Development of a new method for identification of FSH glycoforms involved in human fertility

Follicle Stimulating Hormone (FSH) is one of the most important glycohormones involved in human fertility and it is widely used to assess the ovarian reserve in women for in vitro fertilization. FSH is a heterodimeric glycohormone composed by two polypeptide subunits, the alpha-subunit (common to all gonadotropins) and a FSH specific beta-subunit. The molecule has 4 glycosylation site, Asn52-Asn78 of alpha-subunit and Asn7-Asn24 of beta-subunit. Each glycosite hosts a wide microheterogeneity of glycans, thus generating a heterogeneous population of different glycoforms. The biological activity of human hFSH is highly regulated by its glycosylation, but the role of the different glycoforms in FSH mechanism of action is still poorly understood. The development of new methodologies aimed at characterizing and identifying the site-specific glycosidic profile of hFSH could contribute to understanding how the glycosylation pattern modulates hFSH function. Glycomic studies on serum hFSH are very challenging because of the huge heterogeneity of glycoforms and their very low concentration in serum. The current reference method for serum hFSH dosage is ELISA, but it fails in distinguishing proteoforms because of the lack of specific antibodies. A combination of mass spectrometry approaches was used here to characterise recombinant r-FSH and ultimately quantify endogenous hFSH proteoforms in serum. Site-specific glycosylation profiles of different commercialised r-FSH molecules (GONAL-F ®, BEMFOLA, OVALEAP, FOSTIMON) were determined by untargeted proteomics and glycan analysis. The MS analysis led to the identification of at least 5 known glycoforms for each of 4 glycosylation sites of the molecule. In order to find higher efficiency r-FSH isoforms of the molecule, artificial glycovariant were produced by stress conditions (temperature, pH, etc.) and analysed in the same way. Glycosylation of the molecule subject to pH stress condition shows a slight decrease in the sialic acid content as well as in hyper O-Acetylated species. AEX-pH acidic fraction was characterised by highly sialylated end-capping species, while the basic fraction showed the presence of neutral galactosylated species and a lower content of sialylated moieties. Totally de-sialylated and partially de-galactosylated FSH glycovariants were obtained by enzymatic treatments. In vivo and in vitro experiments will be conducted by Merck (www.merckgroup.com) to determine the efficiency of these glycovariants in fertility therapy. On the basis of mass spectral r-FSH characterisation, two innovative targeted MS methods were developed to identify and quantify the global amount of hFSH as well as different glycoforms in woman serum. A Multiple Reaction Monitoring (MRM) MS-based method for the absolute quantification of circulating hFSH molecule was optimised, and it was successfully applied to the analysis of 10 women sera samples of different ages showing a great variability of the amount of the hormone. Results show comparable sensitivity (>1 ng/ml) and higher selectivity and specificity than conventional ELISA assays, and therefore this MRM-MS method could be proposed as an alternative for the quantification of hFSH. As for glycoforms detection and dosage, a method based on high-resolution mass spectrometry in Parallel Reaction Monitoring (PRM-MS) mode was developed to monitor the glycosylation profile of hFSH in pooled sera sample. The sensitivity was boosted by an optimised sample preparation involving immunoaffinity purification of hFSH from serum. The 9 most abundant hFSH site-specific glycoforms were identified in a very complex matrix such as serum. Moreover, a relative quantification of the identified glycoforms was obtained by post acquisition data processing using Skyline software. In both recombinant and serum hFSH the same most abundant glycoforms were found, the biantennary fully sialylated (A2G2S2) on alpha-Asn52, the mono-sialylated (A2G2S1) on alpha-Asn78 and fucosylated biantennary (FA2G2S2 and FA2G2S1) structures on beta-Asn 24. Glycopeptide spectra containing beta-Asn 7 glycosite didn't pass our quality criteria for quantitative analysis. Although the importance of glycosylation in the biological activity of FSH is well documented, this is the first study reporting site-specific characterization of circulating h-FSH glycoforms by MS. The proposed strategy, if applied to a large cohort of women, can be useful to investigate the existing relations between the hFSH glycan moieties and the hormone function. Such knowledge will bring new insights on the complex mechanisms in which hFSH glycoforms are involved and, ultimately, will improve its usage in infertility treatments. Furthermore, these targeted MS methods can be easily implemented for a simultaneous analysis of several serum proteins, making the PRM/MRM-MS methodology advantageous in terms of time and cost

Caustics in gravitational lensing by mixed binary systems

We investigate binary lenses with $1/r^n$ potentials in the asymmetric case with two lenses with different indexes $n$ and $m$. These kinds of potentials have been widely used in several contexts, ranging from galaxies with halos described by different power laws to lensing by wormholes or exotic matter. In this paper, we present a complete atlas of critical curves and caustics for mixed binaries, starting from the equal-strength case, and then exploring unequal-strength systems. We also calculate the transitions between all different topology regimes. Finally we find some useful analytic approximations for the wide binary case and for the extreme unequal-strength case.Comment: 25 pages, 18 figures, in press on Universe, special issue Gravitational Lensing and Optical Geometry: A Centennial Perspectiv

Neighbors-Aware Proportional Fair scheduling for future wireless networks with mixed MAC protocols

Abstract In this paper, we consider a beyond-5G scenario, where two types of users, denoted as scheduled and uncoordinated nodes, coexist on the same set of radio resources for sending data to a base station. Scheduled nodes rely solely on a centralized scheduler within the base station for the assignment of resources, while uncoordinated nodes use an unslotted Carrier Sense Multiple Access (CSMA) protocol for channel access. We propose and evaluate through simulations: (a) a novel centralized resource scheduling algorithm, called Neighbors-Aware Proportional Fair (N-PF) and (b) a novel packet length adaptation algorithm, called Channel-Aware (CA) Packet Length Adaptation algorithm for the scheduled nodes. The N-PF algorithm considers the uplink channel state conditions and the number of uncoordinated nodes neighboring each scheduled node in the aggregate scheduling metric, in order to maximize packet transmission success probability. The CA algorithm provides an additional degree of freedom for improving the performance, thanks to the fact that scheduled nodes with lower number of hidden terminals, i.e., having higher packet capture probability, are assigned longer packet transmission opportunities. We consider two benchmark schemes: Proportional Fair (PF) algorithm, as a resource scheduling algorithm, and a discrete uniform distribution (DUD) scheme for packet lengths distribution. Simulation results show that the proposed schemes can result in significant gain in terms of network goodput, without compromising fairness, with respect to two benchmark solutions taken from the literature

Mass spectrometry based proteomics for the molecular fingerprinting of Fiano, Greco and Falanghina cultivars

Abstract The official methodologies used for the identification and comparison of vine cultivars are ampelography and ampelometry. These methodologies are essentially based on qualitative assessments or biometric dependent morphological features of the plant. The heterogeneity of cultivars and consequently the increasing demand for a more detailed product typization, led to the introduction of new methodologies for the varietal characterization. In this scenario, proteomics has already proved to be a very useful discipline for the typization of many kinds of edible products. In this paper, we present a proteomic study carried out on three cultivars of Vitis vinifera peculiar of south Italy (Campania) used for white wine production (Fiano, Greco and Falanghina) by advanced biomolecular mass spectrometry approach. Our data highlight variations in the proteomic profiles during ripening for each cultivar and between analyzed cultivars, thus suggesting a new way to outline the biomolecular signature of vines

D22.1 Definition of EuWIn@CNIT/Bologna testbed interfaces and preliminary plan of activities

Preprin

Proteomic Characterization of Collagen-Based Animal Glues for Restoration

Animal glues are widely used in restoration as adhesives, binders, and consolidants for organic and inorganic materials. Their variable performances are intrinsically linked to the adhesive properties of collagen, which determine the chemical, physical, and mechanical properties of the glue. We have molecularly characterized the protein components of a range of homemade and commercial glues using mass spectrometry techniques. A shotgun proteomic analysis provided animal origin, even when blended, and allowed us to distinguish between hide and bone glue on the basis of the presence of collagen type III, which is abundant in connective skin/leather tissues and poorly synthetized in bones. Furthermore, chemical modifications, a consequence of the preparation protocols from the original animal tissue, were thoroughly evaluated. Deamidation, methionine oxidation, and backbone cleavage have been analyzed as major collagen modifications, demonstrating their variability among different glues and showing that, on average, bone glues are less deamidated than hide glues, but more fragmented, and mixed-collagen glues are overall less deamidated than pure glues. We believe that these data may be of general analytical interest in the characterization of collagen-based materials and may help restorers in the selection of the most appropriate materials to be used in conservation treatments

CATASAN Is a New Anti-Biofilm Agent Produced by the Marine Antarctic Bacterium Psychrobacter sp. TAE2020

The development of new approaches to prevent microbial surface adhesion and biofilm formation is an emerging need following the growing understanding of the impact of biofilm-related infections on human health. Staphylococcus epidermidis, with its ability to form biofilm and colonize biomaterials, represents the most frequent causative agent involved in infections of medical devices. In the research of new anti-biofilm agents against S. epidermidis biofilm, Antarctic marine bacteria represent an untapped reservoir of biodiversity. In the present study, the attention was focused on Psychrobacter sp. TAE2020, an Antarctic marine bacterium that produces molecules able to impair the initial attachment of S. epidermidis strains to the polystyrene surface. The setup of suitable purification protocols allowed the identification by NMR spectroscopy and LC-MS/MS analysis of a protein–polysaccharide complex named CATASAN. This complex proved to be a very effective anti-biofilm agent. Indeed, it not only interferes with cell surface attachment, but also prevents biofilm formation and affects the mature biofilm matrix structure of S. epidermidis. Moreover, CATASAN is endowed with a good emulsification activity in a wide range of pH and temperature. Therefore, its use can be easily extended to different biotechnological applications

Marine phycotoxin levels in shellfish-14 years of data gathered along the Italian coast

Along the Italian coasts, toxins of algal origin in wild and cultivated shellfish have been reported since the 1970s. In this study, we used data gathered by the Veterinary Public Health Institutes (IZS) and the Italian Environmental Health Protection Agencies (ARPA) from 2006 to 2019 to investigate toxicity events along the Italian coasts and relate them to the distribution of potentially toxic species. Among the detected toxins (OA and analogs, YTXs, PTXs, STXs, DAs, AZAs), OA and YTX were those most frequently reported. Levels exceeding regulatory limits in the case of OA (≤2,448 μg equivalent kg-1) were associated with high abundances of Dinophysis spp., and in the case of YTXs (≤22 mg equivalent kg-1) with blooms of Gonyaulax spinifera, Lingulodinium polyedra, and Protoceratium reticulatum. Seasonal blooms of Pseudo-nitzschia spp. occur all along the Italian coast, but DA has only occasionally been detected in shellfish at concentrations always below the regulatory limit (≤18 mg kg-1). Alexandrium spp. were recorded in several areas, although STXs (≤13,782 μg equivalent kg-1) rarely and only in few sites exceeded the regulatory limit in shellfish. Azadinium spp. have been sporadically recorded, and AZAs have been sometimes detected but always in low concentrations (≤7 μg equivalent kg-1). Among the emerging toxins, PLTX-like toxins (≤971 μg kg-1 OVTX-a) have often been detected mainly in wild mussels and sea urchins from rocky shores due to the presence of Ostreopsis cf. ovata. Overall, Italian coastal waters harbour a high number of potentially toxic species, with a few HAB hotspots mainly related to DSP toxins. Nevertheless, rare cases of intoxications have occurred so far, reflecting the whole Mediterranean Sea conditions

Fitting Computer Science orientation in K12 education - Supplemental material

Dataset and supplemental material of the pubblication "Fitting Computer Science orientation in K12 education"