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First we prove a comparison result for a nonlinear divergence structure elliptic partial differential equation. Next we find an estimate of the solution of a boundary value problem in a domain Ω in terms of the solution of a related symmetric boundary value problem in a ball B having the same measure as Ω. For p-Laplace equations, the corresponding result is due to Giorgio Talenti. In a special (radial) case we also prove a reverse comparison result.

Opening of the cardiac voltage-gated Na+ channel (Nav1.5) is responsible for robust depolarization of the cardiac action potential, while inactivation, which rapidly follows, allows for repolarization. Regulation of both the voltage- and time-dependent kinetics of Nav1.5 inactivation can alter the ability of the heart to initiate and sustain a re-entrant arrhythmia. The C-terminal domain (CTD) of Nav1.5 has been shown to modulate fast inactivation of the channel, and multiple auxiliary proteins bind to the CTD, including calmodulin (CaM) and intracellular fibroblast growth factor 12A (FGF12A). Recently, a non-canonical CaM-binding site was also discovered on the N-terminal of A-splice variants of iFGFs. We performed cut-open Vaseline gap (COVG) voltage-clamp to test whether FGF12A with and without CaM regulates Nav1.5 gating. In WT Nav1.5 channels, FGF12A with and without CaM present had a minimal effect on the voltage dependence of both activation and inactivation. Conversely, when CaM is absent on the Nav1.5 CTD (IQ/AA), a dramatic shift in steady-state inactivation (SSI) occurred, regardless of whether CaM was present on FGF12A. These two distinct mechanisms are operative in Nav1.5 LQT3 mutations where FGF12A requires CaM to shift in the voltage-dependence of inactivation, but not to inhibit the persistent late current. We conclude that there are two distinct mechanisms by which FGF12A modulates the Nav1.5 channel: CaM-regulated alteration of the voltage dependence of inactivation and CaM-independent inhibition of persistent late current.

Missense variants in calmodulin (CaM) predispose patients to arrhythmias associated with high mortality rates ("calmodulinopathy"). As CaM regulates many key cardiac ion channels, an understanding of disease mechanism associated with CaM variant arrhythmias requires elucidating individual CaM variant effects on distinct channels. One key CaM regulatory target is the KCNQ1 (K V 7.1) voltage-gated potassium channel that carries the I Ks current. Yet, relatively little is known as to how CaM variants interact with KCNQ1 or affect its function. Here, we take a multipronged approach employing a live-cell fluorescence resonance energy transfer binding assay, fluorescence trafficking assay, and functional electrophysiology to characterize >10 arrhythmia-associated CaM variants for effect on KCNQ1 CaM binding, membrane trafficking, and channel function. We identify one variant (G114W) that exhibits severely weakened binding to KCNQ1 but find that most other CaM variants interact with similar binding affinity to KCNQ1 when compared with CaM wild-type over physiological Ca 2+ ranges. We further identify several CaM variants that affect KCNQ1 and I Ks membrane trafficking and/or baseline current activation kinetics, thereby delineating KCNQ1 dysfunction in calmodulinopathy. Lastly, we identify CaM variants with no effect on KCNQ1 function. This study provides extensive functional data that reveal how CaM variants contribute to creating a proarrhythmic substrate by causing abnormal KCNQ1 membrane trafficking and current conduction. We find that CaM variant regulation of KCNQ1 is not uniform with effects varying from benign to significant loss of function, suggesting how CaM variants predispose patients to arrhythmia via the dysregulation of multiple cardiac ion channels. Classification : Biological, Health, and Medical Sciences, Physiology., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Pitt-Hopkins syndrome (PTHS) is a rare, neurodevelopmental genetic disorder caused by mutations in the TCF4 gene. This gene encodes a ubiquitous, class I, basic helix-loop-helix factor, which is implicated in various developmental and regulatory processes. Predominant clinical manifestations of PTHS include facial dysmorphisms, intellectual disability, absence of expressive language, epilepsy, as well as visual and musculoskeletal impairments. Gastrointestinal (GI) complications, such as chronic intestinal pseudo-obstruction, gastroparesis with delayed bowel transit, chronic constipation culminating in failure to thrive, and gastroesophageal reflux disease (GERD), are also prevalent in these patients. The early identification of pain etiology in PTHS patients poses a significant clinical challenge. This report presents two cases of PTHS patients suffering from gastrointestinal dysmotility, evaluated at our Pediatrics Clinic at the "Microcitemico" Hospital. A review of existing literature was conducted via the PubMed database to elucidate the current understanding of the GI phenotype in PTHS. Twenty articles were deemed most relevant and selected for this purpose. In both patients, severe constipation and abdominal distension resulted in persistent agitation and inconsolable crying. These distress symptoms were completely ameliorated following prompt pharmacological intervention., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Comisi et al.)

Voltage-gated sodium (NaV) channels are responsible for the initiation and propagation of action potentials. In the heart, the predominant NaV1.5 α subunit is composed of four homologous repeats (I-IV) and forms a macromolecular complex with multiple accessory proteins, including intracellular fibroblast growth factors (iFGF). In spite of high homology, each of the iFGFs, iFGF11-iFGF14, as well as the individual iFGF splice variants, differentially regulates NaV channel gating, and the mechanisms underlying these differential effects remain elusive. Much of the work exploring iFGF regulation of NaV1.5 has been performed in mouse and rat ventricular myocytes in which iFGF13VY is the predominant iFGF expressed, whereas investigation into NaV1.5 regulation by the human heart-dominant iFGF12B is lacking. In this study, we used a mouse model with cardiac-specific Fgf13 deletion to study the consequences of iFGF13VY and iFGF12B expression. We observed distinct effects on the voltage-dependences of activation and inactivation of the sodium currents (INa), as well as on the kinetics of peak INa decay. Results in native myocytes were recapitulated with human NaV1.5 heterologously expressed in Xenopus oocytes, and additional experiments using voltage-clamp fluorometry (VCF) revealed iFGF-specific effects on the activation of the NaV1.5 voltage sensor domain in repeat IV (VSD-IV). iFGF chimeras further unveiled roles for all three iFGF domains (i.e., the N-terminus, core, and C-terminus) on the regulation of VSD-IV, and a slower time domain of inactivation. We present here a novel mechanism of iFGF regulation that is specific to individual iFGF isoforms and that leads to distinct functional effects on NaV channel/current kinetics., (© 2023 Angsutararux et al.)

BK type Ca 2+ -activated K + channels activate in response to both voltage and Ca 2+ . The membrane-spanning voltage sensor domain (VSD) activation and Ca 2+ binding to the cytosolic tail domain (CTD) open the pore across the membrane, but the mechanisms that couple VSD activation and Ca 2+ binding to pore opening  are not clear. Here we show that a compound, BC5, identified from in silico screening, interacts with the CTD-VSD interface and specifically modulates the Ca 2+ dependent activation mechanism. BC5 activates the channel in the absence of Ca 2+ binding but Ca 2+ binding inhibits BC5 effects. Thus, BC5 perturbs a pathway that couples Ca 2+ binding to pore opening to allosterically affect both, which is further supported by atomistic simulations and mutagenesis. The results suggest that the CTD-VSD interaction makes a major contribution to the mechanism of Ca 2+ dependent activation and is an important site for allosteric agonists to modulate BK channel activation., (© 2022. The Author(s).)

Background: Organs at risk (OARs) delineation is a crucial step of radiotherapy (RT) treatment planning workflow. Time-consuming and inter-observer variability are main issues in manual OAR delineation, mainly in the head and neck (H & N) district. Deep-learning based auto-segmentation is a promising strategy to improve OARs contouring in radiotherapy departments. A comparison of deep-learning-generated auto-contours (AC) with manual contours (MC) was performed by three expert radiation oncologists from a single center., Methods: Planning computed tomography (CT) scans of patients undergoing RT treatments for H&N cancers were considered. CT scans were processed by Limbus Contour auto-segmentation software, a commercial deep-learning auto-segmentation based software to generate AC. H&N protocol was used to perform AC, with the structure set consisting of bilateral brachial plexus, brain, brainstem, bilateral cochlea, pharyngeal constrictors, eye globes, bilateral lens, mandible, optic chiasm, bilateral optic nerves, oral cavity, bilateral parotids, spinal cord, bilateral submandibular glands, lips and thyroid. Manual revision of OARs was performed according to international consensus guidelines. The AC and MC were compared using the Dice similarity coefficient (DSC) and 95% Hausdorff distance transform (DT)., Results: A total of 274 contours obtained by processing CT scans were included in the analysis. The highest values of DSC were obtained for the brain (DSC 1.00), left and right eye globes and the mandible (DSC 0.98). The structures with greater MC editing were optic chiasm, optic nerves and cochleae., Conclusions: In this preliminary analysis, deep-learning auto-segmentation seems to provide acceptable H&N OAR delineations. For less accurate organs, AC could be considered a starting point for review and manual adjustment. Our results suggest that AC could become a useful time-saving tool to optimize workload and resources in RT departments.

Purpose: As a discipline in its infancy, online adaptive RT (ART) needs new ontologies and ad hoc criteria to evaluate the appropriateness of its use in clinical practice. In this experience, we propose a predictive model able to quantify the dosimetric impact due to daily inter-fraction variability in a standard RT breast treatment, to identify in advance the treatment fractions where patients might benefit from an online ART approach., Methods: The study was focused on right breast cancer patients treated using standard adjuvant RT on an artificial intelligence (AI)-based linear accelerator. Patients were treated with daily CBCT images and without online adaptation, prescribing 40.05 Gy in 15 fractions, with four IMRT tangential beams. ESTRO guidelines were followed for the delineation on planning CT (pCT) of organs at risk and targets. For each patient, all the CBCT images were rigidly aligned to pCT: CTV and PTV were manually re-contoured and the original treatment plan was recalculated. Various radiological parameters were measured on CBCT images, to quantify inter-fraction variability present in each RT fraction after the couch shifts compensation. The variation of these parameters was correlated with the variation of V95% of PTV (ΔV95%) using the Wilcoxon Mann-Whitney test. Fractions where ΔV95% > 2% were considered as adverse events. A logistic regression model was calculated considering the most significant parameter, and its performance was quantified with a receiver operating characteristic (ROC) curve., Results: A total of 75 fractions on 5 patients were analyzed. The body variation between daily CBCT and pCT along the beam axis with the highest MU was identified as the best predictor ( p = 0.002). The predictive model showed an area under ROC curve of 0.86 (95% CI, 0.82-0.99) with a sensitivity of 85.7% and a specificity of 83.8% at the best threshold, which was equal to 3 mm., Conclusion: A novel strategy to identify treatment fractions that may benefit online ART was proposed. After image alignment, the measure of body difference between daily CBCT and pCT can be considered as an indirect estimator of V95% PTV variation: a difference larger than 3 mm will result in a V95% decrease larger than 2%. A larger number of observations is needed to confirm the results of this hypothesis-generating study., 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 Iezzi, Cusumano, Piccari, Menna, Catucci, D’Aviero, Re, Di Dio, Quaranta, Boschetti, Marras, Piro, Tomei, Votta, Valentini and Mattiucci.)

Introduction: Recent published data demonstrated how subjects born preterm are at higher risk of developing early atrial fibrillation (AF)., Materials and Methods: The surface ECG of twenty-four adults, former preterm infants born with an extremely low birth weight (ex-ELBW; mean age at study: 23.2±3.3 years; mean gestational age: 27.8±2.3 weeks; mean birth weight: 840±120.1 grams), were compared with those of 24 healthy counterparts born at term (C). A few parameters known to be capable of predicting a predisposition to develop AF were examined: P wave duration and dispersion, P terminal force, isoelectric interval length, PR interval length, and advanced interatrial blocks., Results: A shorter PR interval length was found in ex-ELBW compared to C (p<0.0003) as well as longer P wave duration and dispersion, p terminal force, and isoelectric interval (p<0.0001, p<0.0001, p<0.01, and p<0.0004, respectively). Four cases of advanced interatrial block were detected in ex-ELBW, and none in C (p<0.0001). P wave duration, PR interval length, and P wave dispersion were significantly correlated with birth weight (r=0.51 p<0.01, r=0.46 p<0.02, and r=0.42 p<0.04, respectively). When excluding the possible influence of gestational age on birth weight, P wave duration and dispersion were found to be the only statistically significant determinants of abnormal atrial electrical activation (p<0.03 and p<0.04, respectively). On the contrary, when excluding the possible influence of birth weight on gestational age, only P wave duration remained statistically significant (p<0.05)., Conclusion: Surface ECG findings of abnormal atrial activity in ex-ELBW may explain their previously reported predisposition to developing AF.

Atrial fibrillation (AF) is a really uncommon arrhythmia in newborns. Here, we report the case of a 1-day-old infant who was recovered in Neonatal Intensive Care Unit for a twin-to-twin transfusion. The appearance of an unexpected AF was registered at his continuous electrocardiographic monitoring. Both chest X-ray and echocardiographic examination revealed the anomalous insertion of the tip of a central venous catheter (CVC) into the heart ('umbilical artery → inferior vena cava → right atrium → patent foramen ovale → left atrium'). AF ceased as soon as the CVC was extracted from the heart. The mechanical irritation of both atria caused by the malposition of the CVC might be the underlying mechanism responsible for the triggering of AF in this unique case report.

Ricotta fresca cheese is the product of Sardinian dairy industry most exposed to microbial post-process contamination. Due to its technological characteristics, intrinsic parameters, pH (6.10-6.80) and water activity (0.974-0.991), it represents an excellent substrate for the growth of spoilage and pathogenic microorganisms, which are usually resident in cheese-making plants environments. Generally, ricotta fresca has a shelf life of 5-7 days. For this reason, at industrial level, modified atmosphere packaging (MAP) is used to extend the durability of the product. However, few investigations have been conducted to validate the use of MAP in ricotta fresca. The aim of this work is to evaluate the shelf life of ricotta fresca under MAP. A total of 108 samples were collected from three Sardinian industrial cheese-making plants and analysed within 24 h after packaging and after 7, 14 and 21 days of refrigerated storage. Aerobic mesophilic bacteria, mesophilic and thermophilic cocci and lactobacilli, Enterobacteriaceae and E. coli, L. monocytogenes, Pseudomonas spp, Bacillus cereus , yeasts and moulds, and the chemical-physical parameters and composition of the product were determined. At the end of the shelf life, Pseudomonas spp. and Enterobacteriaceae reached high concentrations, 5 to 7 and 3 to 6 log 10 colony forming unit g -1 , respectively. The presence of environmental contaminants indicates that the use of MAP without the appropriate implementation of prerequisite programmes is not sufficient to extend the durability of ricotta fresca. Gas mixture and packaging material should be selected only on the basis of scientific evidence of their effectiveness., Competing Interests: the authors declare no potential conflict of interest.

Purpose: The aim of the study was to evaluate the efficacy of an injectable and partly absorbable calcium bone cement (CERAMENT™, Bone Support, Sweden) in the treatment of osteoporotic or traumatic vertebral fractures by percutaneous vertebroplasty., Methods: From March 2009 to October 2010 an open, prospective study in two centres was performed. 33 patients with symptomatic vertebral fractures were enrolled. Patients were included based on evaluation by X-ray, CT, and MRI. Clinical evaluation by Visual Analogue Scale (VAS, 0-10) and Oswestry Disability index test (ODI, 0-100 %) was performed before the operation as well as 1, 6 and 12 months after the procedure. Radiology assessment post-procedure was carried out by X-ray, CT, and MRI at 1, 6 and 12 months post-op. Intake of analgesic medications pre- and post-procedure was monitored., Results: 66 vertebral bodies underwent percutaneous vertebroplasty. VAS score demonstrated a significant decrease from 8.61 (SD 19.8) pre-operatively to 2.48 (SD 2.36) at 1 month. The score was 2.76 (SD 2.68) at 6 months and 1.36 (SD 1.33) at the latest follow up. ODI score dropped significantly from 58.86 pre-op to 26.94 at 6 months and further down to 7.61 at 12 months. No re-fractures or adjacent level fractures were reported., Conclusion: Data show that CERAMENT can be a substitute of PMMA in the treatment of osteoporotic and traumatic vertebral fractures, especially in young patients.

Background: Neonatal death in full-term infants who suffer from perinatal asphyxia (PA) is a major subject of investigation, since few tools exist to predict patients at risk of ominous outcome. We studied the possibility that urine S100B measurement may identify which PA-affected infants are at risk of early postnatal death., Methodology/principal Findings: In a cross-sectional study between January 1, 2001 and December 1, 2006 we measured S100B protein in urine collected from term infants (n = 132), 60 of whom suffered PA. According to their outcome at 7 days, infants with PA were subsequently classified either as asphyxiated infants complicated by hypoxic ischemic encephalopathy with no ominous outcome (HIE Group; n = 48), or as newborns who died within the first post-natal week (Ominous Outcome Group; n = 12). Routine laboratory variables, cerebral ultrasound, neurological patterns and urine concentrations of S100B protein were determined at first urination and after 24, 48 and 96 hours. The severity of illness in the first 24 hours after birth was measured using the Score for Neonatal Acute Physiology-Perinatal Extension (SNAP-PE). Urine S100B levels were higher from the first urination in the ominous outcome group than in healthy or HIE Groups (p<0.001 for all), and progressively increased. Multiple logistic regression analysis showed a significant correlation between S100B concentrations and the occurrence of neonatal death. At a cut-off >1.0 microg/L S100B had a sensitivity/specificity of 100% for predicting neonatal death., Conclusions/significance: Increased S100B protein urine levels in term newborns suffering PA seem to suggest a higher risk of neonatal death for these infants.

Background: Intrauterine growth restriction (IUGR) is associated with perinatal mortality and with neurologic damage from intraventricular hemorrhage (IVH). We investigated whether S100B, a neural protein found in high concentrations after cell injury in the nervous system, is increased in serum of women whose pregnancies are complicated by IUGR and whose newborns develop IVH. We also explored the prognostic accuracy of maternal serum S100B for IVH in the newborn., Methods: We conducted a case-control study of 106 pregnancies complicated by IUGR, including a subgroup (n = 26) who developed IVH after birth, and 212 unaffected pregnancies matched for gestational age. Ultrasound examination, Doppler velocimetry patterns (in the utero-placental vessels and middle cerebral artery), and maternal blood collection were performed before birth; cerebral ultrasound and neurologic examinations were performed after birth., Results: S100B was higher (P <0.001) in IUGR pregnancies complicated by IVH than in those that were not and in controls. At a cutoff of 0.72 microg/L, sensitivity was 100% [95% confidence interval (95% CI), 87%-100%] and specificity was 99.3% (97.5%-99.9%) for prediction of IVH (area under the ROC curve, 0.999). The prevalence of IVH was 8.2% in the whole study population, 93% (95% CI, 83.6%-100%) in those with maternal S100B >0.72 microg/L, and 0% (0%-2.5%) in those with maternal S100B <0.72 microg/L., Conclusion: For prediction of IVH, measurements of maternal S100B may be useful at times before clinical, laboratory, and ultrasound patterns can identify risk of IVH.