Fine tuning of a sequencing protocol for species identification of Mytilus spp. specimens

The genus Mytilus comprises eight species of mussels of commercial relevance, which have different geographic origin and distribution and can hybridize in case of coexistence (1). To date, events of habitat contamination with allochthonous species occur with increasing frequency, due mainly to anthropogenic transfer, posing threats to local ecosystems (2). Moreover, such events favour species mislabeling, which is reported as the most frequent issue affecting seafood products (3). To prevent these problems, EU Regulation 1379/2013 promotes the use of DNA-based methods. PCR-RFLP is to date one of the most efficient techniques used in Mytilus spp. species identification (4-5). However, it has some drawbacks linked to limited resolution and subjective interpretation of the results. To improve such aspects, an optimized protocol for the sequencing of short fragments was developed. A total of 50 samples were analyzed. Of them, 12 were DNA samples belonging to various Mytilus spp. that were already identified by PCR-RFLP in a previous study (5). The other 38 were tissue samples, of which 10 belonging to specimens directly collected from production sites in Chile, and 28 belonging to fresh and pre-cooked specimens collected from national market. For these samples, the total DNA was extracted using the DNeasy Mericon Food kit (Qiagen). The target PAP region was amplified from all the 50 samples (DNA and tissue) using the primer pair designed by Satto et al. (4). All PCR products were analyzed through capillary electrophoresis to verify the successful amplification, and then sequenced using Sanger technique. Twenty samples (41%), of which 10 from production site and 10 from market samples, were randomly selected to perform the RFLP analysis to be compared to the sequencing results. Ten out of the 12 species (83.3%) identified by PCR-RFLP in the previous study were confirmed by sequencing. The remaining 2 samples, identified as M. chilensis x M. edulis by PCR-RFLP were instead identified as M. chilensis x M. trossulus by capillary electrophoresis and confirmed by sequencing. For the remaining 38 samples, sequencing identified 20 tissue samples (52.6%) as M. galloprovincialis, 7 as M. chilensis (18.4%), 6 as hybrids M. chilensis x M. galloprovincialis (15.8%), 2 as M. edulis (5.3%), 2 as hybrids M. chilensis x M. trossulus (5.3%) and 1 as hybrid M. galloprovincialis x M. edulis (2.6%). The PCR-RFLP confirmed the sequencing results for 19 of the tested samples and failed the unambiguous identification of one sample which was identified as hybrid by sequencing. Overall, the species identification by PCRRFLP failed in 3 out 32 (9.4%) tested samples (12 DNA and 20 tissue). These findings suggest that the optimized protocol relying on Sanger sequencing has some practical advantages. First, the use of capillary electrophoresis to visualize the PCR products allowed the clear distinction of M. edulis and M. trossulus which, having a difference in amplicon length of only 12 bp (177 bp vs 165 bp), are very hard to discriminate with a standard agarose gel electrophoresis. Moreover, the use of sequencing allowed the unambiguous identification of pure species and hybrids, especially in the case of M. galloprovincialis x M. edulis, in which a double peak at the SNP site is clearly visible. Considering the ever-dropping costs linked to sequence-based technology, we propose this sequencing protocol as a valid, consistent, and reliable alternative to the currently used methods

The demersal fish assemblages of the infra and circalittoral coastal rocky bottoms of the Aeolian Archipelago (Central Mediterranean Sea) studied by Remotely Operated Vehicle (ROV)

Demersal fish assemblages on the rocky bottoms of the Aeolian Archipelago were investigated using a Remotely Operated Vehicle (ROV) within the framework of research activities aimed at drawing up the zoning proposal for a new Italian national marine protected area. Visual assessments were conducted around the seven main islands by means of 36 ROV transects. The video material was divided into 3 parts belonging to 3 Archipelago sectors (Western, Central and Eastern) and into 3 depth ranges (20-50, 51-120, 121-190). Thirty taxa of teleosts (29 species and 1 genus) belonging to 16 families were recorded. The assemblages werenumerically dominated by some schooling fishes, such as Anthias anthias, Callanthias ruber and Chromis chromis that exhibited adepth-related partitioning of space, and three non-gregarious species, namely Serranus cabrilla, Coris julis and Lappanella fasciata.In terms of species composition, the assemblages observed in the sectors of the Archipelago largely overlapped. No significant sector-related differences were detected in fish species richness, diversity and total density. Species composition and the investigated assemblage parameters were significantly affected by depth. The pattern of variation in species richness among depth rangesdiffered from one archipelago sector to another. No significant interaction between the depth range and sector factors was observed in terms of species diversity and total density. Diversity values at 20-50 and 121-190 m depth were similar and significantly higher than at 51-120 m depth. Fish total density showed a clear downward trend with increasing depth, although significant differences were detected between the 20-50 and 51-120 depth layers and the deepest one. Overall, the demersal fish assemblage of the Aeolian Archipelago was poorly diversified and depleted, most likely due to overfishing. This information highlights the importanceof adopting specific measures aimed at the recovery of overexploited resources and the restoration of entire marine ecosystems

COVID-19-Related Myocarditis: Are We There Yet? A Case Report of COVID-19-Related Fulminant Myocarditis

There is increasing evidence of cardiac involvement in COVID-19 cases, with a broad range of clinical manifestations spanning from acute life-threatening conditions such as ventricular dysrhythmias, myocarditis, acute myocardial ischemia and pulmonary thromboembolism to long-term cardiovascular sequelae. In particular, acute myocarditis represents an uncommon but frightening complication of SARS-CoV-2 infection. Even if many reports of SARS CoV-2 myocarditis are present in the literature, the majority of them lacks histological confirmation of cardiac injury. Here, we report a case of a young lady, who died suddenly a few days after testing positive for SARS-CoV-2, whose microscopic and genetics features suggested a direct cardiac involvement compatible with fulminant myocarditis

Facies created by the yellow coral Dendrophyllia cornigera (Lamarck, 1816): Origin, substrate preferences and habitat complexity

The yellow coral Dendrophyllia cornigera (Lamarck, 1816) is a NE Atlantic-Mediterranean scleractinian. It is considered a typical hard bottom species, generally reported on outcropping rocks from mesophotic to upper bathyal depths. Several evidences suggest that this species is able to tolerate a broad range of temperatures, which allows it to colonize numerous environments in a wide depth range. In the present study, we first provided a detailed ecological characterization of the D. cornigera dense aggregation thriving on the Mantice Shoal (NW Ligurian Sea, Mediterranean Sea). Information on substrate type and inclination, average extension and density, colonies size-class frequency distribution and associated fauna are reported. Then, we presented an extensive review of the available information on the ecology of this species, including 142 new ROV records from the Italian coast (40–1820 m). Results indicated that D. cornigera occurs on a wide range of substrates, including soft bottoms and hardgrounds (outcropping rocks, coralligenous rock and dead cold-water coral frameworks), with significant differences in colony density and size among different substrates. Dendrophyllia cornigera creates three main facies, each characterized by a specific combination of substrate, inclination, depth, and associated fauna. Scattered living colonies, as well as large thanatocoenoses, display a wide geographical and bathymetric distribution. Differently, the facies represented by dense meadows on horizontal soft-bottoms results rare, being reported only from the Mantice Shoal and the Amendolara Bank (Ionian Sea). The radiocarbon age of the thanatocoenoses varies between 400 (Corsica Channel) and 13000 (Vercelli Seamount) years before present. This study highlights the wide adaptability of D. cornigera in terms of environmental settings, changing the current view on the ecology of this species and providing essential insights for the implementation of international deep-sea habitat classification schemes and conservation measures

The ACTyourCHANGE study protocol: promoting a healthy lifestyle in patients with obesity with Acceptance and Commitment Therapy-a randomized controlled trial

BackgroundAs treatment of choice in promoting psychological flexibility, Acceptance and Commitment Therapy (ACT) was found to be effective in several conditions, and among different populations, including weight management in individuals with obesity. However, the mechanism of action of psychological flexibility is less known. The aim of the present study is, within the context of a brief ACT intervention for behavioral change and behavioral maintenance of a healthy lifestyle in a sample of inpatients with obesity, to explore the effect of each subcomponent of the psychological flexibility model on treatment processes and outcomes.MethodsA randomized controlled trial will be conducted. Ninety Italian adult inpatients with obesity attending a rehabilitation program for weight loss will be randomly allocated into three experimental conditions targeting respectively each subcomponent of the psychological flexibility model: group Engage focused on values-oriented behaviors, group Openness focused on acceptance and cognitive defusion, and group Awareness focused on being present and aware of thoughts, feelings, and behaviors at every moment. Weight, BMI (kg/m(2)), the Psychological General Well-Being Inventory (PGWBI), the Outcome Questionnaire-45.2 (OQ-45.2), the Depression Anxiety and Stress Scale (DASS-21), the Difficulties in Emotion Regulation Scale (DERS), the Dutch Eating Behaviors Questionnaire (DEBQ), the Brief Values Inventory (BVI), the Committed Action Questionnaire (CAQ), the Italian-Cognitive Fusion Questionnaire (I-CFQ), the Five Facet Mindfulness Questionnaire (FFMQ), and the Acceptance and Action Questionnaire (AAQ-II) will be assessed at the beginning (time 0), at the end of psychological intervention (time 1), and after 3 (time 2) and 6months (time 3) and 9months (time 4) from discharge. During the following month after discharge, outpatients will be monitored in their adherence to a healthy lifestyle, using a wearable device.To assess the effectiveness of the intervention, mixed between-within 3 (conditions) x4 (times) repeated measure ANOVAs will be conducted to examine changes from time 0 to time 1, 2, 3, and 4 in means of weight, BMI, and means of scores PGWBI, OQ-45.2, DASS, DERS, DEBQ, AAQ-II, BVI, CAQ, I-CFQ, and FFMQ, between three groups.DiscussionThis study will contribute to clarify the mechanism of action of each subcomponent of the psychological flexibility model and understand its impact on the promotion of a healthy lifestyle.Trial registrationClinicalTrials.govNCT04474509. Registered on July 4, 202

New advances in metabolic syndrome, from prevention to treatment. The role of diet and food

The definition of metabolic syndrome (MetS) has undergone several changes over the years due to the difficulty in establishing universal criteria for it. Underlying the disorders related to MetS is almost invariably a pro-inflammatory state related to altered glucose metabolism, which could lead to elevated cardiovascular risk. Indeed, the complications closely related to MetS are cardiovascular diseases (CVDs) and type 2 diabetes (T2D). It has been observed that the predisposition to metabolic syndrome is modulated by complex interactions between human microbiota, genetic factors, and diet. This review provides a summary of the last decade of literature related to three principal aspects of MetS: (i) the syndrome’s definition and classification, pathophysiology, and treatment approaches; (ii) prediction and diagnosis underlying the biomarkers identified by means of advanced methodologies (NMR, LC/GC-MS, and LC, LC-MS); and (iii) the role of foods and food components in prevention and/or treatment of MetS, demonstrating a possible role of specific foods intake in the development of MetS

Bone sarcoma patient-derived xenografts are faithful and stable preclinical models for molecular and therapeutic investigations

Standard therapy of osteosarcoma (OS) and Ewing sarcoma (EW) rests on cytotoxic regimes, which are largely unsuccessful in advanced patients. Preclinical models are needed to break this impasse. A panel of patient-derived xenografts (PDX) was established by implantation of fresh, surgically resected osteosarcoma (OS) and Ewing sarcoma (EW) in NSG mice. Engraftment was obtained in 22 of 61 OS (36%) and 7 of 29 EW (24%). The success rate in establishing primary cell cultures from OS was lower than the percentage of PDX engraftment in mice, whereas the reverse was observed for EW; the implementation of both in vivo and in vitro seeding increased the proportion of patients yielding at least one workable model. The establishment of in vitro cultures from PDX was highly efficient in both tumor types, reaching 100% for EW. Morphological and immunohistochemical (SATB2, P-glycoprotein 1, CD99, caveolin 1) studies and gene expression profiling showed a remarkable similarity between patient's tumor and PDX, which was maintained over several passages in mice, whereas cell cultures displayed a lower correlation with human samples. Genes differentially expressed between OS original tumor and PDX mostly belonged to leuykocyte-specific pathways, as human infiltrate is gradually replaced by murine leukocytes during growth in mice. In EW, which contained scant infiltrates, no gene was differentially expressed between the original tumor and the PDX. A novel therapeutic combination of anti-CD99 diabody C7 and irinotecan was tested against two EW PDX; both drugs inhibited PDX growth, the addition of anti-CD99 was beneficial when chemotherapy alone was less effective. The panel of OS and EW PDX faithfully mirrored morphologic and genetic features of bone sarcomas, representing reliable models to test therapeutic approaches

Small Area Estimation of Latent Economic Well-being

© The Author(s) 2019. Small area estimation (SAE) plays a crucial role in the social sciences due to the growing need for reliable and accurate estimates for small domains. In the study of well-being, for example, policy makers need detailed information about the geographical distribution of a range of social indicators. We investigate data dimensionality reduction using factor analysis models and implement SAE on the factor scores under the empirical best linear unbiased prediction approach. We contrast this approach with the standard approach of providing a dashboard of indicators or a weighted average of indicators at the local level. We demonstrate the approach in a simulation study and a real data application based on the European Union Statistics for Income and Living Conditions for the municipalities of Tuscany

Surgical and survival outcomes with perioperative or neoadjuvant immune-checkpoint inhibitors combined with platinum-based chemotherapy in resectable NSCLC: A systematic review and meta-analysis of randomised clinical trials

: The use of neoadjuvant or perioperative anti-PD(L)1 was recently tested in multiple clinical trials. We performed a systematic review and meta-analysis of randomised trials comparing neoadjuvant or perioperative chemoimmunotherapy to neoadjuvant chemotherapy in resectable NSCLC. Nine reports from 6 studies were included. Receipt of surgery was more frequent in the experimental arm (odds ratio, OR 1.39) as was pCR (OR 7.60). EFS was improved in the experimental arm (hazard ratio, HR 0.55) regardless of stage, histology, PD-L1 expression (PD-L1 negative, HR 0.74) and smoking exposure (never smokers, HR 0.67), as was OS (HR 0.67). Grade > = 3 treatment-related adverse events were more frequent in the experimental arm (OR 1.22). The experimental treatment improved surgical outcomes, pCR rates, EFS and OS in stage II-IIIB, EGFR/ALK negative resectable NSCLC; confirmatory evidence is warranted for stage IIIB tumours and with higher maturity of the OS endpoint

An Italian Multicenter Perspective Harmonization Trial for the Assessment of MET Exon 14 Skipping Mutations in Standard Reference Samples

Lung cancer remains the leading cause of cancer deaths worldwide. International societies have promoted the molecular analysis of MET proto-oncogene, receptor tyrosine kinase (MET) exon 14 skipping for the clinical stratification of non-small cell lung cancer (NSCLC) patients. Different technical approaches are available to detect MET exon 14 skipping in routine practice. Here, the technical performance and reproducibility of testing strategies for MET exon 14 skipping carried out in various centers were evaluated. In this retrospective study, each institution received a set (n = 10) of a customized artificial formalin-fixed paraffin-embedded (FFPE) cell line (Custom METex14 skipping FFPE block) that harbored the MET exon 14 skipping mutation (Seracare Life Sciences, Milford, MA, USA), which was previously validated by the Predictive Molecular Pathology Laboratory at the University of Naples Federico II. Each participating institution managed the reference slides according to their internal routine workflow. MET exon 14 skipping was successfully detected by all participating institutions. Molecular analysis highlighted a median Cq cut off of 29.3 (ranging from 27.1 to 30.7) and 2514 (ranging from 160 to 7526) read counts for real-time polymerase chain reaction (RT-PCR) and NGS-based analyses, respectively. Artificial reference slides were a valid tool to harmonize technical workflows in the evaluation of MET exon 14 skipping molecular alterations in routine practice