Quantitative analysis in proteomics and metabolomics

The quantification of differences between two or more states, physiological or pathological, in order to identify differentially expressed analytes in complex mixtures is among the most important objectives of proteomics and metabolomics. Mass spectrometry (MS) is one of the key analytical technology on which the emerging ‘‘-omics’’ approaches are based. It may provide detection and quantization of thousands of proteins and biologically active metabolites from a tissue, body fluid or cell culture working in a ‘‘global’’ or ‘‘targeted’’ manner, down to ultra-trace levels. Selected reaction monitoring (SRM) performed on triple quadrupole mass spectrometers has been actually the reference quantitative technique to analyze different molecules with high selectivity, sensitivity and a wide dynamic range. In SRM analysis, two stages of mass selection, i.e., selection of a precursor ion and monitoring of specific product ion(s) derived from the precursor, provide the highest analytical specificity applicable to complex mixture analysis. The combination of m/z setting for the first and third quadrupole is referred to as ‘transition'. New methodologies for selective quantitative analysis of targeted metabolites and proteins based on LC-MS/MS in Selected Reaction Monitoring scan mode. are developed. This approach has shown to be a valuable tool to identify altered pathways in pathological conditions and useful for quantitative measurements of a specific subset of known analytes such as same hormone and TDC, oxylipins, H2S sulfur metabolites, D, L Asp and NMDA and proteins as IDE and TSH. The SRM strategies optimized in this thesis focused on 1) identification of a set of proteins and metabolites of interest capable of satisfying a specific biological or clinic request 2) selection of transitions maximizing sensitivity and selectivity 3) optimization of SRM transitions by tuning acquisition parameters of the mass spectrometer 4) validation of the transitions in biological matrix to account to unspecific contributions of fragment ions background 5) quantification by the external standard method with the realization of specific calibration curves. The analysis in SRM mode (Selected Reaction Monitoring), has allowed us to achieve the specificity and selectivity to quantify the analytes of interest in relatively complex matrices, also at very low concentration with good reproducibility and accuracy

Greenhouse application of light-drone imaging technology for assessing weeds severity occurring on baby-leaf red lettuce beds approaching fresh-cutting

Aim of study: For baby-leaf lettuces greenhouse cultivations the absence of weeds is a mandatory quality requirement. One of the most promising and innovative technologies in weed research, is the use of Unmanned Aerial Vehicles (or drones) equipped with acquisition systems. The aim of this study was to provide an estimation of the exact weed amount on baby-sized red lettuce beds using a light drone equipped with an RGB microcamera.Area of study: Trials were performed at specialized organic farm site in Eboli (Salerno, Italy), under polyethylene multi-tunnel greenhouse.Material and methods: The RGB images acquired were processed with specific algorithms distinguishing weeds from crop yields, estimating the weeds covered surface and the severity of weed contamination in terms of biomass. A regression between the percentage of the surface covered by weed (with respect to the image total surface) and the weight of weed (with respect to the total harvested biomass) was calculated.Main results: The regression between the total cover values of the 25 calibration images and the total weight measured report a significant linear correlation. Digital monitoring was able to capture with accuracy the highly variable weed coverage that, among the different grids positioned under real cultivation conditions, was in the range 0-16.4% of the total cultivated one.Research highlights: In a precision weed management context, with the aim of improving management and decreasing the use of pesticides, this study provided an estimation of the exact weed amount on baby-sized red lettuce beds using a light drone

Growth Conditions Influence Lactobacillus Cell-Free Supernatant Impact on Viability, Biofilm Formation, and Co-Aggregation of the Oral Periodontopathogens Fusobacterium nucleatum and Porphyromonas gingivalis

Fusobacterium nucleatum and Porphyromonas gingivalis human periodontopathogens play a leading part in oral squamous cell carcinoma through cell proliferation, invasion, and persistent inflammation promotion and maintenance. To explore how the activity of Lactobacillus-derived cell-free supernatants (CFSs) can be influenced by growth medium components, CFSs were produced both in the standard MRS and the novel animal-derivative-free "Terreno Industriale Lattobacilli" (TIL) media, and in vitro screened for the containment of F. nucleatum and P. gingivalis both single and co-cultured and also for the interference on their co-aggregation. The viability assay demonstrated that the Limosilactobacillus reuteri LRE11 and Ligilactobacillus salivarius LS03 MRS-produced CFSs were significantly more effective against single and co-cultured pathogens. All the other CFSs significantly improved their efficacy when produced in TIL. Both MRS- and TIL-produced CFSs significantly inhibited the single and co-cultured pathogen biofilm formation. Only Levilactobacillus brevis LBR01 CFS in MRS specifically reduced F. nucleatum and P. gingivalis co-aggregation, while viable LBR01, LS03, and LRE11 in MRS significantly co-aggregated with the pathogens, but only LS03 cultivated in TIL improved this effect. This work paves the way to better consider environmental growth conditions when screening for probiotic and postbiotic efficacy as crucial to pathogen aggregation, adhesion to the host's niches, and exclusion

Gold-based drug encapsulation within a ferritin nanocage: X-ray structure and biological evaluation as a potential anticancer agent of the Auoxo3-loaded protein

Auoxo3, a cytotoxic gold(iii) compound, was encapsulated within a ferritin nanocage. Inductively coupled plasma mass spectrometry, circular dichroism, UV-Vis absorption spectroscopy and X-ray crystallography confirm the potential-drug encapsulation. The structure shows that naked Au(i) ions bind to the side chains of Cys48, His49, His114, His114 and Cys126, Cys126, His132, His147. The gold-encapsulated nanocarrier has a cytotoxic effect on different aggressive human cancer cells, whereas it is significantly less cytotoxic for non-tumorigenic cells

Molecular Coancestry and Classical Genetic Distances Depict Different Patterns of Relationship Among Sheep Breeds from Southern Italy

Several molecular-based parameters, such as similarity indexes, can be adopted to optimize the management of genetic diversity in conservation programmes. From simulated data, Oliehoek et al. (2006) showed that molecular coancestry (Toro et al., 2002) is, among the possible relatedness estimators, the one that performs better in structured populations, such as populations in need of conservation usually are. Several studies have, therefore, proposed the use of molecular coancestry coefficients as a measure of genetic variability and as a useful tool for conservation of endangered breeds (Ciampolini et al., 2007; Glowatzki-Mullis et al., 2009). Here we report the results obtained evaluating within- and between-breed molecular coancestry (Toro et al., 2002), together with other classical genetic parameters, for two insular sheep breeds (Sarda from Sardinia and Comisana from Sicily), recently spread almost all over Italy, and for five local rare sheep breeds from Southern Italy

A hypothesis of sudden body fluid vaporization in the 79 AD victims of Vesuvius.

In AD 79 the town of Herculaneum was suddenly hit and overwhelmed by volcanic ash-avalanches that killed all its remaining residents, as also occurred in Pompeii and other settlements as far as 20 kilometers from Vesuvius. New investigations on the victims' skeletons unearthed from the ash deposit filling 12 waterfront chambers have now revealed widespread preservation of atypical red and black mineral residues encrusting the bones, which also impregnate the ash filling the intracranial cavity and the ash-bed encasing the skeletons. Here we show the unique detection of large amounts of iron and iron oxides from such residues, as revealed by inductively coupled plasma mass spectrometry and Raman microspectroscopy, thought to be the final products of heme iron upon thermal decomposition. The extraordinarily rare preservation of significant putative evidence of hemoprotein thermal degradation from the eruption victims strongly suggests the rapid vaporization of body fluids and soft tissues of people at death due to exposure to extreme heat

Artificial Intelligence Application to Screen Abdominal Aortic Aneurysm Using Computed tomography Angiography

The aim of our study is to validate a totally automated deep learning (DL)-based segmentation pipeline to screen abdominal aortic aneurysms (AAA) in computed tomography angiography (CTA) scans. We retrospectively evaluated 73 thoraco-abdominal CTAs (48 AAA and 25 control CTA) by means of a DL-based segmentation pipeline built on a 2.5D convolutional neural network (CNN) architecture to segment lumen and thrombus of the aorta. The maximum aortic diameter of the abdominal tract was compared using a threshold value (30 mm). Blinded manual measurements from a radiologist were done in order to create a true comparison. The screening pipeline was tested on 48 patients with aneurysm and 25 without aneurysm. The average diameter manually measured was 51.1 ± 14.4 mm for patients with aneurysms and 21.7 ± 3.6 mm for patients without aneurysms. The pipeline correctly classified 47 AAA out of 48 and 24 control patients out of 25 with 97% accuracy, 98% sensitivity, and 96% specificity. The automated pipeline of aneurysm measurements in the abdominal tract reported a median error with regard to the maximum abdominal diameter measurement of 1.3 mm. Our approach allowed for the maximum diameter of 51.2 ± 14.3 mm in patients with aneurysm and 22.0 ± 4.0 mm in patients without an aneurysm. The DL-based screening for AAA is a feasible and accurate method, calling for further validation using a larger pool of diagnostic images towards its clinical use

In Vitro Selection of Lactobacillus and Bifidobacterium Probiotic Strains for the Management of Oral Pathobiont Infections Associated to Systemic Diseases

: The human oral pathobionts Aggregatibacter actinomycetemcomitans, Streptococcus mitis and Streptococcus mutans, in dysbiosis-promoting conditions, lead to oral infections, which also represent a threat to human systemic health. This scenario may be worsened by antibiotic misuse, which favours multi-drug resistance, making the research on pathogen containment strategies more than crucial. Therefore, we aimed to in vitro select the most promising probiotic strains against oral pathogen growth, viability, biofilm formation, and co-aggregation capacity, employing both the viable probiotics and their cell-free supernatants (CFSs). Interestingly, we also assessed probiotic efficacy against the three-pathogen co-culture, mimicking an environment similar to that in vivo. Overall, the results showed that Lactobacillus CFSs performed better than the Bifidobacterium, highlighting Limosilactobacillus reuteri LRE11, Lacticaseibacillus rhamnosus LR04, Lacticaseibacillus casei LC04, and Limosilactobacillus fermentum LF26 as the most effective strains, opening the chance to deeper investigation of their action and CFS composition. Altogether, the methodologies presented in this study can be used for probiotic efficacy screenings, in order to better focus the research on a viable probiotic, or on its postbiotics, suitable in case of infections

Effects of Probiotics Administration on Human Metabolic Phenotype

The establishment of the beneficial interactions between the host and its microbiota is essential for the correct functioning of the organism, since microflora alterations can lead to many diseases. Probiotics improve balanced microbial communities, exerting substantial healthpromoting effects. Here we monitored the molecular outcomes, obtained by gut microflora modulation through probiotic treatment, on human urine and serum metabolic profiles, with a metabolomic approach. Twenty-two subjects were enrolled in the study and administered with two different probiotic types, both singularly and in combination, for 8 weeks. Urine and serum samples were collected before and during the supplementation and were analyzed by nuclear magnetic resonance (NMR) spectroscopy and statistical analyses. After eight weeks of treatment, probiotics deeply influence the urinary metabolic profiles of the volunteers, without significantly altering their single phenotypes. Anyway, bacteria supplementation tends to reduce the differences in metabolic phenotypes among individuals. Overall, the effects are recipient-dependent, and in some individuals, robust effects are already well visible after four weeks. Modifications in metabolite levels, attributable to each type of probiotic administration, were also monitored. Metabolomic analysis of biofluids turns out to be a powerful technique to monitor the dynamic interactions between the microflora and the host, and the individual response to probiotic assumption