Revealing the Hidden Diagnostic Clues of Male Infertility from Human Seminal Plasma by Dispersive Solid Phase Extraction and MALDI-TOF MS.

Seminal plasma (SP) mirrors the local pathophysiology of the male reproductive system and represents a non-invasive fluid for the study of infertility. Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) provides a high-throughput platform to rapidly extrapolate the diagnostic profiles of information-rich patterns. In this study, dispersive solid phase extraction (d-SPE) combined with MALDI-TOF-MS was applied for the first time to the human SP, with the aim of revealing a diagnostic signature for male infertility. Commercially available octadecyl (C18)-, octyl (C8)-bonded silica sorbents and hexagonal mesoporous silica (HMS) were tested and the robustness of MALDI-TOF peptide profiling was evaluated. Best performances were obtained for C18-bonded silica with the highest detection of peaks and the lowest variation of spectral features. To assess the diagnostic potential of the method, C18-bonded silica d-SPE and MALDI-TOF-MS were used to generate enriched endogenous peptide profiles of SP from 15 fertile and 15 non-fertile donors. Principal component analysis (PCA) successfully separated fertile from non-fertile men into two different clusters. An array of seven semenogelin-derived peptides was found to distinguish the two groups, with high statistical significance. These findings, while providing a rapid and convenient route to selectively enrich native components of SP peptidome, strongly reinforce the prominent role of semenogelins in male infertility.

Mapping the SARS-CoV-2-Host Protein-Protein Interactome by Affinity Purification Mass Spectrometry and Proximity-Dependent Biotin Labeling: A Rational and Straightforward Route to Discover Host-Directed Anti-SARS-CoV-2 Therapeutics.

Protein-protein interactions (PPIs) are the vital engine of cellular machinery. After virus entry in host cells the global organization of the viral life cycle is strongly regulated by the formation of virus-host protein interactions. With the advent of high-throughput -omics platforms, the mirage to obtain a "high resolution" view of virus-host interactions has come true. In fact, the rapidly expanding approaches of mass spectrometry (MS)-based proteomics in the study of PPIs provide efficient tools to identify a significant number of potential drug targets. Generation of PPIs maps by affinity purification-MS and by the more recent proximity labeling-MS may help to uncover cellular processes hijacked and/or altered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), providing promising therapeutic targets. The possibility to further validate putative key targets from high-confidence interactions between viral bait and host protein through follow-up MS-based multi-omics experiments offers an unprecedented opportunity in the drug discovery pipeline. In particular, drug repurposing, making use of already existing approved drugs directly targeting these identified and validated host interactors, might shorten the time and reduce the costs in comparison to the traditional drug discovery process. This route might be promising for finding effective antiviral therapeutic options providing a turning point in the fight against the coronavirus disease-2019 (COVID-19) outbreak.

Rapid Detection and Identification of Antimicrobial Peptide Fingerprints of Nasal Fluid by Mesoporous Silica Particles and MALDI-TOF/TOF Mass Spectrometry: From the Analytical Approach to the Diagnostic Applicability in Precision Medicine.

BACKGROUND: Antimicrobial peptides (AMP) play a pivotal role in innate host defense and in immune response. The delineation of new MS-based profiling tools, which are able to produce panels of AMP of the nasal fluid (NF), may be attractive for the discovery of new potential diagnostic markers of respiratory disorders. METHODS: Swabs collected NF from healthy patients and from patients with respiratory disorders. We used a fast procedure based on mesoporous silica particles (MPS) to enrich NF in its AMP component in combination with MALDI-TOF/TOF MS as a key tool for rapidly analyzing clinical samples. RESULTS: Reproducible MS peptide fingerprints were generated for each subject and several AMP were detected including (Human Neutrophil Peptides) HNPs, Statherin, Thymosin-ß4, Peptide P-D, II-2, ß-MSP, SLPI, Lysozyme-C, and their proteo-forms. In particular, Statherin, Thymosin-ß4, and Peptide P-D were accurately identified by direct MS/MS sequencing. Examples of applicability of this tool are shown. AMP fingerprints were obtained before and after a nasal polypectomy as well as before and post-treatment with azelastine/fluticasone in one case of allergic rhinitis. CONCLUSION: The potential of our platform to be implemented by new mesoporous materials for capturing a wider picture of AMP might offer an amazing opportunity for diagnostic clinical studies on individual and population scales.

Seminal plasma untargeted metabolomic and lipidomic profiling for the identification of a novel panel of biomarkers and therapeutic targets related to male infertility.

Male infertility occurs approximately in about 50% of all infertility cases and represents a serious concern worldwide. Traditional semen analysis alone is insufficient to diagnose male infertility. Over the past two decades, advances in omics technologies have led to the widespread application of metabolomics profiling as a valuable diagnostic tool for various diseases and disorders. Seminal plasma represents a rich and easily accessible source of metabolites surrounding spermatozoa, a milieu that provides several indispensable nutrients to sustain sperm motility and fertilization. Changes of metabolic profiles in seminal plasma reflect male reproductive tract disorders. Here, we performed seminal plasma metabolomics and lipidomics profiling to identify a new pattern of biomarkers of male infertility. Seminal plasma samples from unfertile subjects (n = 31) and fertile controls (n = 19) were analyzed using an untargeted metabolomics/lipidomics integrated approach, based on Ultra-High-Pressure Liquid Chromatography-tandem Mass Spectrometry. Partial Least Squares-Discriminant Analysis showed a distinct separation between healthy fertile men and infertile subjects. Among the 15 selected candidate biomarkers based on Variable Importance in Projection scores, phosphatidylethanolamine (PE) (18:1; 18:1) resulted with the highest score. In total, 40 molecular species showed statistically significant variations between fertile and infertile men. Heat-map and volcano plot analysis indicated that acylcarnitines, phosphatidylserine (PS) (40:2) and lactate were decreased, while PE (18:1; 18:1), Phosphatidic acid (PA) (O-19:2; 18:1), Lysophosphatidylethanolamine (LPE) (O-16:1) and Phosphatidylcholine (PC) (O-16:2; 18:1)-CH3 were increased in the infertile group. The present study is the first one to analyze the metabolomics/lipidomics dysregulation in seminal plasma between fertile and infertile individuals regardless of sub-infertility condition. Association of several metabolites/lipids dysregulation with male infertility reinforced data of previous studies performed with different approaches. In particular, we confirmed significantly decreased levels of PS and carnitines in infertile patients as well as the positive correlation with sperm motility and morphology. If validated on a larger prospective cohort, the metabolite biomarkers of infertility in seminal plasma we identified in the present study might inform novel strategies for diagnosis and interventions to overcome male infertility.

Nanoparticle microinjection and Raman spectroscopy as tools for nanotoxicology studies.

Microinjection techniques and Raman spectroscopy have been combined to provide a new methodology to investigate the cytotoxic effects due to the interaction of nanomaterials with cells. In the present work, this novel technique has been used to investigate the effects of Ag and Fe(3)O(4) nanoparticles on Hela cells. The nanoparticles are microinjected inside the cells and these latter ones are probed by means of Raman spectroscopy after a short incubation time, in order to highlight the first and impulsive mechanisms developed by the cells to counteract the presence of the nanoparticles. The results put in evidence a different behaviour of the cells treated with nanoparticles in comparison with the control cells; these differences are supposed to be generated by an emerging oxidative stress due to the nanoparticles. The achieved results demonstrate the suitability of the proposed method as a new tool for nanotoxicity studies.

Detection and functional analysis of an SNP in the promoter of the human ferritin H gene that modulates the gene expression.

The H ferritin promoter spans approximately 150 bp, upstream of the transcription start and is composed by two cis-elements in position -132 (A box) and -62 (B-box), respectively. The A box is recognized by the transcription factor Sp1, and the B-box by a protein complex called Bbf, which includes the CAAT binding factor NF-Y. In this study we performed a functional analysis of an H ferritin promoter allele carrying a G to T substitution adjacent to the Bbf binding site, in position -69. In vitro studies with reporter constructs revealed a significantly reduced transcriptional activity of this allele compared to that of the w.t. promoter that was mirrored by a decrease in Bbf binding. In vivo, this variant genotype is accompanied by a reduced amount of the H mRNA in peripheral blood lymphocytes.

Bilateral cataract in a subject carrying a C to A transition in the L ferritin promoter region.

OBJECTIVES: The aim of this study is to evaluate the potential impact of mutations in the promoter region of the L ferritin gene on its transcriptional activity. DESIGN AND METHODS: To search for the presence of mutations in the promoter of the L gene, we amplified by PCR a DNA region of about 385 n.t. in 100 healthy subjects from Southern Italy. RESULTS: A subject carrying a C to A transition in position -216 was identified. This transition causes an increased transcriptional activity in vitro. This finding was substantiated by Real Time Quantitative PCR, which showed increased levels of L ferritin mRNA. CONCLUSIONS: A previously unidentified mutation in the promoter region of the L ferritin gene was detected in an individual. Interestingly, this subject is affected by bilateral cataract, a disease that has been correlated, in a subset of patients, with high levels of circulating ferritin. We hypothesize that changes in the expression of the L ferritin might be linked, at least to a certain extent, to the pathogenesis of this rare eye disease.