Evaluation of an ELISA method to detect Listeria monocytogenes in fresh-cut rocket

Leafy vegetables are consumed fresh after harvest with bland washes, not always efficient in removing pathogenic bacteria that may be attached to the external skin or surfaces. In this work, an optimized ELISA based method developed in our laboratory was used to detect the presence of Listeria monocytogenes in fresh-cut rocket leaves. From a certain quantity (5 g) of fresh rocket leaves contaminated with Listeria monocytogenes, bacteria were isolated and the pathogen was detected using an ELISA protocol. The preliminary results are promising in the use of antibody-antigen interaction to verify the presence of L. monocytogenes in the minimally processed vegetable distribution chain

Gene expression analysis of rocket salad under pre-harvest and postharvest stresses : a transcriptomic resource for Diplotaxis tenuifolia

Diplotaxis tenuifolia L. is of important economic value in the fresh-cut industry for its nutraceutical and sensorial properties. However, information on the molecular mechanisms conferring tolerance of harvested leaves to pre- and postharvest stresses during processing and shelf-life have never been investigated. Here, we provide the first transcriptomic resource of rocket by de novo RNA sequencing assembly, functional annotation and stress-induced expression analysis of 33874 transcripts. Transcriptomic changes in leaves subjected to commercially-relevant pre-harvest (salinity, heat and nitrogen starvation) and postharvest stresses (cold, dehydration, dark, wounding) known to affect quality and shelf-life were analysed 24h after stress treatment, a timing relevant to subsequent processing of salad leaves. Transcription factors and genes involved in plant growth regulator signaling, autophagy, senescence and glucosinolate metabolism were the most affected by the stresses. Hundreds of genes with unknown function but uniquely expressed under stress were identified, providing candidates to investigate stress responses in rocket. Dehydration and wounding had the greatest effect on the transcriptome and different stresses elicited changes in the expression of genes related to overlapping groups of hormones. These data will allow development of approaches targeted at improving stress tolerance, quality and shelf-life of rocket with direct applications in the fresh-cut industries

Validation of ELISA-based detection of L. monocytogenes and E. coli O157:H7 in fresh cut vegetables

Innovative diagnostic methods were developed for the detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed fresh cut fruits and vegetables. The aim of the present study was to validate the technical efficiency of these methods and evaluate their efficacy and viability for routine analysis. To this purpose, ready-to-eat fresh fruits and vegetables were collected throughout the production chain. A multidisciplinary approach, including a newly developed ELISA method compared to ISO procedures, was applied to detect the pathogenic bacteria after harvesting, processing and shelf-life. Results obtained exhibited the technical efficiency of the developed methods showing similar sensitivity, specificity, negative predictive values and negative likelihood ratios

Detection and enumeration of Listeria monocytogenes in fresh cut vegetables using MPN-Real-Time PCR

Listeria monocytogenes is a gram positive, rod shaped, pathogenic bacterium, causative agent of a severe infection generally known as listeriosis. Packaging and storage conditions of fresh cut vegetables may favour the growth of this psychrotrophic pathogen leading to potential health threat. Detection and enumeration of L. monocytogenes in concentrations up to 103 CFU/g, usually implies use of the most-probable-number technique (MPN) which may take up to seven days for verified identification of the pathogen. We developed a fast and reliable protocol combining MPN with a Real-Time quantitative PCR (qPCR) approach. Samples of fresh cut salads (25 g) purchased at local shops were spiked with 1 to 105 CFU/g of L. monocytogenes. Samples were homogenized, and triplicate series of tubes containing 10-5 to 10 g of food were incubated in Fraser broth at 30\ub0C for 48 h for standard MPN analysis. After incubation, broth samples were taken from each tube and DNA was extracted. DNA from enrichment tubes was used as template in a qPCR assay targeting a 64 bp hlyA gene sequence of L. monocytogenes. Results of this assay were than compared with those of standard MPN analysis and a complete accordance was observed. Furthermore, we tested an enrichment free approach using the same qPCR assay. Samples were prepared as described for MPN-qPCR while DNA extraction was performed prior to enrichment of inoculated salads. This approach allowed us to identify L. monocytogenes in samples spiked with 10-105 CFU/g. The whole process, including DNA extraction, required less than four hours, thus providing a fast and reliable tool for detection of L. monocytogenes in fresh cut vegetables

Effect of temperature and cut size on the volatile organic compound profile, and expression of Chorismate synthase in fresh-cut melon

The postharvest quality of fresh-cut melon is strongly affected by storage conditions to which it is subjected. During postharvest, fruit undergoes several stresses and its physiology is similar to that in senescent tissues. This affects both its biochemistry and the expression of genes involved in secondary metabolite biosynthesis. Volatile organic compounds (VOCs) have been used previously to assess quality of fresh cut melon as they reflect changes in flavor and also changes in overall metabolism. Chorismate synthase (CS) is a key enzyme in the shikimate pathway and catalyzes the formation of chorismate, which is the precursor of numerous aromatic compounds in plants. In this work the effects of different storage temperatures and cut-sizes were studied, with the aim of identifying effects on flavor through changes in VOCs, and molecular responses of the CmCS gene to different postharvest conditions. Melon (Cucumis melo L. 'Macigno') fruits were harvested at a fully ripened commercial stage, were washed in a chlorine water solution, and the mesocarp (pulp) was cut in cube-shaped portions; two sizes were chosen, 1×1 and 3×2 cm. Melon cubes were then stored at 20 or 4°C. VOCs were assessed in the 3×2 cut size at both temperatures and showed clear changes during storage. A subset of VOCs were shown to correlate negatively with storage time and temperature and from these, specific compounds can be identified that act as markers for an overall change in VOC profiles. CmCS was more affected by temperature, showing decreased levels of expression during storage at 20°C with respect to harvest and to cold storage. On the other hand, cut-size did not result in changes in its expression in 3×2 cut size

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Background: Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. // Methods: We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung's disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. // Findings: We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung's disease) from 264 hospitals (89 in high-income countries, 166 in middle-income countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in low-income countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. // Interpretation: Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between low-income, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

MOLECULAR AND GENETIC ANALYSIS OF RNA SILENCING PATHWAYS IN VITIS VINIFERA

Plants contain a heterogeneous and complex population of small RNAs (sRNAs) with regulatory function at the transcriptional and post-transcriptional level. sRNAs are involved in regulation of endogenous genes, in defence response and in maintaining genome integrity. Their biosynthetic pathways require different combinations of RNA silencing proteins such as DICER-LIKE protein (DCL), ARGONAUTE (AGO) as well as other components such as RNA dependent RNA polymerase (RDR). The aim of this Ph.D. research was to study the role of the RNA silencing proteins in the generation of sRNAs classes and to understand their biological function in plant development and viral resistance in grape (Vitis vinifera). A total of 4 DCLs, 6 RDRs and 10 AGO proteins were identified in grape. In order to identify sRNAs classes according to their specific requirement, hairpin-RNA interference (hpRNAi) technology was applied to silence VvDCL4 and VvRDR6. These two genes cooperate in the generation pathway of the trans-acting short interfering RNAs (tasiRNAs) and in viral defence. Transgenic plants carrying hpRNAi constructs were affected in growth and development and showed alteration in leaf development compare to control plants. Deep sequencing of small RNAs of the most highly silenced plants revealed the reduction in the accumulation of specific tasiRNAs involved in leaf development Furthermore, RNA silencing mechanism in grape upon viral infection were better characterized. Small RNAs deriving from plants infected with Grape leaf roll virus (GRLaV) and Grape fleck virus (GFKV) were sequenced using the Illumina next generation sequencing technology and their expression profile studied. Our results suggest an inter-complementation of DCLs in grape upon infection with GfKV and possibly the involvement of only VvDCL4 upon infection with GLRaV1. Our work provides a better understanding of the role of the RNA silencing machinery in grape during development and in response to viral infection. In the future, our results will help to develop novel cultivars resistant to GLRaV1 to prevent heavy losses in grape cultures worldwide

Isolamento di marcatori molecolari per la valutazione della qualit&#224; in rucola

Metodi rapidi e non distruttivi sono necessari per valutare la qualit\ue0 dei prodotti di quarta gamma lungo la catena produttiva che va dalla raccolta alla conservazione. La qualit\ue0 dei prodotti vegetali in post-raccolta non pu\uf2 essere migliorata, ma soltanto preservata, l\u2019identificazione di marcatori molecolari pu\uf2 essere utile nel determinare la minima variazione di qualit\ue0 fin dal primo giorno di conservazione. L\u2019obiettivo del nostro lavoro \ue8 quello di isolare marcatori molecolari utilizzando una strategia di sequenziamento dell\u2019RNA (RNA-seq). La rucola (Diplotaxis tenuifolia L.) coltivata in floating system \ue8 stata sottoposta a diversi tipi di stress abiotici. In pre-raccolta sono stati imposti stress salino (200\u2009mM NaCl), stress radicale da alte temperature (40\ub0C), carenza di nitrati (assenza di NH4+/NO3-), mentre in post-raccolta \ue8 stato imposto stress da freddo (4\ub0C), taglio e perdita di acqua. Il campionamento delle piante \ue8 stato effettuato dopo 24 ore. L\u2019RNA totale \ue8 stato estratto dai campioni stressati e dal controllo e sequenziato tramite Illumina HiSeq\u2122 2000. L\u2019RNA-seq ha permesso di isolare quei geni la cui espressione \ue8 specifica per ogni stress oltre a quelli la cui espressione \ue8 comune a tutti gli stress. Tali geni codificano per proteine associate alla senescenza, a processi degenerativi e a perdita di qualit\ue0 e, pertanto, vengono considerati come marcatori di qualit\ue0. I risultati di RNA-seq saranno validati tramite PCR quantitativa e i livelli di espressione genica saranno monitorati in diverse condizioni durante il periodo di conservazione della rucola. L\u2019espressione dei potenziali marcatori durante la conservazione sar\ue0 correlata con le variazioni dei principali parametri qualitativi. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n. 289719 (Project QUAFETY

Design and simulation of a DC/DC converter with dynamic thermal motor control for racing application

In this paper, a DC/DC power converter devoted to electrical motor drive in motorbike racing application is designed and simulated in MATLAB Simulink environment, along with its control logic strategy. In particular, we conceived, developed and simulated a thermal control strategy, to be implemented as the logic core of the power converter, which dynamically and automatically fits the electric power fed to motor, depending on its actual temperature. As a result, motor burnout during the race is avoided, improving the power train reliability without affecting the racing performance. Simulation results, in which the whole power train system was modeled in Simulink, confirmed the proposed algorithm effectiveness