High IGFBP2 expression correlates with tumor severity in pediatric rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common childhood sarcoma and is identified as either the embryonal or alveolar (ARMS) subtype. In approximately 75% of cases, ARMSs are characterized by specific chromosomal translocations that involve PAX and FKHR genes. ARMS gene expression signatures vary, depending on the presence or absence of the translocations. Insulin-like growth factor-binding protein 2 (IGFBP2) is strongly overexpressed in translocation-negative RMS. Because IGFBP2 is associated with tumorigenesis, we investigated its functional role in RMS. An analysis of IGFBP2 distribution in RMS cell lines revealed a strong accumulation in the Golgi complex, in which morphological characteristics appeared peculiarly modified. After silencing IGFBP2 expression, our microarray analysis revealed mostly cell cycle and actin cytoskeleton gene modulations. In parallel, IGFBP2-silenced cells showed reduced cell cycle and rates of invasion and decreased seeding in the lungs after tail vein injections in immunodeficient mice. An analysis of IGFBP2 mRNA and protein localization in human tumors showed abnormal protein accumulation in the Golgi complex, mostly in PAX/FKHR-negative RMS. Moreover, an analysis of patients with RMS revealed the presence of conspicuous circulating levels of IGFBP2 proteins in children with highly aggressive RMS tumors. Taken together, our data provide evidence that IGFBP2 contributes to tumor progression and that it could be used as a marker to better classify clinical and biological risks in RMS

ATRT–SHH comprises three molecular subgroups with characteristic clinical and histopathological features and prognostic significance

Atypical teratoid/rhabdoid tumor (ATRT) is an aggressive central nervous system tumor characterized by loss of SMARCB1/INI1 protein expression and comprises three distinct molecular groups, ATRT–TYR, ATRT–MYC and ATRT–SHH. ATRT–SHH represents the largest molecular group and is heterogeneous with regard to age, tumor location and epigenetic profile. We, therefore, aimed to investigate if heterogeneity within ATRT–SHH might also have biological and clinical importance. Consensus clustering of DNA methylation profiles and confirmatory t-SNE analysis of 65 ATRT–SHH yielded three robust molecular subgroups, i.e., SHH-1A, SHH-1B and SHH-2. These subgroups differed by median age of onset (SHH-1A: 18 months, SHH-1B: 107 months, SHH-2: 13 months) and tumor location (SHH-1A: 88% supratentorial; SHH-1B: 85% supratentorial; SHH-2: 93% infratentorial, often extending to the pineal region). Subgroups showed comparable SMARCB1 mutational profiles, but pathogenic/likely pathogenic SMARCB1 germline variants were over-represented in SHH-2 (63%) as compared to SHH-1A (20%) and SHH-1B (0%). Protein expression of proneural marker ASCL1 (enriched in SHH-1B) and glial markers OLIG2 and GFAP (absent in SHH-2) as well as global mRNA expression patterns differed, but all subgroups were characterized by overexpression of SHH as well as Notch pathway members. In a Drosophila model, knockdown of Snr1 (the fly homologue of SMARCB1) in hedgehog activated cells not only altered hedgehog signaling, but also caused aberrant Notch signaling and formation of tumor-like structures. Finally, on survival analysis, molecular subgroup and age of onset (but not ASCL1 staining status) were independently associated with overall survival, older patients (> 3 years) harboring SHH-1B experiencing relatively favorable outcome. In conclusion, ATRT–SHH comprises three subgroups characterized by SHH and Notch pathway activation, but divergent molecular and clinical features. Our data suggest that molecular subgrouping of ATRT–SHH has prognostic relevance and might aid to stratify patients within future clinical trials. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00401-022-02424-5

Design and fabrication of a 3D-structured gold film with nanopores for local electric field enhancement in the pore

Three-dimensionally structured gold membrane films with nanopores of defined, periodic geometries are designed and fabricated to provide the spatially localised enhancement of electric fields by manipulation of the plasmons inside nanopores. Square nanopores of different size and orientation relative to the pyramid are considered for films in aqueous and air environments, which allow for control of the position of electric fields within the structure. Designs suitable for use with 780 nm light were created. Here, periodic pyramidal cavities produced by potassium hydroxide etching to the {111} planes of (100) silicon substrates are used as templates for creating a periodic, pyramidal structured, free-standing thin gold film. Consistent with the findings from the theoretical studies, a nano-sized hole of 50 nm square was milled through the gold film at a specific location in the cavity to provide electric field control which can subsequently used for enhancement of fluorescence or Raman scattering of molecules in the nanopore

Magnetron Sputtering of Au-Based Alloys on NiTi Elements: Surface Investigation for New Products in SMA-Based Fashion and Luxury Accessories and Watchmaking

A novel approach for the deposition of Au-based coatings on NiTi components was proposed to give rise to innovative SMA-based products for the fashion, luxury, and watchmaking fields. Different Au-Cu and Au-Ag-Cu alloys (with confidential compositions within the color designations 2N, 4N, and 5N) were deposited by magnetron sputtering on superelastic and shape-memory NiTi ribbons. After preliminary morphological and microstructural characterizations, the influence of the film deposition on the functional, mechanical, and tribological behavior was deeply investigated. The macroscopic mechanical properties, including the damping, superelastic, and shape recovery characteristics, were not affected since the behavior upon both small and severe deformations was unchanged and the coatings were not damaged. Indeed, both the film adhesion and the precious aspect were maintained. Furthermore, a more detailed surface characterization, through nanoindentation, tribocorrosion, and scratch and wear tests, was performed. This experimental investigation evidenced the ductile behavior of the Au-based films and their good adhesion on NiTi substrates. Moreover, the coatings exhibited a good wear resistance, both in dry conditions and simulated body fluids, which proved to be suitable for fashion and watchmaking fields. Despite slight differences being observed within the films’ responses, all of them could be considered suitable and interesting for the design of smart luxury accessories, proving that the chosen deposition process is sound and reliable for these applications

TiO2-HA bi-layer coatings for improving the bioactivity and service-life of Ti dental implants

A titania (TiO\u20602) - hydroxyapatite (HA) bi-layered material, with a target surface design, was obtained by means of a two-step process on the surface of Ti substrates with three different pristine morphologies and topographies. A dense, compact and crystalline titania inter-layer was obtained via Metal Organic Chemical Vapor Deposition, and a homogeneously spread discontinuous calcium phosphate ceramic (hydroxyapatite) top-layer, with particular chemical composition, crystallinity, and morphology, was deposited by means of spray pyrolysis. A final thermal treatment at 600\ub0C was found as the best compromise between an adequate crystallinity, bioactivity and composite material features. The novelty of this work is that this synergic two-step approach allows the co-existence of both TiO\u20602 and HA ceramics on the implant surface. This can help in increasing the surface bioactivity and improving the short(HA)- and long(TiO\u20602)-term implant service-life. The influence of the two-step modification process was investigated. Coated surfaces exhibited better electrochemical performances in artificial saliva and reduced metallic ion release; also the mechanical properties at the nano-scale level of the composite materials were improved thanks to the functionalization. The composite material wettability was also studied: the freshly prepared surfaces always showed hydrophilicity. However, in air environments, the wettability decreased with ageing time due to hydrocarbon contamination. Acellular in-vitro bioactivity of all species was tested evaluating the ability of the materials to form a bone-like apatite layer after immersion in Dulbecco's Phosphate Buffer Saline solution at 37\ub0C. The obtained results showed that a bone-like apatite layer was effectively formed on the two-step functionalized Ti substrates, giving significant results just after 30min incubation time

Assessment of synergistic effects of LP-MOCVD TiO2 and Ti surface finish for dental implant purposes

Three differently treated titanium substrates to be used in dental implant applications and with different surficial morphology were coated with titanium dioxide (TiO2) films by using Low-Pressure Metal Organic Chemical Vapor Deposition (MOCVD). No literature references were found on the performance evaluation of the TiO2 MOCVD coatings on substrates with different pristine morphology; in this work the influence of the pristine Ti surface characteristics on TiO2 crystalline structure, morphology, wettability as well as on ion release, electrochemical behavior, tribocorrosion performance, and nano-mechanical properties were studied and discussed. It was shown that the pristine substrate influenced both the crystalline phases' formation and crystallite size. Scanning electron microscopy analyses and roughness evaluation showed the optimal conformal coverage of all the MOCVD coatings for all substrates, with grain size depending on the substrate morphology and topography. The wettability of the TiO2 coated Ti substrates highlighted a superhydrophilic behavior and, if stored in air, decreased as a function of the time aging. Ions release tests, nanoindentation measurements, tribocorrosion, and potentiodynamic polarization experiments suggest the enhancement of functional properties of the coated samples. Titania sandblasted/acid etched coated Ti substrates generally showed the best performance of the functional properties for their use in dental implant fabrications

Liquid Biopsy in Pediatric Renal Cancer: Stage I and Stage IV Cases Compared

Pediatric renal cancer is rare, and robust evidence for treatment recommendations is lacking. In the perspective of personalized medicine, clinicians need new biomarkers to improve risk stratification and patients’ follow-up. Herein, we analyzed some liquid biopsy tools, which have been never tested in pediatric renal cancer: namely, circulating tumor cells (CTCs); the expression of M30, an apoptosis marker, to test CTC metastatic potential; and c-MET expression in CTCs, because of its role in renal cancer progression and drug-resistance. Furthermore, we evaluated the Circulating Endothelial Cells (CECs), whose utility we previously demonstrated in adult metastatic renal cancer treated with anti-angiogenic therapy. We compared two renal cell carcinomas of clear-cell type, stage I and IV, which underwent surgery and surgery plus Sunitinib, respectively. Baseline CTC level and its changes during follow-up were consistent with patients’ outcome. In case 2, stage IV, the analysis of CECs performed during Sunitinib revealed a late response to treatment consistent with poor outcome, as the finding of M30-negative, viable cells. Noteworthily, few CTCs were MET-positive in both cases. Our study highlights the feasibility for a change in the prognostic approach and follow-up of childhood renal cancer, with a view to guide a better treatment design

A nanoporous gold membrane for sensing applications

Design and fabrication of three-dimensionally structured, gold membranes containing hexagonally close-packed microcavities with nanopores in the base, are described. Our aim is to create a nanoporous structure with localized enhancement of the fluorescence or Raman scattering at, and in the nanopore when excited with light of approximately 600 nm, with a view to provide sensitive detection of biomolecules. A range of geometries of the nanopore integrated into hexagonally close-packed assemblies of gold micro-cavities was first evaluated theoretically. The optimal size and shape of the nanopore in a single microcavity were then considered to provide the highest localized plasmon enhancement (of fluorescence or Raman scattering) at the very center of the nanopore for a bioanalyte traversing through. The optimized design was established to be a 1200 nm diameter cavity of 600 nm depth with a 50 nm square nanopore with rounded corners in the base. A gold 3D-structured membrane containing these sized microcavities with the integrated nanopore was successfully fabricated and ‘proof of concept’ Raman scattering experiments are described. Keywords: Nanopore, Polymer sphere, Gold membrane, Plasmons, Sensing, SER

Inhibition of nuclear export restores nuclear localization and residual tumor suppressor function of truncated SMARCB1/INI1 protein in a molecular subset of atypical teratoid/rhabdoid tumors

Loss of nuclear SMARCB1 (INI1/hSNF5/BAF47) protein expression due to biallelic mutations of the SMARCB1 tumor suppressor gene is a hallmark of atypical teratoid/rhabdoid tumors (ATRT), but the presence of cytoplasmic SMARCB1 protein in these tumors has not yet been described. In a series of 102 primary ATRT, distinct cytoplasmic SMARCB1 staining on immunohistochemistry was encountered in 19 cases (19%) and was highly over-represented in cases showing pathogenic sequence variants leading to truncation or mutation of the C-terminal part of SMARCB1 (15/19 vs. 4/83; Chi-square: 56.04, p = 1.0E−10) and, related to this, in tumors of the molecular subgroup ATRT-TYR (16/36 vs. 3/66; Chi-square: 24.47, p = 7.6E−7). Previous reports have indicated that while SMARCB1 lacks a bona fide nuclear localization signal, it harbors a masked nuclear export signal (NES) and that truncation of the C-terminal region results in unmasking of this NES leading to cytoplasmic localization. To determine if cytoplasmic localization found in ATRT is due to unmasking of NES, we generated GFP fusions of one of the SMARCB1 truncating mutations (p.Q318X) found in the tumors along with a p.L266A mutation, which was shown to disrupt the interaction of SMARCB1-NES with exportin-1. We found that while the GFP-SMARCB1(Q318X) mutant localized to the cytoplasm, the double mutant GFP-SMARCB1(Q318X;L266A) localized to the nucleus, confirming NES requirement for cytoplasmic localization. Furthermore, cytoplasmic SMARCB1(Q318X) was unable to cause senescence as determined by morphological observations and by senescence-associated β-galactosidase assay, while nuclear SMARCB1(Q318X;L266A) mutant regained this function. Selinexor, a selective exportin-1 inhibitor, was effective in inhibiting the nuclear export of SMARCB1(Q318X) and caused rapid cell death in rhabdoid tumor cells. In conclusion, inhibition of nuclear export restores nuclear localization and residual tumor suppressor function of truncated SMARCB1. Therapies aimed at preventing nuclear export of mutant SMARCB1 protein may represent a promising targeted therapy in ATRT harboring truncating C-terminal SMARCB1 mutations. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00401-021-02328-w