Validation of a 1-Day Analytical Diagnostic Real-Time PCR for the Detection of Salmonella in Different Food Meat Categories

Foodborne disease caused by Salmonella represents a worldwide public health problem. In Europe, salmonellosis is still the second most commonly recorded zoonosis. Since the standard culture method for detecting Salmonella (ISO 6579:2002) requires up to 5 days to produce results, the need to develop rapid methods represents an important issue for the authorities and the producers. The aim of the present study was the in-house validation, according to ISO 16140, of an open-formula diagnostic real-time PCR for the detection of Salmonella in all the different meat categories reported in the EU Regulations relative to microbiological criteria for food safety. The assay employed specific primers and a probe target within the ttrRSBCA locus, which allows the tetrathionate respiration in Salmonella. Selectivity, relative accuracy, relative sensitivity and relative specificity were established by testing 110 bacterial strains and 175 various edible meat samples. Results showed 100 % selectivity, 100 % relative accuracy, 100 % relative sensitivity and 100 % relative specificity of the real-time PCR when compared to the standard culture method used as reference. In addition, in order to minimize the effect of the competitive micro-flora naturally present on meat samples, a highly nutritious and selective commercial medium (ONE Broth Salmonella, Oxoid) was evaluated in comparison with the classical non-selective pre-enrichment broth (buffered peptone water). Results demonstrated that the ONE Broth Salmonella medium increases the growth of Salmonella in the presence of competitive micro-flora.     

Isolation and characterization of copolymers of beta-lactoglobulin, alpha-lactalbumin, kappa-casein, and alphas1-casein generated by pressurization and thermal treatment of raw milk

Raw skim milk was submitted to high pressure (300 to 600 MPa) and temperature (4 to 70 degrees C) treatments for 2 or 5 min. The combined effects of pressure and temperature on milk proteins induced structural changes and polymer and copolymer formation characterized by anion-exchange and size-exclusion fast protein liquid chromatography and electrophoretic techniques. Approximately half of the beta-lactoglobulin formed polymers, and the other half formed large copolymers, mainly with kappa-casein, alpha-lactalbumin via intermolecular disulfide bond exchange, and alpha(s1)-casein via physicochemical interactions, in proportions of 1.0:0.7:0.3:0.1, respectively. Minor whey proteins (serum albumin, immunoglobulins, and lactoferrin) also participated in the formation of the copolymers but to a lesser extent. Two populations of the copolymers were found with apparent molecular masses ranging from 440 to 2000 kDa for the first and more than 2000 kDa for the second. On the contrary, for heated milks the aggregation kinetics obtained by combination of high pressure and thermal treatment were very fast, as no intermediates such as dimers and small size oligomers were observed after pressurization, whatever the temperature studied. Lactosylation of proteins as well as proteolysis were very limited. A beta-casein amino-terminal peptide of 22 kDa was specifically recovered in milk samples treated under the more drastic conditions (500 MPa/55 degrees C per 5 min and 600 MPa/70 degrees C per 5 min) and might have been generated by neutral proteases such as elastase released from somatic cells present in milk. No casein was released from the micelle whatever the combination of high pressure and temperature studied.     

Quantitative Assessment of Affinity Selection Performance by Using DNA-Encoded Chemical Libraries

DNA-encoded chemical libraries are often used for the discovery of ligands against protein targets of interest. These large collections of DNA-barcoded chemical compounds are typically screened by using affinity capture methodologies followed by PCR amplification and DNA sequencing procedures. However, the performance of individual steps in the selection procedures has been scarcely investigated, so far. Herein, the quantitative analysis of selection experiments, by using three ligands with different affinity to carbonic anhydrase IX as model compounds, is described. In the first set of experiments, quantitative PCR (qPCR) procedures are used to evaluate the recovery and selectivity for affinity capture procedures performed on different solid-phase supports, which are commonly used for library screening. In the second step, both qPCR and analysis of DNA sequencing results are used to assess the recovery and selectivity of individual carbonic anhydrase IX ligands in a library, containing 360 000 compounds. Collectively, this study reveals that selection procedures can be efficient for ligands with sub-micromolar dissociation constants to the target protein of interest, but also that selection performance dramatically drops if 10⁴ copies per library member are used as the input.     

Genes and Pathways Involved in Adult Onset Disorders Featuring Muscle Mitochondrial DNA Instability

Replication and maintenance of mtDNA entirely relies on a set of proteins encoded by the nuclear genome, which include members of the core replicative machinery, proteins involved in the homeostasis of mitochondrial dNTPs pools or deputed to the control of mitochondrial dynamics and morphology. Mutations in their coding genes have been observed in familial and sporadic forms of pediatric and adult-onset clinical phenotypes featuring mtDNA instability. The list of defects involved in these disorders has recently expanded, including mutations in the exo-/endo-nuclease flap-processing proteins MGME1 and DNA2, supporting the notion that an enzymatic DNA repair system actively takes place in mitochondria. The results obtained in the last few years acknowledge the contribution of next-generation sequencing methods in the identification of new disease loci in small groups of patients and even single probands. Although heterogeneous, these genes can be conveniently classified according to the pathway to which they belong. The definition of the molecular and biochemical features of these pathways might be helpful for fundamental knowledge of these disorders, to accelerate genetic diagnosis of patients and the development of rational therapies. In this review, we discuss the molecular findings disclosed in adult patients with muscle pathology hallmarked by mtDNA instability.     

A Critical Review of Biological Processes and Technologies for Landfill Leachate Treatment

The most common method to deal with the huge production of municipal wastes is still the disposal in landfills which, however, generates a leachate with high contents of organic and nitrogen pollutants. Requirements must be fixed by law to control leachate release from such landfills, and effective treatment processes and technologies must be applied to reduce its impact on the environment. Biological processes for leachate treatment are discussed in this review, in the former part of which attention is paid to leachate formation and its qualitative and quantitative features, while the latter deals with the state‐of‐the‐art of the most effective biotechnological treatments presently employed. Processes, alternative methods, and technological improvements are compared, with special focus on novel technologies to remove nitrogen pollutants from leachate, highlighting advantages and drawbacks of each treatment.     

Surface chemistry of SnO2 nanowires on Ag-catalyst-covered Si substrate studied using XPS and TDS methods

In this paper we investigate the surface chemistry, including surface contaminations, of SnO₂ nanowires deposited on Ag-covered Si substrate by vapor phase deposition (VPD), thanks to x-ray photoelectron spectroscopy (XPS) in combination with thermal desorption spectroscopy (TDS). Air-exposed SnO₂ nanowires are slightly non-stoichiometric, and a huge amount of C contaminations is observed at their surface. After the thermal physical desorption (TPD) process, SnO₂ nanowires become almost stoichiometric without any surface C contaminations. This is probably related to the fact that C contaminations, as well as residual gases from air, are weakly bounded to the crystalline SnO₂ nanowires and can be easily removed from their surface. The obtained results gave us insight on the interpretation of the aging effect of SnO₂ nanowires that is of great importance for their potential application in the development of novel chemical nanosensor devices.     

CMOS Non‐tailed differential pair

A continuous‐time complementary metal–oxide–semiconductor differential pair that does not require the traditional tail current source as a way to control the direct current and common‐mode current is presented. Compared with a p‐channel long‐tailed pair, the proposed non‐tailed solution operates under a higher maximum input common‐mode voltage that includes (V_DD + V_SS)/2 even under low supply voltages. Experimental measurements on a prototype fabricated in a 0.35‐µm technology (with metal – oxide – semiconductor thresholds greater than 0.6 V) confirm this behavior for supply voltages as low as 1.2 V, whereas the long‐tailed pair with the same technology offers the same capability only for supplies higher than 1.6 V. Copyright © 2015 John Wiley & Sons, Ltd.     

Luminescent Mononuclear and Dinuclear Iridium(III) Cyclometalated Complexes Immobilized in a Polymeric Matrix as Solid-State Oxygen Sensors

Oxygen quenching of the luminescence of mononuclear and dinuclear Ir(III) cyclometalated complexes immobilized in the pPEGMA matrixes has been studied. Linear Stern-Volmer plots, even when experiments at different emission wavelengths have been performed, were evidenced. Despite the different luminescence lifetimes of the chromophores in the absence of quencher, similar Stern-Volmer slopes have been calculated. This behavior was tentatively interpreted by taking into account the size and charge of the chromophores. Increased sizes and lower charges seem to enhance the sensitivity of the systems. Such findings could be of interest for the design of new solid-state luminescent oxygen sensors with improved performance.     

Identification of rockfall source areas using the seed cell concept and bivariate susceptibility modelling

The objective of this research was to prepare a rockfall susceptibility map. Explorations were conducted in the Dubra?ina River basin (Croatia). The input data included a geological map, an orthophoto and a 1-m digital terrain model (DTM). After a talus inventory was prepared, the seed cell concept was applied to define the rockfall source areas. The contributing factors (predictors) of rockfalls were evaluated by the chi-squared test. The analysis confirmed the following predictors: CORINE land cover, lithology, slope, aspect, distance from a spring, distance from a road, distance from a fault, distance from a stream, and distance from the rock-soil geological boundary. A matrix pairwise comparison of the predictor ratings was used to define the most significant contributing factors. The predictors that affected the susceptibility map in the share of 86.3% were the slope (61.6%), lithology (13.4%), CORINE land cover (6.2%), and distance from the rock-soil geological boundary (5.1%). Two susceptibility maps were prepared: one using all nine contributing factors and another using the four most significant factors. The analysis showed that both maps were good, with the same areas under the receiver operating characteristic (ROC) curves. The map prepared with only four contributing factors can be considered a better map due to its more precise spatial definition of critical areas. It can be concluded that geological map, 1-m DTM and orthophoto provide enough data to prepare reliable rockfall susceptibility map. The application of the bivariate statistical zonation method called the “frequency ratio method” was proven to be successful. This research demonstrates that the application of the seed cell concept can be useful to speed up the process of rockfall source area detections in large research regions.

     

Computing the energy balance of a statue of bronze: The San Marco's horses as a case study

The energy balance is discussed for the complex case of bronze or marble statuary horses and particularly the four horses of San Marco in Venice. Having computed the coefficient of exchange between bronze and the atmosphere, the daytime overheating is evaluated for different seasons and orientations of the statues. The equations can be solved with reasonable approximation and both the past and future overheatings can be evaluated as a function of the growing surface weathering and blackening. Therefore, the present analysis points out the chief characteristics of both the diurnal cycle and the variation over the centuries of the response of the bronzes to atmospheric variables.