Advancing liquid atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry toward ultra-high-throughput analysis

Label-free high-throughput screening using mass spectrometry has the potential to provide rapid large-scale sample analysis at a speed of more than one sample per second. Such speed is important for compound library, assay and future clinical screening of millions of samples within a reasonable time frame. Herein, we present a liquid atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) setup for high-throughput large-scale sample analysis (>5 samples per second) for three substance classes (peptides, antibiotics and lipids). Liquid support matrices (LSM) were used for the analysis of standard substances as well as complex biological fluids (milk). Throughput and analytical robustness were mainly dependent on the complexity of the sample composition and the current limitations of the commercial hardware. However, the ultimate limits of liquid AP-MALDI in sample throughput can be conservatively estimated to be beyond 10-20 samples per second. This level of analytical speed is highly competitive compared with other label-free MS methods, including electrospray ionization and solid state MALDI, as well as MS methods using multiplexing by labelling, which in principle can also be used in combination with liquid AP-MALDI MS

Palmitoylethanolamide and related ALIAmides: prohomeostatic pipid compounds for animal health and wellbeing

Virtually every cellular process is affected by diet and this represents the foundation of dietary management to a variety of small animal disorders. Special attention is currently being paid to a family of naturally occurring lipid amides acting through the so-called autacoid local injury antagonism, i.e., the ALIA mechanism. The parent molecule of ALIAmides, palmitoyl ethanolamide (PEA), has being known since the 1950s as a nutritional factor with protective properties. Since then, PEA has been isolated from a variety of plant and animal food sources and its proresolving function in the mammalian body has been increasingly investigated. The discovery of the close interconnection between ALIAmides and the endocannabinoid system has greatly stimulated research efforts in this field. The multitarget and highly redundant mechanisms through which PEA exerts prohomeostatic functions fully breaks with the classical pharmacology view of “one drug, one target, one disease”, opening a new era in the management of animals’ health, i.e., an according-to-nature biomodulation of body responses to different stimuli and injury. The present review focuses on the direct and indirect endocannabinoid receptor agonism by PEA and its analogues and also targets the main findings from experimental and clinical studies on ALIAmides in animal health and wellbeing

Monitoring dynamics in bacterial competition by Imaging Mass Spectrometry

Microbial competition is a mechanism that occurs when two or more microbial species compete for ecological niches to support their survival and growth (Hibbing et al. 2010). Different factors can contribute to the outcome of microbial competition, such as molecules exchanged between the competing organisms for the regulation of cell densities and the initial spatial configuration of the microbe–microbe interaction. Specifically, production of compounds that kill or limit the growth of competing strains or species can promote niche monopolization (Gonzalez et al. 2011). The released compounds include secondary metabolite antibiotics, bacterial peptides or low-molecular-mass organic compounds. In that sense, it is very important to develop tools that could capture metabolic interactions between two or more bacterial populations. Imaging Mass Spectrometry (IMS) enables the visualization of both spatial and temporal production of a huge number of metabolites from a single bacterial species and can observe the effects of multiple microbial signals in an interspecies interaction without using tags or labels (Yang et al. 2009). This technique has the potential to be used for identification of novel metabolites and peptides that were previously undetected by other analytical methods. In this work, a combination of IMS and LC-MS/MS was used to study the competition between Listeria monocytogenes (LM) and Lactococcus lactis (LAC) to investigate the metabolic profile of each bacterium in the interacting microbial colonies. IMS analysis revealed several interesting compounds during interaction of microbial colonies. At least six compounds are uniquely expressed during the interaction between LM and LAC. These results could be useful to setup new molecular strategies in the control of bacterial species for a better food safety

Food Safety: Secretome of Lactococcus lactis and Listeria monocytogenes in competition.

Listeria monocytogenes (LM) is a foodborne pathogen responsible of listeriosis. In the spreading of this pathology, milk and dairy products are key reservoir for this pathogen1. Food processing represents one of the major steps that could be linked to LM growth. Inhibition of LM growth through competition of Lactococcus lactis (LAC) could represent a solution to this problem. Exoproteome of LM and two different strains of Lactic Acid Bacteria in co-culture have been studied in order to highlight mechanisms of bacterial competition useful to improve food safety. Two different strains of LAC and one strain of LM were cultivated in appropriate medium cultures (BHI), also in competition. Filtrated cultures (SECRETOME) were lyophilized and resuspended for proteomics analysis. Shotgun analysis on each secretome was performed on nano UPLC-MS system. Obtained data reveal, during competition, the higher production by LM of moonlighting protein Enolase and Glucose 6 Phosphate isomerase, of Septation ring formation regulator EzrA, involved into cell replication and the lower secretion of Endopeptidase P60. In parallel, L. lactis produced higher amounts of Secreted 45 kDa protein and switched from lantibiotic Nisin A production to Nisin Z production. In competition with LM, LAC strain investigated produce higher amounts of Secreted 45 kDa protein with peptidoglycan lytic activity and the selective secretion of Nisin Z probably to improve lantibiotic solubility in less acidic environment. Next step will be validation of obtained results in dairy products. These results are of interesting to design new strategies of fighting LM as contaminant in food from animal origin.Work supported by Ministry of Health-CCM “Milano EXPO 2015 Project: Garantire la sicurezza alimentare- Valorizzare le produzioni

Rapid liquid AP-MALDI MS profiling of lipids and proteins from goat and sheep milk for speciation and colostrum analysis

Rapid profiling of the biomolecular components of milk can be useful for food quality assessment and for food fraud detection. Differences in commercial value and availability of milk from specific species are often the reasons for the illicit and fraudulent sale of milk whose species origin is wrongly declared. In this study, a fast, MS-based speciation method is presented to distinguish sheep from goat milk and sheep colostrum at different phases. Using liquid atmospheric pressure (AP)-matrix-assisted laser desorption/ionisation (MALDI) MS, it was possible to classify samples of goat and sheep milk with 100% accuracy in one minute of data acquisition per sample. Moreover, an accuracy of 98% was achieved in classifying pure sheep milk samples and sheep milk samples containing 10% goat milk. Evaluating colostrum quality and postnatal stages represents another possible application of this technology. Classification of sheep colostrum samples that were collected within 6 hours after parturition and 48 hours later was achieved with an accuracy of 84.4%. Our data show that substantial changes in the lipid profile can account for the accurate classification of colostrum collected at the early and late time points. This method applied to the analysis of protein orthologs of different species can, as in this case, allow unequivocal speciation analysis

Peptide nanotubes self-assembled from leucine-rich alpha helical surfactant-like peptides

The designed arginine-rich surfactant-like peptide R3L12 (arginine3–leucine12) is shown to form a remarkable diversity of self-assembled nanostructures in aqueous solution, depending on pH, including nanotubes, mesh-like tubular networks in three-dimensions and square planar arrays in two-dimensions. These structures are built from α-helical antiparallel coiled–coil peptide dimers arranged perpendicular to the nanotube axis, in a “cross-α” nanotube structure. The aggregation behavior is rationalized based on the effects of dimensionality, and the balance of hydrophobic and electrostatic interactions. The nanotube and nanomesh structures display arginine at high density on their surfaces, which may be valuable for future applications

DART: the distributed agent based retrieval toolkit

The technology of search engines is evolving from indexing and classification of web resources based on keywords to more sophisticated techniques which take into account the meaning and the context of textual information and usage. Replying to query, commercial search engines face the user requests with a large amount of results, mostly useless or only partially related to the request; the subsequent refinement, operated downloading and examining as much pages as possible and simply ignoring whatever stays behind the first few pages, is left up to the user. Furthermore, architectures based on centralized indexes, allow commercial search engines to control the advertisement of online information, in contrast to P2P architectures that focus the attention on user requirements involving the end user in search engine maintenance and operation. To address such wishes, new search engines should focus on three key aspects: semantics, geo-referencing, collaboration/distribution. Semantic analysis lets to increase the results relevance. The geo-referencing of catalogued resources allows contextualisation based on user position. Collaboration distributes storage, processing, and trust on a world-wide network of nodes running on users’ computers, getting rid of bottlenecks and central points of failures. In this paper, we describe the studies, the concepts and the solutions developed in the DART project to introduce these three key features in a novel search engine architecture

Phenotypic and genomic identification of Staphylococcus epidermidis GOI1153754-03-14 isolated from an infected orthopedic prosthesis

Introduction: Staphylococcus epidermidis GOI1153754-03-14 is able to colonize orthopedic implants and to cause septic non-unions, as validated in a recent in vivo study (Lovati, 2016). To pore over the mechanisms leading to the biofilm formation on metallic implants, in the present study, we carried out the phenotypic and genotypic characterization of the clinical isolate S. epidermidis GOI1153754-03-14.Materials and Methods: The antimicrobial susceptibility and minimum inhibitory concentration (MIC) of the strain were evaluated through the Vitek2 System (Biomerieux), as well as its ability to form biofilm in vitro through a spectrophotometric assay (Stepanovich, 2000).The genomic DNA was extracted by Bacterial Genomic DNA Isolation Kit (Norgen Biotek Corp.). Libraries were prepared with the ThruPLEX DNA-seq (Rubicon Genomics) and then sequenced on the Illumina MiSeq platform through the MiSeq Reagent Kit v3 (600-cycles) to produce 300 bp paired-end reads (Illumina Inc.). Reads were quality-trimmed and gene annotated thanks to the RAST software (Aziz, 2008).Results: The antimicrobial susceptibility along with the MIC values are reported in Table 1. The outputs resulted in 51 contigs (Average = 50,720.6 Mb) with 396X fold average coverage. The total genome is 2,586,753 bp long with a GC content of 31.84% and an N50 value of 7 bp. The whole genome is composed by 2,467 protein-encoding genes and 64 RNAs (55 tRNAs and 9 rRNAs). The entire genome sequence has been deposited in the European Nucleotide Archive (ENA) under the accession no. FWCG01000000 (Bottagisio, 2017).Discussion: The genotypic and phenotypic characterization of the S. epidermidis GOI1153754-03-14 will enable a better comprehension of the mechanisms involved in the biofilm formation on orthopedic implants paving the way for innovative preventative and therapeutic strategies. Moreover, the sequence of this clinical strain is mandatory to develop dedicated proteomics analysis in order to highlight functional mechanism of biofilm formation

A collaborative, semantic and context-aware search engine

Search engines help people to find information in the largest public knowledge system of the world: the Web. Unfortunately its size makes very complex to discover the right information. The users are faced lots of useless results forcing them to select one by one the most suitable. The new generation of search engines evolve from keyword-based indexing and classification to more sophisticated techniques considering the meaning, the context and the usage of information. We argue about the three key aspects: collaboration, geo-referencing and semantics. Collaboration distributes storage, processing and trust on a world-wide network of nodes running on users’ computers, getting rid of bottlenecks and central points of failures. The geo-referencing of catalogued resources allows contextualisation based on user position. Semantic analysis lets to increase the results relevance. In this paper, we expose the studies, the concepts and the solutions of a research project to introduce these three key features in a novel search engine architecture.213-21