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Background: L1CAM (L1 cell adhesion molecule) is a member of the L1 family of neural adhesion molecules, involved in the development of multiple organs and tissues, including kidneys, the enteric nervous system, and adrenal glands. The aim of this study was to analyze, at the immunohistochemical level, the expression of L1CAM in the human tongue, parotid glands, and the different segments of the gastrointestinal tract during human development. Design and method: Immunohistochemical analysis for L1CAM was performed in the human tongue, parotid glands, and in the different segments of the gastrointestinal tract during development, starting from the 8th up to the 32nd week of gestation. Results: Our results were given by the expression of the L1CAM protein in different segments of the gastrointestinal tract during development, starting from the 8th week up to the 32nd week of gestation. L1CAM-reactive cells appeared aggregated in small bodies, irregular in shape, showing L1CAM storage in the cytoplasm. L1CAM expressing bodies were frequently found to be connected one to the next by thin fibers, a finding suggestive of the existence of an L1CAM network inside the developing tissue. Conclusion: Our study confirms that L1CAM is involved in gut development, as well as in tongue and salivary gland development. These findings confirm that the role of L1CAM in fetal development is not restricted to the central nervous system and are necessary for further studies on the role of this molecule in human development.

Thymosin β4 (Tβ4) was extracted forty years ago 1 from calf thymus. Since then, it has been identified as a G-actin binding protein involved in blood clothing, tissue regeneration, angiogenesis, and anti-inflammatory processes. Tβ4 has also been implicated in tumor metastasis and neurodegeneration. However, the precise roles and mechanism(s) of action of Tβ4 in these processes remain largely unknown, with the binding of the G-actin protein being insufficient to explain these multi-actions. Here we identify for the first time the important part of Tβ4 mechanism in ferroptosis, an iron-dependent form of cell death, which leads to neurodegeneration and somehow protects cancer cells against cell death. Specifically, we demonstrate four iron2+and iron3+ binding regions along the peptide and show that the presence of Tβ4 in cell growing medium inhibits erastin and glutamate-induced ferroptosis in macrophage cell line. Moreover, Tβ4 increases the expression of oxidative stress-related genes, namely BAX, hem oxygenase-1, Heat shock protein 70 and Thioredoxin reductase 1, which are downregulated during ferroptosis. We state the hypothesis that Tβ4 is an endogenous iron chelator and take part of iron homeostasis in ferroptosis process. We discuss the literature data of parallel involvement of Tβ4 and ferroptosis in different human pathologies, mainly cancer and neurodegeneration. Our findings confronted with literature data shows that controlled Tβ4 release could command on/off switching of ferroptosis, and may provide novel therapeutic opportunities in pathologies of cancer and tissue degeneration.

Endometriosis is a chronic gynecological inflammatory disease characterized by the presence of functional endometrial glands...

Copper homeostasis, including intestinal absorption, blood transport, uptake, trafficking, and excretion, is regulated by multiple genes encoding for specific copper transporters, which coordinate copper bioavailability. Mutations in genes coding for copper pumps or copper chaperons are responsible for copper overload or deficiency, with relevant consequences on cell structureand human health. The spectrum of genetic disorders of copper metabolism includes multiple entities, characterized by different clinical presentations, with liver and brain as target organs. Wilson disease and Menkes disease are the most widely studied copper-related genetic disorders in humans. Wilson disease is an autosomal recessive disorder caused by a mutation in the ATP7B gene, encoding for a copper pump localized in the Trans-Golgi Network (TGN) or at the biliary pole of the hepatocyte, that is strongly expressed in the normal liver. Malfunction or dislocation of ATP7B is at the basis of copper storage in the liverand in the brain, in which copper overload is associated with storage of other trace elements such as iron. Menkes disease, also known as kinky hair disease, is an X-linked, often fatal, neurodegenerative disease caused by a mutation in the ATP7A gene, encoding for another copper pump, ATP7A, whose malfunction is responsible for impaired copper absorption in the gut and decreased copper passage through the blood-brain barrier. The Occipital Horn Syndrome is the milder variant of Menkes syndrome. Moreover, a distal motor neuropathy caused by a novel missense mutation in the ATP7A gene has been recently described [8]. In recent years, a new autosomal recessive mucocutaneous syndrome characterized by Mental retardation, Enteropathy, Deafness, peripheral Neuropathy, Ichthyosis, and Keratodermia (MEDNIK) has been described in several French-Canadian families. Previously defined as atypical erythrokeratodermia in 1972, and as erythrodermia variabilis-3 in 2005, in 2008 MEDNIK syndrome has been associated with a splice mutation in the Adaptor Protein-1 S1 (AP1S1) gene, encoding for the small subunit σ1A of the adaptor protein (AP)-1 complex.MEDNIK syndrome opens a new field of human pathology, that has been called “adaptinopathies” , including all congenital diseases associated with mutations in genes coding for adaptor complexes subunits. Moreover, the report of MEDNIK syndrome induces to focus on the developing spectrum of inborn errors of copper metabolism, where at least eight diseases may be included. Nonetheless, the high number of copper chaperons involved in copper uptake and transport in human cells, including hCTR1, hCTR2, ATOX1, hCOX17, CCS, MURR1/COMMD1, induces to hypothesize an enlargement, in the next future, of this complex and fascinating field of human pathology. Deep knowledge of the molecular events at the basis of these diseases could ensure an early diagnosis in the perinatal period, allowing proper therapy and avoiding the insurgence of severe pathological complications in affected newborns

Adamantinomatous craniopharyngioma (ACP) is still often burdened by a poor prognosis in children as far as the risk of recurrence and the quality of life are concerned. Therefore, many efforts are now dedicated to investigate the molecular characteristics of this tumor aiming at finding new therapeutic options. ACP is prevalently a cystic lesion so that an increasing number of researches are focused on the analysis of its cystic content. In the present article, the main results of the current proteomic analysis (PA) on the ACP fluid are summarized. Both "bottom-up" and "top-down" approaches have been utilized. In the bottom-up approach, proteins and peptides are enzymatically or chemically digested prior to liquid chromatography and mass spectrometry analyses. The bottom-up approach pointed out several proteins of the inflammation (namely, α2-HS-glycoprotein, α1-antichymotrypsin and apolipoproteins) as possibly involved in the genesis and growth of the cystic component of ACP. The top-down strategy analyzes proteins and peptides in the intact state, making it particularly suitable for the identification of peptides and low molecular weight proteins and for the characterization of their possible isoforms and post-translational modifications. The top-down approach disclosed the presence of the thymosin β family. Thymosin β4, in particular, which is involved in the cytoskeleton organization and migration of several tumors, could play a role in the progression of ACP. Finally, PA was utilized to investigate alterations in cyst fluid character after treatment with interferon-α. The analyzed samples showed a progressive reduction of the levels of α-defensins (proteins involved in the inflammatory-mediated response) after the intracystic injection of interferon-α, thus reinforcing the hypothesis that inflammation contributes to ACP cyst pathogenesis. Additional studies on the solid component of ACP are still necessary to further validate the previous results and to identify possible markers for targeted therapy.

Additional file 1: Table S1. Identified proteins in CM in the group of controls and in patients affected by endometriosis.

In the present study, we show that the heterogeneous mixture of glycoforms of the basic salivary proline-rich protein 3M, encoded by PRB3-M locus, is a major component of the acidic soluble fraction of human whole saliva in the first years of life. Reversed-phase high-performance liquid chromatography with high-resolution electrospray ionization mass spectrometry analysis of the intact proteoforms before and after N-deglycosylation with Peptide-N-Glycosidase F and tandem mass spectrometry sequencing of peptides obtained after Endoproteinase GluC digestion allowed the structural characterization of the peptide backbone and identification of N- and O-glycosylation sites. The heterogeneous mixture of the proteoforms derives from the combination of 8 different neutral and sialylated glycans O-linked to Threonine 50, and 33 different glycans N-linked to Asparagine residues at positions 66, 87, 108, 129, 150, 171, 192, and 213.

In the present study, we show that the heterogeneous mixture of glycoforms of the basic salivary proline-rich protein 3M, encoded by PRB3-M locus, is a major component of the acidic soluble fraction of human whole saliva in the first years of life. Reversed-phase high-performance liquid chromatography with high-resolution electrospray ionization mass spectrometry analysis of the intact proteoforms before and after N-deglycosylation with Peptide-N-Glycosidase F and tandem mass spectrometry sequencing of peptides obtained after Endoproteinase GluC digestion allowed the structural characterization of the peptide backbone and identification of N- and O-glycosylation sites. The heterogeneous mixture of the proteoforms derives from the combination of 8 different neutral and sialylated glycans O-linked to Threonine 50, and 33 different glycans N-linked to Asparagine residues at positions 66, 87, 108, 129, 150, 171, 192, and 213.

Alzheimer's disease (AD) is a progressive form of dementia characterized by increased production of amyloid-β plaques and hyperphosphorylated tau protein, mitochondrial dysfunction, elevated oxidative stress (OS), reduced protein clearance among others. Several studies showed systemic modifications of immune and inflammatory systems due, in part, to decreased levels of T cells in peripheral blood during AD. Considering that oxidative stress, both in the brain and in the periphery, can influence the activation and differentiation of T cells, we investigated the nitro-oxidized proteome of blood T cells derived from AD patients compared to non-demented subjects (NSD) by using a redox proteomic approach. We identified eleven proteins showing increasing levels of 3-nitrotyrosine (3-NT) in AD T cells compared with NSD. These proteins are involved in energy metabolism, transcriptional regulation and antioxidant response and can help to clarify molecular mechanism that impact on the reduced T cells differentiation in AD. Our results highlight the role of peripheral OS in T cells related to immune-senescence during AD pathology focusing on the specific target of protein nitration that can be suitable to further therapies.

In 1620 Francis Bacon published the Novum Organum, opening a new era in the experimental research of natural phenomena based on the principle of induction. When describing this principle, he anticipated the effects of advances in science, engineering, and technology. Such a principle opposed to the a priori deductive knowledge has been the corner stone for proteomics investigations based on the unsupervised observation of protein modulation in a given biological background. This approach has led to contrasting evidence in the scientific literature from the last 20 years, primarily based on the discoveries of unexpected molecular association, to the complexity of protein modulation and often to the strong will of the investigators to potentially extend the application of their experimental observation beyond the given experimental framework. A wellknown example of this experience is available in the quest for protein biomarker discovery in clinical proteomics campaigns. Unfortunately inductive methods may only provide a probability for a given argument in a well-defined framework and their wide extension has been proved by a number of philosophers, e.g. Karl Popper and Bertrand Russell, to potentially lead to false conclusions. Although the introduction of a priori based proteomics investigations by applying targeted SRM (Selective Reaction Monitoring) design on proteotypic transition brought a new impulse, many researchers still prefer inductive experimental methods in order to obtain novel evidences. Inductive proteomics still provides a passage through the Pillars of Hercules, which for the ancients, symbolized the limits of possible human explorations. Beyond the pillars lay the edge of current knowledge. Thus, their crossing represents our aim toward new uncharted biological molecular mechanisms. These open-platform investigations are presently achieving a higher experimental confidence, providing large dataset collections for a more rigorous description of the experimental framework for the application of the reported results. In this themed issue dedicated to proteomics we have a clear example of such an extended description. The paper from Claudia Desiderio et al., dedicated to ‘Integrated proteomic platforms for the comparative characterization ofmedulloblastoma and pilocytic astrocytoma pediatric brain tumors’ (DOI: 10.1039/ C5MB00076A), provides a good example of such a definition. Following the research on medulloblastoma, Maurizio Ronci et al. (DOI: 10.1039/C5MB00034C) addressed the protein determinants associated with the stemness phenotype in hedgehog models. Glioblastoma response to nitric oxide releasing compounds is addressed in the paper by Roberta Leone et al. (DOI: 10.1039/ C4MB00725E) by applying 2DE-DIGE approaches. Redox modifications of proteins are further addressed in a welldefined model for microglia exposure to beta amyloid peptide in the work of Virginia Correani et al. (DOI: 10.1039/C4MB00703D) and in the paper by Claudia D’Anna et al. (DOI: 10.1039/C5MB00188A) on the impact of cigarette smoke on fibroblasts. While clinical proteomics investigations based on bottom-up approaches have been proving their limits, top-down proteomics investigations represent a fundamental approach to achieve the high specificity required. The current FDA 510k and CE-IVD approved proteomics-based platforms for clinical microbiology (MALDI Biotyper and Vitek MS) are in fact both based on a top-down experimental set-up. In this light, the work from Monica Sanna et al. (DOI: 10.1039/C4MB00719K) may a Department of Experimental Medicine and Surgery, University of Rome ‘‘Tor Vergata’’, Via Montpellier 1, Rome, Italy. E-mail: andrea.urbani@uniroma2.it b IRCCS-Fondazione S. Lucia, Rome, Italy c Section of Proteomics, Istituto Sperimentale Italiano L. Spallanzani, University of Milano, Milano, Italy d Department of Biomedical, Experimental and Clinical Sciences, University of Florence, Italy e Dipartimento di Scienze Ambientali, Universita della Tuscia, Viterbo, Italy f Department of Life Sciences, University of Siena, Italy g Department of Theoretical and Applied Sciences, and Center of Neuroscience, University of Insubria, Busto Arsizio, Italy h Institute of Biochemistry and Clinical Biochemistry, Faculty of Medicine, Catholic University, Piazza A. Gemelli, Rome, Italy. E-mail: massimo.castagnola@icrm.cnr.it i Istituto di Chimica del Riconoscimento Molecolare, CNR, Rome, Italy DOI: 10.1039/c5mb90021b

The combination of top-down and bottom-up platforms was utilized for the LC-MS proteomic characterization of the intracystic fluid of adamantinomatous craniopharyngioma pediatric brain tumor disease. Proteins and peptides characterization was achieved by high-resolution LC-ESI-LTQ-Orbitrap-MS analysis while low-resolution LC-ESI-IT-MS was applied for the complete screening of the samples and the evaluation of the protein distribution within patients. Top-down analyses were applied to liquid/liquid extracted samples while bottom-up analyses were performed after trypsin digestion of both untreated and pretreated samples. The two proteomic approaches were complementary for the characterization of the proteome of craniopharyngioma intracystic fluid. Proteins and peptides involved in inflammation, mineralization processes and lipid transport were identified, in agreement with the calcium flecks, cholesterol granules and bone residues characteristic of this fluid. Apolipoprotein A-I, A-II, C-I and J, hemoglobin fragments, ubiquitin, α-2-HS-glycoprotein or fetuin A, α-1-antichymotrypsin, vitamin D binding protein, and α-1-acid glycoprotein were characterized. These data could be relevant for the comprehension of the processes involved in the pathogenesis of the disease and the development of the cyst and could contribute to the individuation of therapeutic targets for the reduction of the cyst volume delaying and/or avoiding invasive surgical treatments.

This study describes the characterization of the glycan moieties and the peptide backbone of six glycoforms of IB-8a CON1(+), a basic proline-rich protein present in human saliva. MS analyses on the intact glycoproteins before and after N-deglycosylation with PNGase F and high-resolution MS/MS sequencing by LTQ Orbitrap XL of peptides and glycopeptides from tryptic digests allowed the structural characterization of the glycan moieties and the polypeptide backbone, as well as to establish the glycosylation site at the asparagine residue at 98th position. Five of the glycoforms carry a biantennary N-linked glycan fucosylated in the innermost N-acetylglucosamine of the core and showing from zero to four additional fucoses in the antennal region. The sixth glycoform carries a monoantennary monofucosylated oligosaccharide. The glycoform cluster was detected on 28 of 71 adult saliva specimens. Level of fucosylation showed interindividual variability with the major relative abundance for the trifucosylated glycoform. Nonglycosylated IB-8a CON1(+) and the variant IB-8a CON1(-), lacking of the glycosylation site, have been also detected in human saliva.