Fabrication of a Macroporous Microwell Array for Surface‐Enhanced Raman Scattering

Here, a colloidal templating procedure for generating high‐density arrays of gold macroporous microwells, which act as discrete sites for surface‐enhanced Raman scattering (SERS), is reported. Development of such a novel array with discrete macroporous sites requires multiple fabrication steps. First, selective wet‐chemical etching of the distal face of a coherent optical fiber bundle produces a microwell array. The microwells are then selectively filled with a macroporous structure by electroless template synthesis using self‐assembled nanospheres. The fabricated arrays are structured at both the micrometer and nanometer scale on etched imaging bundles. Confocal Raman microscopy is used to detect a benzenethiol monolayer adsorbed on the macroporous gold and to map the spatial distribution of the SERS signal. The Raman enhancement factor of the modified wells is investigated and an average enhancement factor of 4 × 10⁴ is measured. This demonstrates that such nanostructured wells can enhance the local electromagnetic field and lead to a platform of ordered SERS‐active micrometer‐sized spots defined by the initial shape of the etched optical fibers. Since the fabrication steps keep the initial architecture of the optical fiber bundle, such ordered SERS‐active platforms fabricated onto an imaging waveguide open new applications in remote SERS imaging, plasmonic devices, and integrated electro‐optical sensor arrays.     

Bio-Functionalized Chitosan for Bone Tissue Engineering

Hybrid biomaterials allow for the improvement of the biological properties of materials and have been successfully used for implantology in medical applications. The covalent and selective functionalization of materials with bioactive peptides provides favorable results in tissue engineering by supporting cell attachment to the biomaterial through biochemical cues and interaction with membrane receptors. Since the functionalization with bioactive peptides may alter the chemical and physical properties of the biomaterials, in this study we characterized the biological responses of differently functionalized chitosan analogs. Chitosan analogs were produced through the reaction of GRGDSPK (RGD) or FRHRNRKGY (HVP) sequences, both carrying an aldehyde-terminal group, to chitosan. The bio-functionalized polysaccharides, pure or “diluted” with chitosan, were chemically characterized in depth and evaluated for their antimicrobial activities and biocompatibility toward human primary osteoblast cells. The results obtained indicate that the bio-functionalization of chitosan increases human-osteoblast adhesion (p < 0.005) and proliferation (p < 0.005) as compared with chitosan. Overall, the 1:1 mixture of HVP functionalized-chitosan:chitosan is the best compromise between preserving the antibacterial properties of the material and supporting osteoblast differentiation and calcium deposition (p < 0.005 vs. RGD). In conclusion, our results reported that a selected concentration of HVP supported the biomimetic potential of functionalized chitosan better than RGD and preserved the antibacterial properties of chitosan.     

TRK Protein Expression in Merkel Cell Carcinoma Is Not Caused by NTRK Fusions

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous malignant tumor with neuroendocrine differentiation, with a rapidly growing incidence rate, high risk of recurrence, and aggressive behavior. The available therapeutic options for advanced disease are limited and there is a pressing need for new treatments. Tumors harboring fusions involving one of the neurotrophin receptor tyrosine kinase (NTRK) genes are now actionable with targeted inhibitors. NTRK-fused genes have been identified in neuroendocrine tumors of other sites; thus, a series of 76 MCCs were firstly analyzed with pan-TRK immunohistochemistry and the positive ones with real-time RT-PCR, RNA-based NGS, and FISH to detect the eventual underlying gene fusion. Despite 34 MCCs showing pan-TRK expression, NTRK fusions were not found in any cases. As in other tumors with neural differentiation, TRK expression seems to be physiological and not caused by gene fusions.     

BACHSCORE. A tool for evaluating efficiently and reliably the quality of large sets of protein structures

In protein structure prediction it is of crucial importance, especially at the refinement stage, to score efficiently large sets of models by selecting the ones that are closest to the native state. We here present a new computational tool, BACHSCORE, that allows its users to rank different structural models of the same protein according to their quality, evaluated by using the BACH++ (Bayesian Analysis Conformation Hunt) scoring function. The original BACH statistical potential was already shown to discriminate with very good reliability the protein native state in large sets of misfolded models of the same protein. BACH++ features a novel upgrade in the solvation potential of the scoring function, now computed by adapting the LCPO (Linear Combination of Pairwise Orbitals) algorithm. This change further enhances the already good performance of the scoring function. BACHSCORE can be accessed directly through the web server: bachserver.pd.infn.it.     

The Molecular Landscape of Primary Acral Melanoma: A Multicenter Study of the Italian Melanoma Intergroup (IMI)

Acral melanoma (AM) is a rare and aggressive subtype of melanoma affecting the palms, soles, and nail apparatus with similar incidence among different ethnicities. AM is unrelated to ultraviolet radiation and has a low mutation burden but frequent chromosomal rearrangements and gene amplifications. Next generation sequencing of 33 genes and somatic copy number variation (CNV) analysis with genome-wide single nucleotide polymorphism arrays were performed in order to molecularly characterize 48 primary AMs of Italian patients in association with clinicopathological and prognostic features. BRAF was the most commonly mutated gene, followed by NRAS and TP53, whereas TERT promoter, KIT, and ARID1A were less frequently mutated. Gains and losses were recurrently found in the 1q, 6p, 7, 8q, 20 and 22 chromosomes involving PREX2, RAC1, KMT2C, BRAF, CCND1, TERT, and AKT3 genes, and in the 6q, 9, 10, 11q and 16q chromosomes including CDKN2A, PTEN, and ADAMTS18 genes, respectively. This study confirmed the variety of gene mutations and the high load of CNV in primary AM. Some genomic alterations were associated with histologic prognostic features. BRAF mutations, found with a higher rate than previously reported, correlated with a low Breslow thickness, low mitotic count, low CNV of the AMs, and with early-stage of disease.     

Phosphorus transformations as affected by sampling date, fertilizer rate and phosphorus uptake in a soil under pasture

A phosphorus (P) fertilization study was conducted in the southeast of the Buenos Aires province in Argentina, to determine the effect of P fertilizer rate and sampling date on microbial biomass P, and of organic and inorganic P extractable with 0.5M NaHCO₃, in a soil under pasture. In addition, Bray-P, dry-matter production and P uptake were measured. Soil was sampled at different times over one year, and two to three years after application of 0, 50, 100 and 200 kg P ha ^–1. The addition of P fertilizer significantly increased total soil P and the labile fraction of P extractable with NaHCO₃, with the greatest change in the labile inorganic P form, but had no effect on microbial biomass P. Fractions of P showed different patterns of seasonal variation. Microbial biomass P had a peak in winter and a lowest value in summer, the opposite occurring with NaHCO₃-extractable organic P, while NaHCO₃-extractable inorganic P remained relatively constant throughout the year. The cumulative dry-matter yield after three years was 31% higher in the fertilized than in the unfertilized treatments; the highest being 27660 kg ha^–1 for 200 kg P ha^–1. Concentration of Bray-P increased by 0.18 mg P kg ^–1 for each additional kg P ha^–1 added, but remained relatively constant over the year. A significant correlation was found between available Bray-P and microbial biomass P (r = 0.53), and NaHCO₃-extractable organic P (r = 0.47), suggesting that these organic fractions may contribute to plant nutrition.     

Effect of the charge state (z = -1, 0, +1) on the nuclear magnetic resonance of monodisperse Au25[S(CH2)2Ph]18(z) clusters

Monodisperse Au(25)L(18)(0) (L = S(CH(2))(2)Ph) and [n-Oct(4)N(+)][Au(25)L(18)(-)] clusters were synthesized in tetrahydrofuran. An original strategy was then devised to oxidize them: in the presence of bis(pentafluorobenzoyl) peroxide, the neutral or the negatively charged clusters react as efficient electron donors in a dissociative electron-transfer (ET) process, in the former case yielding [Au(25)L(18)(+)][C(6)F(5)CO(2)(-)]. As opposed to other reported redox methods, this dissociative ET approach is irreversible, easily controllable, and clean, particularly for NMR purposes, as no hydrogen atoms are introduced. By using this approach, the -1, 0, and +1 charge states of Au(25)L(18) could be fully characterized by (1)H and (13)C NMR spectroscopy, using one- and two-dimensional techniques, in various solvents, and as a function of temperature. For all charge states, the NMR results and analysis nicely match recent structural findings about the presence of two different ligand populations in the capping monolayer, each resonance of the two ligand families displaying distinct NMR patterns. The radical nature of Au(25)L(18)(0) is particularly evident in the (1)H and (13)C NMR patterns of the inner ligands. The NMR behavior of radical Au(25)L(18)(0) was also simulated by DFT calculations, and the interplay between theory and experiments revealed a fundamental paramagnetic contribution coming from Fermi contact shifts. Interestingly, the NMR patterns of Au(25)L(18)(-) and Au(25)L(18)(+) were found to be quite similar, pointing to the latter cluster form as a diamagnetic species.     

Development of Two-Layer Hybrid Scaffolds Based on Oxidized Polyvinyl Alcohol and Bioactivated Chitosan Sponges for Tissue Engineering Purposes

Oxidized polyvinyl alcohol (OxPVA) is a new polymer for the fabrication of nerve conduits (NCs). Looking for OxPVA device optimization and coupling it with a natural sheath may boost bioactivity. Thus, OxPVA/chitosan sponges (ChS) as hybrid scaffolds were investigated to predict in the vivo behaviour of two-layered NCs. To encourage interaction with cells, ChS were functionalized with the self-assembling-peptide (SAP) EAK, without/with the laminin-derived sequences -IKVAV/-YIGSR. Thus, ChS and the hybrid scaffolds were characterized for mechanical properties, ultrastructure (Scanning Electron Microscopy, SEM), bioactivity, and biocompatibility. Regarding mechanical analysis, the peptide-free ChS showed the highest values of compressive modulus and maximum stress. However, among +EAK groups, ChS+EAK showed a significantly higher maximum stress than that found for ChS+EAK-IKVAV and ChS+EAK-YIGSR. Considering ultrastructure, microporous interconnections were tighter in both the OxPVA/ChS and +EAK groups than in the others; all the scaffolds induced SH-SY5Y cells’ adhesion/proliferation, with significant differences from day 7 and a higher total cell number for OxPVA/ChS+EAK scaffolds, in accordance with SEM. The scaffolds elicited only a slight inflammation after 14 days of subcutaneous implantation in Balb/c mice, proving biocompatibility. ChS porosity, EAK 3D features and neuro-friendly attitude (shared with IKVAV/YIGSR motifs) may confer to OxPVA certain bioactivity, laying the basis for future appealing NCs.