Lung function assessment in children with Long-Covid syndrome.

INTRODUCTION: A significant percentage of patients who survived the Coronavirus Infection Disease 2019 (COVID-19) showed persistent general and respiratory symptoms even months after recovery. This condition, called Post-Acute Sequelae of COVID-19 or Long-Covid syndrome (LCS), has been described also in children with positive history for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Little is known about the pathophysiologic mechanisms underlying this syndrome. The aim of this study was to investigate any difference between children with LCS and asymptomatic peers with previous COVID-19 in terms of lung function and lung ultrasound (LUS) patterns. Secondly, we tested associations between lung function abnormalities and LUS findings with Long-Covid. METHODS: We carried out a prospective, descriptive, observational study including 58 children aged 5-17 years: 28 with LCS compared to 30 asymptomatic children with previous COVID-19. We collected demographic data, history of asthma, allergy or smoke exposure, and acute COVID-19 symptoms. After a median period of 4.5 months (1%-95% range 2-21) since the infection, lung function was assessed by spirometry, body plethysmography, diffusion lung capacity for carbon monoxide (DLCO). Airways inflammation was investigated by fractional exhaled nitric oxide (FeNO). LUS was performed independently by two experienced clinicians. RESULTS: We found that children with LCS were older than controls (mean (SD) 12 (4.1) vs. 9.7 (2.6); p = .04). Children with LCS complained more frequently fatigue (46.4%), cough (17.9%), exercise intolerance (14.3%) and dyspnea (14.3%). Lung function was normal and similar between the two groups. The frequency of LUS abnormalities was similar between the two groups (43.3% children with LCS vs. 56.7% controls; p = .436). Children with LCS showed lower FeNO values (log difference -0.30 (CI 95% -0.50, -0.10)), but no association of LCS with a lower lung function and abnormal LUS findings was found. CONCLUSIONS: LCS seems to be more frequent in older age children. Lung functional and structural abnormalities were not different between children with LCS and asymptomatic subjects with previous COVID-19. In addition, children with LCS showed lower FeNO values than controls, suggesting its potential role as a marker in LCS. However, further and larger studies are needed to confirm our findings.

Cationic Calix[4]arene Vectors to Efficiently Deliver AntimiRNA Peptide Nucleic Acids (PNAs) and miRNA Mimics.

One of the most appealing approaches for regulating gene expression, named the "microRNA therapeutic" method, is based on the regulation of the activity of microRNAs (miRNAs), the intracellular levels of which are dysregulated in many diseases, including cancer. This can be achieved by miRNA inhibition with antimiRNA molecules in the case of overexpressed microRNAs, or by using miRNA-mimics to restore downregulated microRNAs that are associated with the target disease. The development of new efficient, low-toxic, and targeted vectors of such molecules represents a key topic in the field of the pharmacological modulation of microRNAs. We compared the delivery efficiency of a small library of cationic calix[4]arene vectors complexed with fluorescent antimiRNA molecules (Peptide Nucleic Acids, PNAs), pre-miRNA (microRNA precursors), and mature microRNAs, in glioma- and colon-cancer cellular models. The transfection was assayed by cytofluorimetry, cell imaging assays, and RT-qPCR. The calix[4]arene-based vectors were shown to be powerful tools to facilitate the uptake of both neutral (PNAs) and negatively charged (pre-miRNAs and mature microRNAs) molecules showing low toxicity in transfected cells and ability to compete with commercially available vectors in terms of delivery efficiency. These results could be of great interest to validate microRNA therapeutics approaches for future application in personalized treatment and precision medicine.

Long COVID in Children: A Multidisciplinary Review.

Long COVID syndrome has emerged as a long-lasting consequence of acute SARS-CoV-2 infection in adults. In addition, children may be affected by Long COVID, with potential clinical issues in different fields, including problems in school performance and daily activities. Yet, the pathophysiologic bases of Long COVID in children are largely unknown, and it is difficult to predict who will develop the syndrome. In this multidisciplinary clinical review, we summarise the latest scientific data regarding Long COVID and its impact on children. Special attention is given to diagnostic tests, in order to help the physicians to find potential disease markers and quantify impairment. Specifically, we assess the respiratory, upper airways, cardiac, neurologic and motor and psychological aspects. Finally, we also propose a multidisciplinary clinical approach.

Multivalent calix[4]arene-based mannosylated dendrons as new FimH ligands and inhibitors.

We report the synthesis and biological characterization of a novel class of multivalent glycoconjugates as hit compounds for the design of new antiadhesive therapies against urogenital tract infections (UTIs) caused by uropathogenic E. coli strains (UPEC). The first step of UTIs is the molecular recognition of high mannose N-glycan expressed on the surface of urothelial cells by the bacterial lectin FimH, allowing the pathogen adhesion required for mammalian cell invasion. The inhibition of FimH-mediated interactions is thus a validated strategy for the treatment of UTIs. To this purpose, we designed and synthesized d-mannose multivalent dendrons supported on a calixarene core introducing a significant structural change from a previously described family of dendrimers bearing the same dendrons units on a flexible pentaerythritol scaffold core. The new molecular architecture increased the inhibitory potency against FimH-mediated adhesion processes by about 16 times, as assessed by yeast agglutination assay. Moreover, the direct molecular interaction of the new compounds with FimH protein was assessed by on-cell NMR experiments acquired in the presence of UPEC cells.

Intra- and Intermolecular Cooperativity in the Catalytic Activity of Phosphodiester Cleavage by Self-Assembled Systems Based on Guanidinylated Calix[4]arenes.

The calix[4]arene scaffold, blocked in the cone conformation through alkylation with long alkyl chains, and decorated at the upper rim with four guanidine or arginine units, effectively catalyzes the cleavage of the phosphodiester bond of DNA and RNA model compounds in water. An exhaustive kinetic investigation unequivocally points to the existence of spontaneous aggregation phenomena, driven by hydrophobic effect, occurring at different critical concentrations that depend on the identity of the compound. A pronounced superiority of the assembled structures compared with the monomers in solution was observed. Moreover, the catalytically active units, clustered on the macrocyclic tetrafunctional scaffold, were proved to efficiently cooperate in the catalytic mechanism and result in improved reaction rates compared to those of the monofunctional model compounds. The kinetic analysis is also integrated and corroborated with further experiments based on fluorescence spectroscopy and light scattering. The advantage of the supramolecular assemblies based on tetrafunctional calixarenes leads to believe that the active units can cooperate not only intramolecularly but also intermolecularly. The molecules in the aggregates can probably mold, flex and rearrange but, at the same time, keep an ordered structure that favors phosphodiester bond cleavage. This dynamic preorganization can allow the catalytic units to reach a better fitting with the substrates and perform a superior catalytic activity.

3-O-Methyltolcapone and Its Lipophilic Analogues Are Potent Inhibitors of Transthyretin Amyloidogenesis with High Permeability and Low Toxicity.

Transthyretin (TTR) is an amyloidogenic homotetramer involved in the transport of thyroxine in blood and cerebrospinal fluid. To date, more than 130 TTR point mutations are known to destabilise the TTR tetramer, leading to its extracellular pathological aggregation accumulating in several organs, such as heart, peripheral and autonomic nerves, and leptomeninges. Tolcapone is an FDA-approved drug for Parkinson's disease that has been repurposed as a TTR stabiliser. We characterised 3-O-methyltolcapone and two newly synthesized lipophilic analogues, which are expected to be protected from the metabolic glucuronidation that is responsible for the lability of tolcapone in the organism. Immunoblotting assays indicated the high degree of TTR stabilisation, coupled with binding selectivity towards TTR in diluted plasma of 3-O-methyltolcapone and its lipophilic analogues. Furthermore, in vitro toxicity data showed their several-fold improved neuronal and hepatic safety compared to tolcapone. Calorimetric and structural data showed that both T4 binding sites of TTR are occupied by 3-O-methyltolcapone and its lipophilic analogs, consistent with an effective TTR tetramer stabilisation. Moreover, in vitro permeability studies showed that the three compounds can effectively cross the blood-brain barrier, which is a prerequisite for the inhibition of TTR amyloidogenesis in the cerebrospinal fluid. Our data demonstrate the relevance of 3-O-methyltolcapone and its lipophilic analogs as potent inhibitors of TTR amyloidogenesis.

Complex Formation between Cytochrome c and a Tetra-alanino-calix[4]arene.

Owing to their remarkable features, calix[n]arenes are being exploited to study different aspects of molecular recognition, including protein complexation. Different complexation modes have been described, depending on the moieties that complement the aromatic cavity, allowing for function regulation and/or controlled assembly of the protein target. Here, a rigid cone calix[4]arene, bearing four anionic alanine units at the upper rim, was tested as a ligand for cytochrome c. Cocrystallization attempts were unfruitful, preventing a solid-state study of the system. Next, the complex was studied using NMR spectroscopy, which revealed the presence of two binding sites at lysine residues with dissociation constants (Kd) in the millimolar range.

A machine learning analysis to predict the response to intravenous and subcutaneous immunoglobulin in inflammatory myopathies. A proposal for a future multi-omics approach in autoimmune diseases.

OBJECTIVE: To evaluate the response to treatment with intravenous (IVIg) and subcutaneous (20%SCIg) immunoglobulin in our series of patients with Inflammatory idiopathic myopathies (IIM) by the means of artificial intelligence. BACKGROUND: IIM are rare diseases mainly involving the skeletal muscle with particular clinical, laboratory and radiological characteristics. Artificial intelligence (AI) represents computer processes which allows to perform complex calculations and data analyses, with the least human intervention. Recently, the use an AI in medicine significantly expanded, especially through machine learning (ML) which analyses huge amounts of information and accordingly makes decisions, and deep learning (DL) which uses artificial neural networks to analyse data and automatically learn. METHODS: In this study, we employed AI in the evaluation of the response to treatment with IVIg and 20%SCIg in our series of patients with IIM. The diagnoses were determined on the established EULAR/ACR criteria. The treatment response was evaluated employing the following: serum creatine kinase levels, muscle strength (MMT8 score), disease activity (MITAX score) and disability (HAQ-DI score). We evaluated all the above parameters, applying, with R, different supervised ML algorithms, including Least Absolute Shrinkage and Selection Operator, Ridge, Elastic Net, Classification and Regression Trees and Random Forest to estimate the most important predictors for a good response to IVIg and 20%SCIg treatment. RESULTS AND CONCLUSION: By the means of AI we have been able to identify the scores that best predict a good response to IVIg and 20%SCIg treatment. The muscle strength as evaluated by MMT8 score at the follow-up is predicted by the presence of dysphagia and of skin disorders, and the myositis activity index (MITAX) at the beginning of the treatment. The relationship between muscle strength and MITAX indicates a better action of IVIg therapy in patients with more active systemic disease. Considering our results, Elastic Net and similar approaches were seen to be the most viable, efficient, and effective ML methods for predicting the clinical outcome (MMT8 and MITAX at most) in myositis.

Detection of Nitroaromatic Explosives in Air by Amino-Functionalized Carbon Nanotubes.

Nitroaromatic explosives are the most common explosives, and their detection is important to public security, human health, and environmental protection. In particular, the detection of solid explosives through directly revealing the presence of their vapors in air would be desirable for compact and portable devices. In this study, amino-functionalized carbon nanotubes were used to produce resistive sensors to detect nitroaromatic explosives by interaction with their vapors. Devices formed by carbon nanotube networks working at room temperature revealed trinitrotoluene, one of the most common nitroaromatic explosives, and di-nitrotoluene-saturated vapors, with reaction and recovery times of a few and tens of seconds, respectively. This type of resistive device is particularly simple and may be easily combined with low-power electronics for preparing portable devices.

A multidisciplinary approach to peritoneal metastasis from hepatocellular carcinoma: clinical features, management and outcomes.

Aim of the study: Hepatocellular carcinoma (HCC) is a lethal malignancy with heterogeneous behavior determined by liver function, clinical presentation and treatment response. Peritoneal metastasis (PM) from HCC is rare and management is challenging. We aim to report a cohort of patients with advanced HCC and describe demographic characteristics, treatment and outcomes of patients with PM. Material and methods: We analyzed data from a retrospective cohort of patients with HCC. Patients with PM were analyzed individually. Baseline characteristics, treatment strategy and median overall survival (OS) with 95% confidence interval (CI) were reported. Results: 238 patients with advanced HCC were evaluated. Eleven patients had PM: 7 patients were treated with systemic treatment and 4 were treated with upfront peritonectomy followed by systemic treatment at recurrence. These 4 patients had well-preserved liver function and low disease burden and were younger compared to the total cohort. The median time to recurrence after peritonectomy was 30.25 months (interquartile range [IQR]: 13.53-46.92): 3 of them presented peritoneal recurrence (2 with diffuse peritoneal spread and 1 with concomitant hepatic recurrence) and 1 presented pulmonary recurrence. Overall, patients with PM showed similar OS compared to patients with other metastatic sites (11.8 months; 95% CI: 1.5-19.8 vs. 8 months; 95% CI: 6.7-10, p = 0.901). Patients with PM treated with upfront surgery had a median OS of 60 months (95% CI: 16.7-not reached). Conclusions: Resection of PM from HCC may provide long-term survival in selected patients. A multidisciplinary approach is the optimal strategy for managing PM from HCC.

Calixarenes Incorporating Sulfonamide Moieties: Versatile Ligands for Carbonic Anhydrases Inhibition.

Carbonic anhydrases (CAs) continue to represent a relevant pharmaceutical target. The need of selective inhibitors and the involvement of these metalloenzymes in many multifaceted diseases boost the search for new ligands able to distinguish among the different CA isoforms, and for multifunctional systems simultaneously able to inhibit CAs and to interfere with other pathological events by interacting with additional targets. In this work, we successfully explored the possibility of preparing new CAs ligands by combining calixarenes with benzensulfonamide units. Inhibition tests towards three human CA isoforms evidenced, for some of the ligands, Ki values in the nanomolar range and promising selectivity. X-ray and molecular modeling studies provided information on the mode of binding of these calixarene derivatives. Thanks to the encouraging results and the structural features typical of the calixarene scaffold, it is then possible to plan for the future the design of multifunctional inhibitors for this class of widely spread enzymes.

Use of lung ultrasound in school-aged children with wheezing.

Background: There is limited information available on fast and safe bedside tools that could help clinicians establish whether the pathological process underlying cases of wheezing is due to asthmatic exacerbation, asthmatic bronchitis, or pneumonia. The study's aim was to characterize Lung Ultrasound (LUS) in school-aged children with wheezing and evaluate its use for their follow-up treatment. Materials and methods: We carried out a cross-sectional study involving 68 consecutive outpatients (mean age 9.9 years) with wheezing and suggestive signs of an acute respiratory infection. An expert sonographer, blinded to all subject characteristics, clinical course, and the study pediatrician's diagnosis, performed an LUS after spirometry and before BDT. The severity of acute respiratory symptoms was determined using the Pediatric Respiratory Assessment Measure (PRAM) score. Results: The LUS was positive in 38.2% (26/68) of patients [12 (46.1%) with multiple B-lines, 24 (92.3%) with consolidation, and 22 (84.6%) with pleural abnormalities]. In patients with pneumonia, asthmatic bronchitis, and asthma, the percentages of those patients with a positive LUS were 100%, 57.7%, and 0%, respectively. Of note, patients with a positive LUS were associated with an increased need for hospital admission (30.8% vs. 2.4%, p = 0.001), administration of oxygen therapy (14.6% vs. 0%, p = 0.009), oral corticosteroids (84.6% vs. 19.0%, p < 0.001), and antibiotics (88.5% vs. 11.9%, p < 0.001); and a higher median value of PRAM score (4.0 (2.0-7.0) vs. 2.0 (1.0-5.0); p < 0.001). Conclusions: Our findings would suggest the use of LUS as a safe and cheap tool used by clinicians to define the diagnosis of school-aged children with wheezing of unknown causes.

Usefulness of Lung Ultrasound in Paediatric Respiratory Diseases.

Respiratory infection diseases are among the major causes of morbidity and mortality in children. Diagnosis is focused on clinical presentation, yet signs and symptoms are not specific and there is a need for new non-radiating diagnostic tools. Among these, lung ultrasound (LUS) has recently been included in point-of-care protocols showing interesting results. In comparison to other imaging techniques, such as chest X-ray and computed tomography, ultrasonography does not use ionizing radiations. Therefore, it is particularly suitable for clinical follow-up of paediatric patients. LUS requires only 5-10 min and allows physicians to make quick decisions about the patient's management. Nowadays, LUS has become an early diagnostic tool to detect pneumonia during the COVID-19 pandemic. In this narrative review, we show the most recent scientific literature about advantages and limits of LUS performance in children. Furthermore, we discuss the major paediatric indications separately, with a paragraph fully dedicated to COVID-19. Finally, we mention potential future perspectives about LUS application in paediatric respiratory diseases.

Calixarene-decorated liposomes for intracellular cargo delivery.

Amphiphilic calix[4]arenes, functionalized with guanidinium groups, are used to decorate the outer surface of liposomes and significantly improve the cellular uptake of a cargo compared to plain liposomes. The improved uptake is elicited and mediated by the interaction between the cationic polar heads of the macrocycle units embedded in the liposome bilayer and anionic heparan-sulfate proteoglycans surrounding the exterior of cells.

Coaggregation of Asthma and Type 1 Diabetes in Children: A Narrative Review.

Asthma and type 1 diabetes mellitus (T1DM) are two of the most frequent chronic diseases in children, representing a model of the atopic and autoimmune diseases respectively. These two groups of disorders are mediated by different immunological pathways, T helper (Th)1 for diabetes and Th2 for asthma. For many years, these two groups were thought to be mutually exclusive according to the Th1/Th2 paradigm. In children, the incidence of both diseases is steadily increasing worldwide. In this narrative review, we report the evidence of the potential link between asthma and T1DM in childhood. We discuss which molecular mechanisms could be involved in the link between asthma and T1DM, such as genetic predisposition, cytokine patterns, and environmental influences. Cytokine profile of children with asthma and T1DM shows an activation of both Th1 and Th2 pathways, suggesting a complex genetic-epigenetic interaction. In conclusion, in children, the potential link between asthma and T1DM needs further investigation to improve the diagnostic and therapeutic approach to these patients. The aim of this review is to invite the pediatricians to consider the potential copresence of these two disorders in clinical practice.

Delivery of Peptide Nucleic Acids Using an Argininocalix[4]arene as Vector.

The importance of peptide nucleic acids (PNAs) for alteration of gene expression is nowadays firmly established. PNAs are characterized by a pseudo-peptide backbone composed of N-(2-aminoethyl)glycine units and have been found to be excellent candidates for antisense and antigene therapies. Recently, PNAs have been demonstrated to alter the action of microRNAs and thus can be considered very important tools for miRNA therapeutics. In fact, the pharmacological modulation of microRNA activity appears to be a very interesting approach in the development of new types of drugs. Among the limits of PNAs in applied molecular biology, the delivery to target cells and tissues is of key importance. The aim of this chapter is to describe methods for the efficient delivery of unmodified PNAs designed to target microRNAs involved in cancer, using as model system miR-221-3p and human glioma cells as in vitro experimental cellular system. The methods employed to deliver PNAs targeting miR-221-3p here presented are based on a macrocyclic multivalent tetraargininocalix[4]arene used as non-covalent vector for anti-miR-221-3p PNAs. High delivery efficiency, low cytotoxicity, maintenance of the PNA biological activity, and easy preparation makes this vector a candidate for a universal delivery system for this class of nucleic acid analogs.

AUTOMA: a wearable device to assess the upper limb muscular activity in patients with neuromuscular disorders.

Inherited muscular dystrophies and congenital myopathies present in early childhood with progressive muscle weakness, determining severe motor limitations. Active surveillance and management of associated complications have improved ambulation, function, quality of life and life expectancy. The need for repeatable, objective and quantitative measures to monitor the clinical course of the disease is a current issue, particularly in the new era where new flows of therapies are proposed to the patients. In this scenario, we designed and tested a wearable device termed AUTOMA that is able to provide quantification of the muscular impairment in the upper limb upon isokinetic tests through the integration of a force sensor and an electric goniometer. This allows qualitatively estimating the muscular functions with a systematic procedure. We carried out a preliminary pilot study on 9 patients that revealed the suitability of AUTOMA as an objective measurement tool for diagnosing and monitoring neuromuscular disorders, and opens to a more extensive clinical study in which to test and validate our platform intensively.

Synthesis, self-assembly and anticancer drug encapsulation and delivery properties of cyclodextrin-based giant amphiphiles.

Cyclodextrin-calixarene giant amphiphiles that can self-assemble into nanospheres or nanovesicles have the ability to encapsulate the anticancer hydrophobic drugs docetaxel, temozolomide and combretastatin A-4 with encapsulation efficiencies >80% and deliver them to tumoral cells, enhancing their therapeutic efficacy by 1-3 orders of magnitude. These amphiphiles were modified by inserting a disulfide bridge confering them redox responsiveness. Disassembly of the resulting nanocompounds and cargo release was favored by high glutathione levels mimicking those present in the tumor microenvironment. Anticancer drug-loaded nanoformulations inhibited prostate, breast, glioblastoma, colon or cervix cancer cell lines proliferation with IC50 values markedly below those observed for the free drugs. Cell-cycle analysis indicated a similar mechanism of action for drug-loaded nanocompounds and free drugs. The results strongly suggest that the cyclodextrin-calixarene heterodimer prototype is an excellent scaffold for nanoformulations aimed to deliver anticancer drugs with limited bioavailability due to low solubility to tumoral cells, markedly increasing their effectivity.

Interactions of tolcapone analogues as stabilizers of the amyloidogenic protein transthyretin.

Transthyretin (TTR) is an amyloidogenic homotetramer involved in the transport of thyroxine and retinol in blood and cerebrospinal fluid. TTR stabilizers, such as tolcapone, an FDA approved drug for Parkinson's disease, are able to interact with residues of the thyroxine-binding sites of TTR, both wild type and pathogenic mutant forms, thereby stabilizing its tetrameric native state and inhibiting amyloidogenesis. Herein, we report on the synthesis of 3-deoxytolcapone, a novel stabilizer of TTR. The high-resolution X-ray analyses of the interactions of 3-O-methyltolcapone and 3-deoxytolcapone with TTR were performed. In the two TTR-ligand complexes the tolcapone analogues establish mainly H-bond and hydrophobic interactions with residues of the thyroxine-binding site of the TTR tetramer. Both compounds are capable of high and selective stabilization of TTR in the presence of plasma proteins, despite their markedly different 'forward' and 'reverse' binding mode, respectively. In fact, the loss or the weakening of stabilizing interactions with protein residues of 3-deoxytolcapone in comparison with tolcapone and 3-O-methyltolcapone is compensated by new interactions established at the dimer-dimer interface. Our data, coupled with previously reported data on the pharmacokinetics properties in humans of tolcapone and 3-O-methyltolcapone, further support the relevance of the latter tolcapone analogue as TTR stabilizer.

Asthma and Obesity in Children.

Asthma and obesity are two major chronic diseases in children and adolescents. Recent scientific evidence points out a causative role of obesity in asthma predisposition. However, studies assessing the real impact of excessive weight gain on lung function in children have shown heterogeneous results. In this review, the pathological mechanisms linking obesity and development of asthma in children are summarized and factors influencing this relationship are evaluated. Common disease modifying factors including age, sex, ethnicity, development of atopic conditions, and metabolic alterations significantly affect the onset and phenotypic characteristics of asthma. Given this, the impact of these several factors on the obesity-asthma link were considered, and from revision of the literature we suggest the possibility to define three main clinical subtypes on the basis of epidemiological data and physiological-molecular pathways: obese-asthmatic and atopy, obese-asthmatic and insulin-resistance, and obese-asthmatic and dyslipidemia. The hypothesis of the different clinical subtypes characterizing a unique phenotype might have an important impact for both future clinical management and research priorities. This might imply the necessity to study the obese asthmatic child with a "multidisciplinary approach", evaluating the endocrinological and pneumological aspects simultaneously. This different approach might also make it possible to intervene earlier in a specific manner, possibly with a personalized and tailored treatment. Surely this hypothesis needs longitudinal and well-conducted future studies to be validated.