An alternative way to perform diagnostic nasopharyngeal swab for SARS-CoV-2 infection

On March 11, 2020, WHO has defined the novel coronavirus disease SARS-CoV-2 (COVID-19) outbreak as a pandemic and still today continues to affect much of the world. Among the reasons for the rapid spread of SARS-CoV-2 infection, there is not only the high transmissibility of the virus, but also the role of asymptomatic or minimally symptomatic carriers. Therefore diagnostic testing is central to contain the global pandemic. Up to now real-time reverse transcriptase polymerase chain reaction (RT-PCR)-based molecular assays for detecting SARSCoV-2 in respiratory specimens is the current reference standard for COVID-19 diagnosis. Nasopharyngeal swab is the preferred choice for SARS-CoV-2 testing; however is not always a free of complications procedure. In patients with severe coagulopathies or diseases such as HHT, the risk of nosebleeding may be high. As in all those conditions like advanced stage sinonasal neoplasms or unfavorable anatomical characteristics, the nasopharyngeal swab may not be feasible. This work reports a safe and effective procedure of nasopharyngeal swab collection for COVID-19 testing, through the transoral way, in patients with contraindication to perform it transnasally. The procedure proved feasible and well tolerated. The discomfort for the patient is comparable with the execution of an oropharyngeal swab without exposing him to additional complications. In selected cases, the procedure described represents a valid alternative to nasopharyngeal swab performed transnasally. In particular, it allows reaching the area with the highest diagnostic sensitivity. Moreover it can be performed by Otolaryngology and, with adequate training, also by non-specialist staff

Preferential solvation of lysozyme in water/ethanol mixtures

We provide a quantitative description of the solvation properties of lysozyme in water/ethanol mixtures, which has been obtained by a simultaneous analysis of small-angle neutron scattering and differential scanning calorimetry experiments. All data sets were analyzed by an original method, which integrates the exchange equilibrium model between water and ethanol molecules at the protein surface and activity coefficients data of water/ethanol binary mixtures. As a result, the preferential binding of ethanol molecules at the protein surface was obtained for both native and thermal unfolded protein states. Excess solvation numbers reveal a critical point at ethanolmolar fraction ≈0.06, corresponding to the triggering of the hydrophobic clustering of alcohol molecules detected in water/ethanol binary mixtures

High pressure small-angle neutron scattering study of the aggregation state of beta-lactoglobulin in water and in water/ethylene-glycol solutions

High-pressure SANS experiments have been performed on acidic dilute solutions of the dimeric protein b-lactoglobulin. To evidence the solvent effect on the protein stability during compression, two different solvents, D2O and a 50% w/w mixture of water and ethylene- glycol have been considered. Data confirm that pressure induces dissociation in both solvents, even if b-lactoglobulin shows an higher stability in 50% ethylene-glycol. An original global fitting procedure has been used to derive the thermodynamic parameters that describe the dissociation equilibrium. As a result, the role of the solvent in protein dissociation has been observed to reflect on volume and compressibility changes

Ethosomes for Coenzyme Q10 Cutaneous Administration: From Design to 3D Skin Tissue Evaluation

Ethosome represents a smart transdermal vehicle suitable for solubilization and cutaneous application of drugs. Coenzyme Q10 is an endogenous antioxidant whose supplementation can counteract many cutaneous disorders and pathologies. In this respect, the present study describes the production, characterization, and cutaneous protection of phosphatidylcholine based ethosomes as percutaneous delivery systems for coenzyme Q10. CoQ10 entrapment capacity in ethosomes was almost 100%, vesicles showed the typical ‘fingerprint’ structure, while mean diameters were around 270 nm, undergoing an 8% increase after 3 months from production. An ex-vivo study, conducted by transmission electron microscopy, could detect the uptake of ethosomes in human skin fibroblasts and the passage of the vesicles through 3D reconstituted human epidermis. Immunofluorescence analyses were carried on both on fibroblasts and 3D reconstituted human epidermis treated with ethosomes in the presence of H2O2 as oxidative stress challenger, evaluating 4-hydroxynonenal protein adducts which is as a reliable biomarker for oxidative damage. Notably, the pretreatment with CoQ10 loaded in ethosomes exerted a consistent protective effect against oxidative stress, in both models, fibroblasts and in reconstituted human epidermis respectively

Berberine and its metabolites: Relationship between physicochemical properties and plasma levels after administration to human subjects

Berberine (1) is an alkaloid used widely in the treatment of several diseases. However, its physicochemical properties, pharmacokinetics, and metabolism remain unclear, and conflicting data have been reported. In this study, the main physicochemical properties of 1 and its metabolites were evaluated, including lipophilicity, solubility, pKa, and albumin binding. A sensitive HPLC-ESIMS/MS method was developed and validated to identify 1 and its main metabolites in human plasma. This method was used to quantify their levels in the plasma of healthy volunteers and hypercholesterolemic patients following a single dose and chronic administration, respectively. In both cases, berberrubine (2) was found to be the main metabolite. Surprisingly, 2 is more lipophilic than 1, which suggests that this compound tautomerizes to a highly conjugated, electroneutral quinoid structure. This was confirmed by NMR studies. These results indicate that the higher plasma concentration of 2 was a consequence of a more efficient intestinal absorption, suggesting that berberrubine is potentially more pharmacologically active than berberine

Experience of an Italian reference laboratory for a rare disease: Hereditary Haemorragic Telangiectasia

Introduction: Hereditary Haemorragic Teleangiectasia (HHT) is an autosomal dominant disorder affecting 1:5000-8000 individuals worldwide. Mucocutaneous telangiectases and Arteriovenous malformations in internal organs (mostly lungs, liver and central nervous system) are the disease hallmarks. HHT is caused by pathogenetic variants in ENG, ACVRL1, SMAD4 and GDF2, belonging to the TGFβ/BMPs pathway. We report the experience of our research laboratory in the last five years (2015-2020), focusing on mutation analysis. Materials and Methods: Patients’ samples were collected by HHT reference centres in Pavia and Crema (CR). Index cases’ samples were analysed by NGS sequencing panel of the four HHT causative genes and MLPA; the molecular investigation in patients’ relatives was performed by Sanger sequencing. Results: We collected 334 patients’ samples; 99/334 were index cases. Results are summarized in the table below. Subjects ACVRL1 ENG SMAD4 Not Found In progress Unaffected Index case 99 39 (39.4%) 26 (26.3%) 1 (1%) 27 (27.3%) 6 (6%) - Patient’s relatives 234 86 60 - - 8 80 Total patients 333 125 86 1 28 13 80 Conclusions: Our data confirm that HHT is mostly underdiagnosed; however, the presence of a reference center enhances the quality of genetic and clinical results. Moreover, we also corroborate the previous observation that in our country ACVRL1 is the major HHT gene. Not found subjects can harbor variants in intronic or regulatory regions rather than in novel genes. However, we are collecting WES data to re-analyse these cases. Grant: CO: Italian Ministry of Education, University and Research to the DMM-University of Pavia “Dipartimenti di Eccellenza (2018-2022)

Circulating microRNAs In Hereditary Hemorrhagic Telangiectasia: Preliminary Results Identify Significant Differences Among Patients

Objectives: We are investigating the role of circulating microRNAs (miRNAs) in HHT as potential disease biomarkers. The main goal is to define an HHT-related miRNAs signature. Particular attention has been paid on miRNAs-genotype and miRNAs-phenotype correlations. Here we present the preliminary results of this study. Methods: We performed a circulating miRNAs profiling in 15 subjects: 5 HHT1, 5 HHT2 Patients, and 5 controls, age and gender matched. miRNAs profile was analysed by qPCR, using serum/plasma microRNA PCR Panel (I + II), V4.M (Exiqon). Each panel contains 752 LNA™ primer sets of human miRNAs, including different controls. Statistical analyses were performed using parametric and non-parametric methods. miRNAs with a p value\0.05 were considered statistically significant and underwent enrichment analysis. Results: The overall result was the detection of 18 deregulated miRNAs. We observed differences between: HHT Patients versus controls; either HHT1 or HHT2 versus controls; HHT1 versus HHT2 Patients and also comparing Patients’ subgroups showing different clinical features. The enrichment analysis identified the top predicted target genes and the related pathways. Among these, we highlighted different pathways already described in association with HHT or angiogenesis. Conclusions: We obtained a preliminary “HHT signature” for circulating miRNAs, underlying, for the first time, differences between the two disease subtypes and a more peculiar miRNAs profile in HHT2. We also described miRNA-PAVMs (Pulmonary Arteriovenous Malformations) correlations. Confirmation of these results in a larger cohort of patients is therefore mandatory, and Patients enrolment for the second step of this study is ongoin

Testing for intrauterine infection [18]

SCOPUS: le.jinfo:eu-repo/semantics/publishe