from Biogen-idec, Denmark., (Copyright © 2020 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.)

Background: The probability of local tumor control after radiotherapy (RT) remains still miserably poor in pediatric rhabdomyosarcoma (RMS). Thus, understanding the molecular mechanisms responsible of tumor relapse is essential to identify personalized RT-based strategies. Contrary to what has been done so far, a correct characterization of cellular radioresistance should be performed comparing radioresistant and radiosensitive cells with the same isogenic background., Methods: Clinically relevant radioresistant (RR) embryonal (RD) and alveolar (RH30) RMS cell lines have been developed by irradiating them with clinical-like hypo-fractionated schedule. RMS-RR cells were compared to parental isogenic counterpart (RMS-PR) and studied following the radiobiological concept of the "6Rs", which stand for repair, redistribution, repopulation, reoxygenation, intrinsic radioresistance and radio-immuno-biology., Results: RMS-RR cell lines, characterized by a more aggressive and in vitro pro-metastatic phenotype, showed a higher ability to i) detoxify from reactive oxygen species; ii) repair DNA damage by differently activating non-homologous end joining and homologous recombination pathways; iii) counteract RT-induced G2/M cell cycle arrest by re-starting growth and repopulating after irradiation; iv) express cancer stem-like profile. Bioinformatic analyses, performed to assess the role of 41 cytokines after RT exposure and their network interactions, suggested TGF-β, MIF, CCL2, CXCL5, CXCL8 and CXCL12 as master regulators of cancer immune escape in RMS tumors., Conclusions: These results suggest that RMS could sustain intrinsic and acquire radioresistance by different mechanisms and indicate potential targets for future combined radiosensitizing strategies.

[This corrects the article DOI: 10.1155/2020/1938704.]., (Copyright © 2020 Iole Macchia et al.)

Purpose: Radiation therapy (RT), by using ionizing radiation (IR), destroys cancer cells inducing DNA damage. Despite several studies are continuously performed to identify the best curative dose of IR, the role of dose-rate, IR delivered per unit of time, on tumor control is still largely unknown. Materials and methods: Rhabdomyosarcoma (RMS) and prostate cancer (PCa) cell lines were irradiated with 2 or 10 Gy delivered at dose-rates of 1.5, 2.5, 5.5 and 10.1 Gy/min. Cell-survival rate and cell cycle distribution were evaluated by clonogenic assays and flow cytometry, respectively. The production of reactive oxygen species (ROS) was detected by cytometry. Quantitative polymerase chain reaction assessed the expression of anti-oxidant-related factors including NRF2, SODs, CAT and GPx4 and miRNAs (miR-22, -126, -210, -375, -146a, -34a). Annexin V and caspase-8, -9 and -3 activity were assessed to characterize cell death. Senescence was determined by assessing β-galactosidase (SA-β-gal) activity. Immunoblotting was performed to assess the expression/activation of: i) phosphorylated H2AX (γ-H2AX), markers of DNA double strand breaks (DSBs); ii) p19 Kip1/Cip1 , p21 Waf1/Cip1 and p27 Kip1/Cip1 , senescence-related-markers; iii) p62, LC3-I and LC3-II, regulators of autophagy; iv) ATM, RAD51, DNA-PKcs, Ku70 and Ku80, mediators of DSBs repair. Results: Low dose-rate (LDR) more efficiently induced apoptosis and senescence in RMS while high dose-rate (HDR) necrosis in PCa. This paralleled with a lower ability of LDR-RMS and HDR-PCa irradiated cells to activate DSBs repair. Modulating the dose rate did not differently affect the anti-oxidant ability of cancer cells. Conclusion: The present results indicate that a stronger cytotoxic effect was induced by modulating the dose-rate in a cancer cell-dependent manner, this suggesting that choose the dose-rate based on the individual patient's tumor characteristics could be strategic for effective RT exposures.

Growing awareness of adverse impacts of artificial light on human health has led to recognize light pollution as a significant global environmental issue. Despite, a large number of studies in rodent and monkey models of Parkinson's disease have reported that near infrared light has neuroprotective effects on dopaminergic neurons, recent findings have shown that prolonged exposure of rodents and birds to fluorescent artificial light results in an increase of neuromelanin granules in substantia nigra and loss of dopaminergic neurons. The observed detrimental effect seems to be dependent on a direct effect of light on the substantia nigra rather than a secondary effect of the alterations of circadian rhythms. Moreover, inferences from animal models to human studies have shown a positive correlation between the prevalence of Parkinson's disease and light pollution. The present article discusses experimental evidence supporting a potentially deleterious impact of light on dopaminergic neurons and highlights the mechanisms whereby light might damage neuronal tissue. Moreover, it analyses epidemiological evidence that suggests light pollution to be an environmental risk factor for Parkinson's disease., (Copyright © 2020 IBRO. All rights reserved.)

Background: Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project., Methods: The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration., Results: Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting., Conclusion: Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options., Competing Interests: Each contributing author declares to have no competing interests., (Copyright © 2020 Iole Macchia et al.)

Obinutuzumab is a glycoengineered tumor-targeting anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for the FcγRIIIA/CD16 receptor, which was recently approved for clinical use in CLL and follicular lymphoma. Here we extend our previous observation that, in human NK cells, the sustained CD16 ligation by obinutuzumab-opsonized targets leads to a markedly enhanced IFN-γ production upon a subsequent cytokine re-stimulation. The increased IFN-γ competence in response to IL-2 or IL-15 is attributable to post-transcriptional regulation, as it does not correlate with the upregulation of IFN-γ mRNA levels. Different from the reference molecule rituximab, we observe that the stimulation with obinutuzumab promotes the upregulation of microRNA (miR)-155 expression. A similar trend was also observed in NK cells from untreated CLL patients stimulated with obinutuzumab-opsonized autologous leukemia. miR-155 upregulation associates with reduced levels of SHIP-1 inositol phosphatase, which acts in constraining PI3K-dependent signals, by virtue of its ability to mediate phosphatidylinositol 3,4,5-trisphosphate (PIP3) de-phosphorylation. Downstream of PI3K, the phosphorylation status of mammalian target of rapamycin (mTOR) effector molecule, S6, results in amplified response to IL-2 or IL-15 stimulation in obinutuzumab-experienced cells. Importantly, NK cell treatment with the PI3K or mTOR inhibitors, idelalisib and rapamycin, respectively, prevents the enhanced cytokine responsiveness, thus, highlighting the relevance of the PI3K/mTOR axis in CD16-dependent priming. The enhanced IFN-γ competence may be envisaged to potentiate the immunoregulatory role of NK cells in a therapeutic setting.