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Rare earth elements (REEs) have become a key component in many technological applications. Due to the rapid increase in their use, the potential environmental exposure has also expanded. However, the effects on the ecosystem have not been yet thoroughly evaluated, leaving many knowledge gaps. To evaluate the effects of REEs, a set of experiments with acute and chronic exposure were performed on photosynthetic organisms. The effects of acute exposure of four elements (cerium, gadolinium, lanthanum and neodymium) in two set upsexperimental scenarios with different pH values (6 and 4), was evaluated on three testing species: Raphidocelis subcapitata, Lepidium sativum and Vicia faba. In the rResults obtained in the acute exposure, a higher toxicity at pH 4 was observedindicated higher toxicity levels, which could be explained by a higher bioavailability of the elements. From the relative calculated median effective concentrations (EC50s), R. subcapitata was the most sensitive species, followed by L. sativum and lastly, V. faba. Further investigations focused on , the chronic exposure of lanthanum and cerium was evaluated on Raphidocelis subcapitata. After a 28 days exposure, results showed a growth inhibition effects effect and increasing a bioaccumulation (from day 7 to 28) with effect at the end of the test. Moreover, endpoints regardedfluctuating values of the relative as biomarkers of stress (ROS, CAT, and SOD)., fluctuated during the sampling days during the exposure (day 7, 14, 21 and 28).

Cystic fibrosis (CF) is the most frequent lethal autosomal recessive disorder among Caucasians (incidence: 1:2,500 newborn). In the last two decades CF prognosis considerably improved and many patients well survive into their adulthood. Furthermore, milder CF with a late onset was described. CF is a challenge for laboratory of molecular genetics that greatly contributes to the natural history of the disease since fetal age. Carrier screening and prenatal diagnosis, also by non-invasive analysis of maternal blood fetal DNA, are now available, and many labs offer preimplantation diagnosis. The major criticism in prenatal medicine is the lack of an effective multidisciplinary counseling that helps the couples to plan their reasoned reproductive choice. Most countries offer newborn screening that significantly reduce CF morbidity but different protocols based on blood trypsin, molecular analysis and sweat chloride cause a variable efficiency of the screening programs. Again, laboratory is crucial for CF diagnosis in symptomatic patients: sweat chloride is the diagnostic golden standard, but different methodologies and the lack of quality control in most labs reduce its effectiveness. Molecular analysis contributes to confirm diagnosis in symptomatic subjects; furthermore, it helps to predict the disease outcome on the basis of the mutation (genotype-phenotype correlation) and mutations in a myriad of genes, inherited independently by CF transmembrane conductance regulator (CFTR), which may modulate the clinical expression of the disease in each single patient (modifier genes). More recently, the search of the CFTR mutations gained a role in selecting CF patients that may benefit from biological therapy based on correctors and potentiators that are effective in patients bearing specific mutations (personalized therapy). All such applications of molecular diagnostics confirm the “uniqueness” of each CF patient, offering to laboratory medicine the opportunity to reposition the patient in the “core” of the medical process. Proceedings of the 11th International Workshop on Neonatology and Satellite Meetings · Cagliari (Italy) · October 26th-31st, 2015 · From the womb to the adult Guest Editors: Vassilios Fanos (Cagliari, Italy), Michele Mussap (Genoa, Italy), Antonio Del Vecchio (Bari, Italy), Bo Sun (Shanghai, China), Dorret I. Boomsma (Amsterdam, the Netherlands), Gavino Faa (Cagliari, Italy), Antonio Giordano (Philadelphia, USA)

Background Reprocessing of endoscopes is key to preventing cross-infection after colonoscopy. Culture-based methods are recommended for monitoring, but alternative and rapid approaches are needed to improve surveillance and reduce turnover times. A molecular strategy based on detection of residual traces from gut microbiota was developed and tested using a multicenter survey. Methods A simplified sampling and DNA extraction protocol using nylon-tipped flocked swabs was optimized. A multiplex real-time polymerase chain reaction (PCR) test was developed that targeted 6 bacteria genes that were amplified in 3 mixes. The method was validated by interlaboratory tests involving 5 reference laboratories. Colonoscopy devices (n = 111) were sampled in 10 Italian hospitals. Culture-based microbiology and metagenomic tests were performed to verify PCR data. Results The sampling method was easily applied in all 10 endoscopy units and the optimized DNA extraction and amplification protocol was successfully performed by all of the involved laboratories. This PCR-based method allowed identification of both contaminated (n = 59) and fully reprocessed endoscopes (n = 52) with high sensibility (98%) and specificity (98%), within 3-4 hours, in contrast to the 24-72 hours needed for a classic microbiology test. Results were confirmed by next-generation sequencing and classic microbiology. Conclusions A novel approach for monitoring reprocessing of colonoscopy devices was developed and successfully applied in a multicenter survey. The general principle of tracing biological fluids through microflora DNA amplification was successfully applied and may represent a promising approach for hospital hygiene.

Inherited liver diseases are a group of metabolic and genetic defects that typically cause early chronic liver involvement. Most are due to a defect of an enzyme/transport protein that alters a metabolic pathway and exerts a pathogenic role mainly in the liver. The prevalence is variable, but most are rare pathologies. We review the pathophysiology of such diseases and the diagnostic contribution of laboratory tests, focusing on the role of molecular genetics. In fact, thanks to recent advances in genetics, molecular analysis permits early and specific diagnosis for most disorders and helps to reduce the invasive approach of liver biopsy.

Key Clinical Message Microvillous inclusion disease (MVID) typically appears with severe chronic diarrhea in the few days after birth and rapidly causes dehydration and metabolic acidosis. In this context, presenting two novel cases, we underline the crucial importance of mutation analysis for the diagnosis of this disease that may be easily misdiagnosed.

Objectives: We aimed to improve the knowledge of pathogenic mutations in sporadic cases of congenital chloride diarrhea (CCD) and emphasize the importance of functional studies to define the effect of novel mutations. Methods: All member 3 of solute carrier family 26 (SLC26A3) coding regions were sequenced in 17 sporadic patients with CCD. Moreover, the minigene system was used to analyze the effect of 2 novel splicing mutations. Results: We defined the SLC26A3 genotype of all 17 patients with CDD and identified 12 novel mutations. Using the minigene system, we confirmed the in silico prediction of a complete disruption of splicing pattern caused by 2 of these novel mutations: the c.971þ3_971þ4delAA and c.735þ4_c.735þ7delAGTA. Moreover, several prediction tools and a structure-function prediction defined the pathogenic role of 6 novel missense mutations. Conclusions: We confirm the molecular heterogeneity of sporadic CDD adding 12 novel mutations to the list of known pathogenic mutations. Moreover, we underline the importance, for laboratories that offer molecular diagnosis and genetic counseling, to perform fast functional analysis of novel mutations

The emerging concern about the increase of antibiotic resistance and associated biofilm has encouraged scientists to look for alternative antibiotics such as antimicrobial peptides (AMPs). This study evaluated the ability of melittin to act as an antibacterial biofilm inhibitor and biofilm remover considering isolates from dairy industry. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), minimum biofilm inhibitory concentrations (MBICs), and biofilm removal activities were studied in polymicrobial biofilms produced from isolates. MIC and MBC were set at 1–3 µg/mL and 25–50 µg/mL for Gram-positive and Gram-negative bacteria, respectively. Results demonstrated a good MBIC reaching 85% inhibition ability and a good activity and better penetration in deeper layers against the mixed preformed biofilm, thereby increasing its activity against all isolates also at the lowest tested concentrations. Melittin showed interesting characteristics suggesting its potential to act as an antimicrobial agent for polymicrobial biofilm from dairy industry even in environmental isolates.

Background:Pneumonias are the most frequent infectious diseases, characterized by a high prevalence especially among children and adults at risk. The socio-economic impact caused by Streptococcus pneumoniae is evaluated in terms of morbidity, death rate and hospitalizations.Objective:The aim of the study was to demonstrate the potential economic advantages by implementation of an active anti-pneumococcal 13-valent vaccine strategy in Campania region (Southern Italy) in two different categories of subjects, children (aged 0-12), and adults (aged 50- 79) at risk (hypertension, nephropathies, COPD and heart diseases).Methods:Vaccination costs were compared with costs necessary to treat avoidable diseases in the presence and absence of a vaccination program.Results:Offering anti-pneumococcal 13-valent vaccine to the paediatric population was quantified as saving one million euros for Italian national health service in two years. In addition, offering anti-pneumococcal vaccine to adults at risk would generate a return of around 29 million euros.Conclusion:In both cases, offering anti-pneumococcal 13-valent vaccine was proven to be a helpful political health strategy, not only in consideration of a reduction of cases but also in view of the favourable economic impacts.

Objective Cystic fibrosis (CF) is the most common inherited, life limiting condition among Caucasians. No healing therapy is currently available for patients with CF. The aim of the study was to define clinical, anthropometric and biochemical effects of regular, supervised physical exercise in a large cohort of patients with CF. Materials and methods Fifty-nine adult patients with CF that performed regularly supervised physical exercise in the last 3 years in comparison to 59 sex and age matched sedentary patients with CF were included in the study. Results Physical exercise had significantly beneficial effects on: (a) FEV1% decline; (b) anthropometric parameters (lower number of cases with altered BMI, waist and arm circumferences); (c) lipid and glucose metabolism; (d) vitamin D serum levels. Of course, some of this improvement may be because of the better adherence to therapy typical of patients with CF that perform physical activity. Conclusions Such clinical and metabolic effects make supervised physical activity one of the hubs in managing patients with CF.

Background The CFTR gene encodes for a chlorine-transporting protein essential for the correct hydration of epithelium in many organs and tissues. The malfunction of CFTR protein causes Cystic Fibrosis, a genetic disease with an incidence of about 1 out of 3000 births. To date, more than 2000 CFTR gene mutations are known and many of them alter the correct splicing of its mRNA. The qualitative evaluation of the effect of a splicing genetic variant is a simple procedure that by an RT-PCR followed by an electrophoretic separation can reveal the alternative splicing products. Conversely, making a quantitative and accurate evaluation of splicing products, in order to evaluate the percentage of proper residual splicing, requires complicated and expensive procedures. Materials and methods We evaluated the use of digital droplet PCR (ddPCR) with EVAGreen technology to quantify the amount of exon skipping due to splicing mutations. In particular, we used total RNA extracted from nasal cells brushed directly from subjects bearing different intron 9 polyT variants, including the 5T-12TG allele. The total RNA was retro-transcribed and amplified by ddPCR using a single primer pair to amplify both PCR products, 305 bp (correct splicing) and 122 bp (Exon 10 skipped). Results Using a single primer pair it was possible to determine different percentage of exon 10 skipping depending on the poly T tract size, as reported in literature. In particular, we obtained about 50% and 95% correct splicing from 5T/7T and 7T/7T subjects. Conclusions These data, even if preliminary, strongly suggest that it is possible to accurately quantify the percentage of correct residual splicing with a simple pair of primers, without the use of special probes. This methodology could be helpful especially in the analysis of composite heterozygous, in which splicing mutations are associated with other severe mutations and it is necessary to know the exact contribution of the splicing variant to the phenotype.

Two natural mixtures, Allium sativum fermented extract (BGE) and cannabinol oil extract (CBD), were assessed for their ability to inhibit and remove Pseudomonas aeruginosa biofilms on soft contact lenses in comparison to a multipurpose Soft Contact Lens-care solution present on the Italian market. Pseudomonas aeruginosa (ATCC 9027 strain) and Pseudomonas aeruginosa clinical strains isolated from ocular swabs were tested. Quantification of the biofilm was done using the microtiter plate assay and the fractional inhibitory concentration index was calculated. Both forms of Pseudomonas aeruginosa generated biofilms. BGE at minimal inhibitory concentration (MIC) showed inhibition percentages higher than 55% for both strains, and CBD inhibited biofilm formation by about 70%. The care solution at MIC inhibited biofilm formation by about 50% for both strains tested. The effect of BGE on the eradication of the microbial biofilm on soft contact lenses at MIC was 45% eradication for P. aeruginosa ATCC 9027 and 36% for P. aeruginosa clinical strain. For CBD, we observed 24% biofilm eradication for both strains. For the care solution, the eradication MICs were 43% eradication for P. aeruginosa ATCC 9027 and 41% for P. aeruginosa clinical strain. It was observed that both the test soft contact lenses solution/BGE (fractional inhibitory concentration index: 0.450) and the test soft contact lenses solution/CBD (fractional inhibitory concentration index: 0.153) combinations exhibited synergistic antibiofilm activity against most of the studied bacteria. The study showed that BGE and CBD have good effect on inhibition of biofilm formation and removal of preformed biofilms, which makes them promising agents that could be exploited to develop more effective care solutions.