Development of a droplet digital PCR assay to detect illicit glucocorticoid administration in bovine

Glucocorticoids are often used illegally in food-producing animals for the growth promotion of livestock animals. In accordance to official chemical methods for glucocorticoid detection, an animal is declared as non-compliant when a residue is identified in the sample. Neverthless, growth promoting molecules can often escape identification due to their rapid elimination or due to the use of non-detectable new generation drugs. Therefore, an indirect screening method able to detect the biological effect of long-term administration of low doses of dexamethasone and prednisolone on livestock has been developed to support official methods. As already described, FKBP5 (FKBP prolyl isomerase 5) expression in bovine thymus is regulated by glucocorticoids, and this specific regulation can be exploited in an indirect screening assay. In the present study, male veal calves and young bulls were considered in three different trials in which estradiol, dexamethasone, and prednisolone were administered alone or in combination with Revalor-200 subcutaneous pellets. Thoracic thymus was sampled from all animals and molecular analysis was performed. A duplex droplet digital PCR assay with EvaGreen(®) was employed to detect the target gene expression using absolute quantification. The developed droplet digital PCR assay was precise, showing intra- and inter-assay mean coefficient of variation values of about 6.16% and 3.17%, respectively. It was also highly specific (100%) with Youden’s index of 76.92% and 53.57% applied to veal calves and young bulls, respectively. The lowest detection limit in which the target gene expression level was kept constant, was 0.05 ng/μl of cDNA with 1 copies/μL and 0.5 copies/μL for target and reference gene, respectively. This study establishes the basis for using a digital PCR-based assay as an efficient test to identify animals illegally treated with glucocorticoids

Inertial bioluminescence rhythms at the Capo Passero (KM3NeT-Italia) site, Central Mediterranean Sea

Peer reviewedPublisher PD

Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia

Key Points CALR mutations occur in half of JAK2 and MPL wt patients with ET and associate with some distinctive phenotypic traits. Patients with ET harboring CALR mutations are at significantly lower risk of thrombosis compared with JAK2- and MPL-mutated patients

Molecular interactions, characterization and photoactivity of Chlorophyll a/chitosan/2-HP-β-cyclodextrin composite films as functional and active surfaces for ROS production

Novel photosensitizing film based on the natural hybrid polymer Chitosan/2-hydroxy-propyl-β-Cyclodextrin (CH/CD) is synthesized introducing Chlorophyll a (CH/CD/Chla) as a photoactive agent for possible application in antimicrobial photodynamic therapy (PDT). The polymer absorbs visible light, in turn able to generate reactive oxygen species (ROS) and, therefore it can be used as environmental friendly and biodegradable polymeric photosensitizer (PS). The modified film is characterized by means of different spectroscopic, calorimetric, diffraction techniques and microscopic imaging methods including time-resolved absorption spectroscopy. UV–Vis, FTIR-ATR and X-ray Photoelectron Spectroscopy (XPS) analyses suggest that Chla shows a strong affinity toward Chitosan introducing interactions with amino groups present on the polymer chains. Nanosecond laser flash photolysis technique provides evidence for the population of the excited triplet state of Chla. Photogeneration of singlet oxygen is demonstrated by both direct detection by using infrared luminescence spectroscopy and chemical methods based on the use of suitable traps. Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and Differential Scanning Calorimetry (DSC) analyses confirm also the occurrence of structural changes both on the film surface and within the film layer induced by the insertion of the pigment. Moreover, X-ray Diffraction data (XRD) shows the existence of an amorphous phase for the chitosan films in all the compared conditions

Proposal for a New Score-Based Approach To Improve Efficiency of Diagnostic Laboratory Workflow for Acute Bacterial Meningitis in Adults

Microbiological tests on cerebrospinal fluid (CSF) utilize a common urgent-care procedure that does not take into account the chemical and cytological characteristics of the CSF, resulting sometimes in an unnecessary use of human and diagnostic resources. The aim of this study was to retrospectively validate a simple scoring system (bacterial meningitis-Careggi score [BM-CASCO]) based on blood and CSF sample chemical/cytological parameters for evaluating the probability of acute bacterial meningitis (ABM) in adults. BM-CASCO (range, 0 to 6) was defined by the following parameters: CSF cell count, CSF protein levels, CSF lactate levels, CSF glucose-to-serum glucose ratio, and peripheral neutrophil count. BM-CASCO was retrospectively calculated for 784 cases of suspected ABM in adult subjects observed during a four-and-a-half-year-period (2010 to 2014) at the emergency department (ED) of a large tertiary-care teaching hospital in Italy. Among the 28 confirmed ABM cases (3.5%), Streptococcus pneumoniae was the most frequent cause (16 cases). All ABM cases showed a BM-CASCO value of ≥3. Most negative cases (591/756) exhibited a BM-CASCO value of ≤1, which was adopted in our laboratory as a cutoff to not proceed with urgent microbiological analysis of CSF in cases of suspected ABM in adults. During a subsequent 1-year follow-up, the introduction of the BM-CASCO in the diagnostic workflow of ABM in adults resulted in a significant decrease in unnecessary microbiological analysis, with no false negatives. In conclusion, BM-CASCO appears to be an accurate and simple scoring system for optimization of the microbiological diagnostic workflow of ABM in adults

Electromyographic signature of isometric squat in the highest refuge in Europe

Reports of electromyography during hypoxic exercise are contrasting, due to protocol and muscle diversity. This work aimed to investigate alterations in muscle activation and myoelectrical fatigue during exercise at high-altitude in those muscles primarily involved in trekking. Twelve young adults balanced by gender and age were tested at low (1,667 m) and high (4,554 m, "Capanna Margherita", Italy) altitude, during an isometric squat lasting 60 seconds. High-density surface electromyography was performed from the quadriceps of right limb. The root mean square (RMS), median frequency with its slope, and muscle fiber conduction velocity (MFCV) were computed. Neither males nor females showed changes in median frequency (Med: 36.13 vs 35.63 Hz) and its slope (Med: -9 vs -12 degree) in response to high-altitude trekking, despite a great inter-individual heterogeneity, nor differences were found for MFCV. RMS was not significantly equivalent, with greater values at low altitude (0.385 ± 0.104 mV) than high altitude (0.346 ± 0.090 mV). Unexpected results can be due either to a postural compensation of the whole body compensating for a relatively greater effort or to the inability to support muscle activation after repeated physical efforts.  Interesting results may emerge by measuring simultaneously electromyography, muscle oxygenation and kinematics comparing trekking at normoxia vs hypoxia

Identifying priorities for the protection of deep Mediterranean Sea ecosystems through an integrated approach

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Fanelli, E., Bianchelli, S., Foglini, F., Canals, M., Castellan, G., Guell-Bujons, Q., Galil, B., Goren, M., Evans, J., Fabri, M.-C., Vaz, S., Ciuffardi, T., Schembri, P. J., Angeletti, L., Taviani, M., & Danovaro, R. Identifying priorities for the protection of deep Mediterranean Sea ecosystems through an integrated approach. Frontiers in Marine Science, 8, (2021): 698890, https://doi.org/10.3389/fmars.2021.698890.Benthic habitats of the deep Mediterranean Sea and the biodiversity they host are increasingly jeopardized by increasing human pressures, both direct and indirect, which encompass fisheries, chemical and acoustic pollution, littering, oil and gas exploration and production and marine infrastructures (i.e., cable and pipeline laying), and bioprospecting. To this, is added the pervasive and growing effects of human-induced perturbations of the climate system. International frameworks provide foundations for the protection of deep-sea ecosystems, but the lack of standardized criteria for the identification of areas deserving protection, insufficient legislative instruments and poor implementation hinder an efficient set up in practical terms. Here, we discuss the international legal frameworks and management measures in relation to the status of habitats and key species in the deep Mediterranean Basin. By comparing the results of a multi-criteria decision analysis (MCDA) and of expert evaluation (EE), we identify priority deep-sea areas for conservation and select five criteria for the designation of future protected areas in the deep Mediterranean Sea. Our results indicate that areas (1) with high ecological relevance (e.g., hosting endemic and locally endangered species and rare habitats),(2) ensuring shelf-slope connectivity (e.g., submarine canyons), and (3) subject to current and foreseeable intense anthropogenic impacts, should be prioritized for conservation. The results presented here provide an ecosystem-based conservation strategy for designating priority areas for protection in the deep Mediterranean Sea.This study was supported by the DG ENV project IDEM (Implementation of the MSFD to the Deep Mediterranean Sea; contract EU No. 11.0661/2017/750680/SUB/EN V.C2). MC and QG-B acknowledge support from Generalitat de Catalunya autonomous government through its funding scheme to excellence research groups (Grant 2017 SGR 315)

Benefits of glucocorticoids in non-ambulant boys/men with Duchenne muscular dystrophy: A multicentric longitudinal study using the Performance of Upper Limb test

The aim of this study was to establish the possible effect of glucocorticoid treatment on upper limb function in a cohort of 91 non-ambulant DMD boys and adults of age between 11 and 26 years. All 91 were assessed using the Performance of Upper Limb test. Forty-eight were still on glucocorticoid after loss of ambulation, 25 stopped steroids at the time they lost ambulation and 18 were GC naive or had steroids while ambulant for less than a year. At baseline the total scores ranged between 0 and 74 (mean 41.20). The mean total scores were 47.92 in the glucocorticoid group, 36 in those who stopped at loss of ambulation and 30.5 in the naive group (p <0.001). The 12-month changes ranged between -20 and 4 (mean -4.4). The mean changes were -3.79 in the glucocorticoid group, -5.52 in those who stopped at loss of ambulation and -4.44 in the naive group. This was more obvious in the patients between 12 and 18 years and at shoulder and elbow levels. Our findings suggest that continuing glucocorticoids throughout teenage years and adulthood after loss of ambulation appears to have a beneficial effect on upper limb function. (C) 2015 The Authors. Published by Elsevier B.V

Timed rise from floor as a predictor of disease progression in Duchenne muscular dystrophy: An observational study

The role of timed items, and more specifically, of the time to rise from the floor, has been reported as an early prognostic factor for disease progression and loss of ambulation. The aim of our study was to investigate the possible effect of the time to rise from the floor test on the changes observed on the 6MWT over 12 months in a cohort of ambulant Duchenne boys.A total of 487 12-month data points were collected from 215 ambulant Duchenne boys. The age ranged between 5.0 and 20.0 years (mean 8.48 ±2.48 DS).The results of the time to rise from the floor at baseline ranged from 1.2 to 29.4 seconds in the boys who could perform the test. 49 patients were unable to perform the test at baseline and 87 at 12 month The 6MWT values ranged from 82 to 567 meters at baseline. 3 patients lost the ability to perform the 6mwt at 12 months. The correlation between time to rise from the floor and 6MWT at baseline was high (r = 0.6, p<0.01).Both time to rise from the floor and baseline 6MWT were relevant for predicting 6MWT changes in the group above the age of 7 years, with no interaction between the two measures, as the impact of time to rise from the floor on 6MWT change was similar in the patients below and above 350 m. Our results suggest that, time to rise from the floor can be considered an additional important prognostic factor of 12 month changes on the 6MWT and, more generally, of disease progression