Stereochemical Behavior of Pyrrolo-Pyrazole Peptidomimetics Promoting Phase-Selective Supramolecular Organogels.

Supramolecular gels were developed by taking advantage of an assembly of small dipeptides containing pyrrolo-pyrazole scaffolds. The dipeptides were prepared through a robust and ecofriendly synthetic approach from the commercially available starting materials of diazoalkanes and maleimides. By playing with the functionalization of the scaffold, the choice of the natural amino acid, and the stereochemistry, we were able to obtain phase-selective gels. In particular, one peptidomimetic showed gelation ability and thermoreversibility in aromatic solvents at very low concentrations. Rheology tests showed a typical viscoelastic solid profile, indicating the formation of strong gels that were stable under high mechanical deformation. NMR studies were performed, allowing us to determine the conformational and stereochemical features at the base of the supramolecular interactions.

Smart Electrospun Nanofibers from Short Peptidomimetics Based on Pyrrolo-pyrazole Scaffold.

We prepared a small library of short peptidomimetics based on 3-pyrrolo-pyrazole carboxylate, a non-coded γ-amino acid, and glycine or alanine. The robust and eco-friendly synthetic approach adopted allows to obtain the dipeptides in two steps from commercial starting materials. This gives the possibility to shape these materials by electrospinning into micro- and nanofibers, in amounts required to be useful for coating surfaces of biomedical relevance. To promote high quality of electrospun fibers, different substitution patterns were evaluated, all for pure peptide fibers, free of any polymer or additive. The best candidate, which affords a homogeneous fibrous matrix, was prepared in larger amounts, and its biocompatibility was verified. This successful work is the first step to develop a new biomaterial able to produce pristine peptide-based nanofibers to be used as helpful component or stand-alone scaffolds for tissue engineering or for the surface modification of medical devices.

Spatial Multiomics of Lipids, N-Glycans, and Tryptic Peptides on a Single FFPE Tissue Section.

Mass spectrometry imaging (MSI) is an emerging technology that is capable of mapping various biomolecules within their native spatial context, and performing spatial multiomics on formalin-fixed paraffin-embedded (FFPE) tissues may further increase the molecular characterization of pathological states. Here we present a novel workflow which enables the sequential MSI of lipids, N-glycans, and tryptic peptides on a single FFPE tissue section and highlight the enhanced molecular characterization that is offered by combining the multiple spatial omics data sets. In murine brain and clear cell renal cell carcinoma (ccRCC) tissue, the three molecular levels provided complementary information and characterized different histological regions. Moreover, when the spatial omics data was integrated, the different histopathological regions of the ccRCC tissue could be better discriminated with respect to the imaging data set of any single omics class. Taken together, these promising findings demonstrate the capability to more comprehensively map the molecular complexity within pathological tissue.

Cytomolecular Classification of Thyroid Nodules Using Fine-Needle Washes Aspiration Biopsies.

Fine-needle aspiration biopsies (FNA) represent the gold standard to exclude the malignant nature of thyroid nodules. After cytomorphology, 20-30% of cases are deemed "indeterminate for malignancy" and undergo surgery. However, after thyroidectomy, 70-80% of these nodules are benign. The identification of tools for improving FNA's diagnostic performances is explored by matrix-assisted laser-desorption ionization mass spectrometry imaging (MALDI-MSI). A clinical study was conducted in order to build a classification model for the characterization of thyroid nodules on a large cohort of 240 samples, showing that MALDI-MSI can be effective in separating areas with benign/malignant cells. The model had optimal performances in the internal validation set (n = 70), with 100.0% (95% CI = 83.2-100.0%) sensitivity and 96.0% (95% CI = 86.3-99.5%) specificity. The external validation (n = 170) showed a specificity of 82.9% (95% CI = 74.3-89.5%) and a sensitivity of 43.1% (95% CI = 30.9-56.0%). The performance of the model was hampered in the presence of poor and/or noisy spectra. Consequently, restricting the evaluation to the subset of FNAs with adequate cellularity, sensitivity improved up to 76.5% (95% CI = 58.8-89.3). Results also suggest the putative role of MALDI-MSI in routine clinical triage, with a three levels diagnostic classification that accounts for an indeterminate gray zone of nodules requiring a strict follow-up.

Ultrashort Peptides and Gold Nanoparticles: Influence of Constrained Amino Acids on Colloidal Stability.

Poor colloidal stability of gold nanoparticles (AuNPs) in physiological environments remains one of the major limitations that contribute to their difficult translation from bench to clinic. For this reason, an active research field is the development of molecules able to hamper AuNPs aggregation tendency in physiological environments. In this context, synthetic peptides are gaining an increased interest as an alternative to the use of biomacromolecules and polymers, due to their easiness of synthesis and their profitable pharmacokinetic profile. In this work, we reported on the use of ultrashort peptides containing conformationally constrained amino acids (AAs) for the stabilization of AuNPs. A small library of non-natural self-assembled oligopeptides were synthesized and used to functionalize spherical AuNPs of 20 nm diameter, via the ligand exchange method. The aim was to investigate the role of the constrained AA, the anchor point (at C- or N-terminus) and the peptide length on their potential use as gold binding motif. Ultrashort Aib containing peptides were identified as effective tools for AuNPs colloidal stabilization. Furthermore, peptide coated AuNPs were found to be storable as powders without losing the stabilization properties once re-dispersed in water. Finally, the possibility to exploit the developed systems for binding proteins via molecular recognition was also evaluated using biotin as model.

Lipidomic Typing of Colorectal Cancer Tissue Containing Tumour-Infiltrating Lymphocytes by MALDI Mass Spectrometry Imaging.

Predicting the prognosis of colorectal cancer (CRC) patients remains challenging and a characterisation of the tumour immune environment represents one of the most crucial avenues when attempting to do so. For this reason, molecular approaches which are capable of classifying the immune environments associated with tumour infiltrating lymphocytes (TILs) are being readily investigated. In this proof of concept study, we aim to explore the feasibility of using spatial lipidomics by MALDI-MSI to distinguish CRC tissue based upon their TIL content. Formalin-fixed paraffin-embedded tissue from human thymus and tonsil was first analysed by MALDI-MSI to obtain a curated mass list from a pool of single positive T lymphocytes, whose putative identities were annotated using an LC-MS-based lipidomic approach. A CRC tissue microarray (TMA, n = 30) was then investigated to determine whether these cases could be distinguished based upon their TIL content in the tumour and its microenvironment. MALDI-MSI from the pool of mature T lymphocytes resulted in the generation of a curated mass list containing 18 annotated m/z features. Initially, subsets of T lymphocytes were then distinguished based on their state of maturation and differentiation in the human thymus and tonsil tissue. Then, when applied to a CRC TMA containing differing amounts of T lymphocyte infiltration, those cases with a high TIL content were distinguishable from those with a lower TIL content, especially within the tumour microenvironment, with three lipid signals being shown to have the greatest impact on this separation (p < 0.05). On the whole, this preliminary study represents a promising starting point and suggests that a lipidomics MALDI-MSI approach could be a promising tool for subtyping the diverse immune environments in CRC.

Peptide-Based Electrospun Fibers: Current Status and Emerging Developments.

Electrospinning is a well-known, straightforward, and versatile technique, widely used for the preparation of fibers by electrifying a polymer solution. However, a high molecular weight is not essential for obtaining uniform electrospun fibers; in fact, the primary criterion to succeed is the presence of sufficient intermolecular interactions, which function similar to chain entanglements. Some small molecules able to self-assemble have been electrospun from solution into fibers and, among them, peptides containing both natural and non-natural amino acids are of particular relevance. Nowadays, the use of peptides for this purpose is at an early stage, but it is gaining more and more interest, and we are now witnessing the transition from basic research towards applications. Considering the novelty in the relevant processing, the aim of this review is to analyze the state of the art from the early 2000s on. Moreover, advantages and drawbacks in using peptides as the main or sole component for generating electrospun nanofibers will be discussed. Characterization techniques that are specifically targeted to the produced peptide fibers are presented.

Peptide grafting strategies before and after electrospinning of nanofibers.

Nanofiber films produced by electrospinning currently provide a promising platform for different applications. Although nonfunctionalized nanofiber films from natural or synthetic polymers are extensively used, electrospun materials combined with peptides are gaining more interest. In fact, the selection of specific peptides improves the performance of the material for biological applications and mainly for tissue engineering, mostly by maintaining similar mechanical properties with respect to the simple polymer. The main drawback in using peptides blended with a polymer is the quick release of the peptides. To avoid this problem, covalent linking of the peptide is more beneficial. Here, we reviewed synthetic protocols that enable covalent grafting of peptides to polymers before or after the electrospinning procedures to obtain more robust electrospun materials. Applications and the performance of the new material compared to that of the starting polymer are discussed.

Ex vivo thyroid fine needle aspirations as an alternative for MALDI-MSI proteomic investigation: intra-patient comparison.

Fine needle aspiration (FNA) is the reference standard for the diagnosis of thyroid nodules. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been successfully used to discriminate the proteomic profiles of benign and malignant thyroid FNAs within the scope of providing support to pathologists for the classification of morphologically borderline cases. However, real FNAs provide a limited amount of material due to sample collection restrictions. Ex vivo FNAs could represent a valuable alternative, increasing sample size and the power of statistical conclusions. In this study, we compared the real and ex vivo MALDI-MSI proteomic profiles, extracted from thyrocyte containing regions of interest, of 13 patients in order to verify their similarity. Statistical analysis demonstrated the mass spectra similarity of the proteomic profiles by performing intra-patient comparison, using statistical similarity systems. In conclusion, these results show that post-surgical FNAs represent a possible alternative source of material for MALDI-MSI proteomic investigations in instances where pre-surgical samples are unavailable or the number of cells is scarce.

Rational Design of a User-Friendly Aptamer/Peptide-Based Device for the Detection of Staphylococcus aureus.

The urgent need to develop a detection system for Staphylococcus aureus, one of the most common causes of infection, is prompting research towards novel approaches and devices, with a particular focus on point-of-care analysis. Biosensors are promising systems to achieve this aim. We coupled the selectivity and affinity of aptamers, short nucleic acids sequences able to recognize specific epitopes on bacterial surface, immobilized at high density on a nanostructured zirconium dioxide surface, with the rational design of specifically interacting fluorescent peptides to assemble an easy-to-use detection device. We show that the displacement of fluorescent peptides upon the competitive binding of S. aureus to immobilized aptamers can be detected and quantified through fluorescence loss. This approach could be also applied to the detection of other bacterial species once aptamers interacting with specific antigens will be identified, allowing the development of a platform for easy detection of a pathogen without requiring access to a healthcare environment.

Molecular trait of follicular-patterned thyroid neoplasms defined by MALDI-imaging.

In the field of thyroid neoplasms, the most interesting recent change regards the introduction of a new terminology for follicular-patterned thyroid tumors, named Noninvasive Thyroid Neoplasm with Papillary-like Nuclear Features (NIFTP). This pre-malignant tumor is considered to be the putative precursor of invasive carcinoma. However, given that several issues are still unresolved, the application of ancillary tools, based on omics-techniques, may improve the clinical management of these challenging cases. The present paper highlights the proteomic profiles of a series of NIFTPs submitted to Fine Needle Aspirations (FNAs) and analysed by MALDI-imaging in order to confirm the heterogeneous phenotype of nodules included in the present NIFTP terminology and to underline the necessity of more accurate biomarkers that can be used for their characterization. Ethical and economic implications in terms of healthcare costs, operative risks, morbidity, as well as the potential need for lifelong hormone replacement therapy, seem to be significant reasons to approach the characterization of NIFTPs using alternative tools such as MALDI-MSI.

Non-natural 3-Arylmorpholino-ß-amino Acid as a PPII Helix Inducer.

A new non-natural ß-amino acid, named 3-Ar-ß-Morph, was designed and synthesized via a regio- and diastereoselective Pd-catalyzed C(sp3)H-arylation of the corresponding 2S,6S-(6-methoxymorpholin-2-yl)carboxylic acid, readily available from glucose. According to the computational prevision and confirmed by IR and NMR data, the insertion of 3-Ar-ß-Morph in a model foldamer represents a way to stabilize a PPII-like helix through the presence of two γ-turns, secondary structure motifs induced by the morpholine ring, and the trans-tertiary amide bond.

Analysis of Hashimoto's thyroiditis on fine needle aspiration samples by MALDI-Imaging.

Matrix-Assisted Laser Desorption/Ionization (MALDI)-Mass Spectrometry imaging (MSI) has been applied in various diseases aimed to biomarkers discovery. In this study diagnosis and prognosis of Hashimoto Thyroiditis (HT) in cytopathology by MALDI-MSI has been investigated. Specimens from a routine series of subjects who underwent UltraSound-guided thyroid Fine Needle Aspirations (FNAs) were used. The molecular classifier trained in a previous study was modified to include HT as a separate entity in the group of benign lesions, in the diagnostic proteomic triage of thyroid nodules. The statistical analysis confirmed the existence of signals that HT shares with hyperplastic lesions and others that are specific and characterize this subgroup. Statistically relevant HT-related peaks were included in the model. Then, the discriminatory capability of the classifier was tested in a second validation phase, showing a good agreement with cytological diagnoses. The possibility to overlap the molecular signatures of both the lymphocytes and epithelial cells components (ROIs or pixel-by-pixel analysis) confirmed the composite proteomic background of HT. These results open the way to their possible translation as alternative serum biomarkers of this autoimmune condition.

Antigen Retrieval and Its Effect on the MALDI-MSI of Lipids in Formalin-Fixed Paraffin-Embedded Tissue.

Formalin-fixed paraffin-embedded (FFPE) tissue represents the primary source of clinical tissue and is routinely used in MALDI-MSI studies. However, it is not particularly suitable for lipidomics imaging given that many species are depleted during tissue processing. Irrespective, a number of solvent-resistant lipids remain, but their extraction may be hindered by the cross-link between proteins. Therefore, an antigen retrieval step could enable the extraction of a greater number of lipids and may provide information that is complementary to that which can be obtained from other biomolecules, such as proteins. In this short communication, we aim to address the effect of performing antigen retrieval prior to MALDI-MSI of lipids in FFPE tissue. As a result, an increased number of lipid signals could be detected and may have derived from lipid species that are known to be implicated in the lipid-protein cross-linking that is formed as a result of formalin fixation. Human renal cancer tissue was used as a proof of concept to determine whether using these detected lipid signals were also able to highlight the histopathological regions that were present. These preliminary findings may highlight the potential to enhance the clinical relevance of the lipidomic information obtained from FFPE tissue.

In-Depth Mapping of the Urinary N-Glycoproteome: Distinct Signatures of ccRCC-related Progression.

Protein N-glycosylation is one of the most important post-translational modifications and is involved in many biological processes, with aberrant changes in protein N-glycosylation patterns being closely associated with several diseases, including the progression and spreading of tumours. In light of this, identifying these aberrant protein glycoforms in tumours could be useful for understanding the molecular mechanism of this multifactorial disease, developing specific biomarkers and finding novel therapeutic targets. We investigated the urinary N-glycoproteome of clear cell renal cell carcinoma (ccRCC) patients at different stages (n = 15 at pT1 and n = 15 at pT3), and of non-ccRCC subjects (n = 15), using an N-glyco-FASP-based method. Using label-free nLC-ESI MS/MS, we identified and quantified several N-glycoproteins with altered expression and abnormal changes affecting the occupancy of the glycosylation site in the urine of RCC patients compared to control. In particular, nine of them had a specific trend that was directly related to the stage progression: CD97, COCH and P3IP1 were up-expressed whilst APOB, FINC, CERU, CFAH, HPT and PLTP were down-expressed in ccRCC patients. Overall, these results expand our knowledge related to the role of this post-translational modification in ccRCC and translation of this information into pre-clinical studies could have a significant impact on the discovery of novel biomarkers and therapeutic target in kidney cancer.

Self-assembled hydrophobic Ala-Aib peptide encapsulating curcumin: a convenient system for water insoluble drugs.

The exploitation of self-assembled systems to improve the solubility of drugs is getting more and more attention. Among the different types of self-assembled biomaterials, peptides and in particular peptides containing non-coded amino acids (NCAPs) are promising because their use opens the door to more stable materials inducing increased stability to proteolysis. New classes of NCAP, Ac-Ala-X-Ala-Aib-AlaCONH2 (X = alpha-aminoisobutyric acid (Aib) or X = cyclopentane amino acid (Ac5c)) have been prepared and the correlation between the different secondary peptide structure and solvent (i.e. CD3CN, CD3OH, H2O/D2O) verified by NMR. Furthermore, the formation of a nanocolloidal system in water was deeply studied by DLS and the morphology of the obtained spherical aggregates with nanometric dimensions was assessed by TEM. Aib containing pentapeptide was selected for greater ease of synthesis. Its ability to encapsulate curcumin, as a model insoluble drug molecule, was investigated using fluorescence emission and confocal microscopy analyses. Two different approaches were used to study the interaction between curcumin and peptide aggregates. In the first approach peptide aggregates were formed in the presence of curcumin, while in the second approach curcumin was added to the already formed peptide aggregates. We succeeded in our challenge by using the second approach and 53.8% of added curcumin had been encapsulated.

Covalent Grafting of Antimicrobial Peptides onto Microcrystalline Cellulose.

The purpose of this work is to set up a general protocol for the production of antimicrobial materials based on cellulose and peptides. We exploited the chemical ligation reaction to achieve the conjugation of peptides to cellulose; to this aim, we produced thioester peptides and cysteine-modified cellulose. As the thioester handle can be inserted at any position of the peptide, the peptide can be immobilized onto the cellulose through its N- or C-terminal end or through any other position within the sequence. Our experiments performed on Escherichia coli cultures show that the cellulose conjugated to the peptides lasioglossin-III and TBKKG6A causes a significant reduction in the concentration of viable cells as compared to unmodified cellulose. In conclusion, antimicrobial peptides bound to cellulose through a covalent bond retain their activity and therefore have the potential to be used as active ingredients in antimicrobial materials.

Matrix-assisted laser desorption/ionization mass spectrometry imaging to uncover protein alterations associated with the progression of IgA nephropathy.

IgA nephropathy (IgAN) is one of the most diffuse glomerulonephrites worldwide, and many issues still remain regarding our understanding of its pathogenesis. The disease is diagnosed by renal biopsy examination, but potential pitfalls still persist with regard to discriminating its primary origin and, as a result, determining patient outcome remains challenging. In this pilot study, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) was performed on renal biopsies obtained from patients with IgAN (n = 11) and other mesangioproliferative glomerulonephrites (MesPGN, n = 6) in order to enlighten proteomic alterations that may be associated with the progression of IgAN. Differences in the proteomic profiles of IgAN and MesPGN tissue could clearly be detected using this approach and, furthermore, 14 signals (AUC ≥ 0.8) were observed to have an altered intensity among the different CKD stages within the IgAN group. In particular, large increases in the intensity of these signals could be observed at CKD stages II and above. These signals primarily corresponded to proteins involved in either inflammatory and healing pathways and their increased intensity was localized within regions of tissue with large amounts of inflammatory cells or sclerosis. Despite much work in recent years, our molecular understanding of IgAN progression remains incomplete. This pilot study represents a promising starting point in the search for novel protein markers that can assist clinicians in better understanding the pathogenesis of IgAN and highlighting those patients who may progress to end-stage renal disease.

MALDI-MSI as a Complementary Diagnostic Tool in Cytopathology: A Pilot Study for the Characterization of Thyroid Nodules.

The present study applies for the first time as Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry Imaging (MSI) on real thyroid Fine Needle Aspirations (FNAs) to test its possible complementary role in routine cytology in the diagnosis of thyroid nodules. The primary aim is to evaluate the potential employment of MALDI-MSI in cytopathology, using challenging samples such as needle washes. Firstly, we designed a statistical model based on the analysis of Regions of Interest (ROIs), according to the morphological triage performed by the pathologist. Successively, the capability of the model to predict the classification of the FNAs was validated in a different group of patients on ROI and pixel-by-pixel approach. Results are very promising and highlight the possibility to introduce MALDI-MSI as a complementary tool for the diagnostic characterization of thyroid nodules.