During aging, neuronal organelles filled with neuromelanin (a dark-brown pigment) and lipid bodies accumulate in the brain, particularly in the substantia nigra, a region targeted in Parkinson’s disease. We have investigated protein and lipid systems involved in the formation of these organelles and in the synthesis of the neuromelanin of human substantia nigra. Membrane and matrix proteins characteristic of lysosomes were found in neuromelanin-containing organelles at a lower number than in typical lysosomes, indicating a reduced enzymatic activity and likely impaired capacity for lysosomal and autophagosomal fusion. The presence of proteins involved in lipid transport may explain the accumulation of lipid bodies in the organelle and the lipid component in neuromelanin structure. The major lipids observed in lipid bodies of the organelle are dolichols with lower amounts of other lipids. Proteins of aggregation and degradation pathways were present, suggesting a role for accumulation by this organelle when the ubiquitin-proteasome system is inadequate. The presence of proteins associated with aging and storage diseases may reflect impaired autophagic degradation or impaired function of lysosomal enzymes. The identification of typical autophagy proteins and double membranes demonstrates the organelle’s autophagic nature and indicates that it has engulfed neuromelanin precursors from the cytosol. Based on these data, it appears that the neuromelanin-containing organelle has a very slow turnover during the life of a neuron and represents an intracellular compartment of final destination for numerous molecules not degraded by other systems.

Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint sig-nals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantita-tive applications of SERS in various fields and several SERS methods have been implemented for each specific applica-tion, ranging in performance characteristics, analytes used, instruments, and analytical matrices. In general, very few methods have been validated according to international guidelines. As a consequence, the application of SERS in high-ly-regulated environments is still considered risky and the perception of a poorly reproducible and insufficiently robust analytical technique has persistently retarded its routine implementation. Collaborative trials are a type of interlabora-tory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quan-tification with SERS arose within the framework of Working Group 1 (WG1) of the COST Action BM1401 Ra-man4Clinics32 in an effort to overcome the problematic perception of quantitative SERS methods. Here we report the first interlaboratory SERS study ever conducted, involving 15 laboratories and 41 researchers. In this study we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method, and to compare differ-ent methods. In our opinion, this is a first important step toward a “standardization” process of SERS protocols, not proposed by a single laboratory but by a larger community.

Cancer-associated fibroblasts (CAF) are the most abundant cells of the tumor stroma and they critically influence cancer growth through control of the surrounding tumor microenvironment (TME). CAF-orchestrated reactive stroma, composed of pro-tumorigenic cytokines and growth factors, matrix components, neovessels, and deregulated immune cells, is associated with poor prognosis in multiple carcinomas, including breast cancer. Therefore, beyond cancer cells killing, researchers are currently focusing on TME as strategy to fight breast cancer. In recent years, nanomedicine has provided a number of smart delivery systems based on active targeting of breast CAF and immune-mediated overcome of chemoresistance. Many efforts have been made both to eradicate breast CAF and to reshape their identity and function. Nano-strategies for CAF targeting profoundly contribute to enhance chemosensitivity of breast tumors, enabling access of cytotoxic T-cells and reducing immunosuppressive signals. TME rearrangement also includes reorganization of the extracellular matrix to enhance permeability to chemotherapeutics, and nano-systems for smart coupling of chemo- and immune-therapy, by increasing immunogenicity and stimulating antitumor immunity. The present paper reviews the current state-of-the-art on nano-strategies to target breast CAF and TME. Finally, we consider and discuss future translational perspectives of proposed nano-strategies for clinical application in breast cancer.

Neuromelanins (NMs) are neuronal pigments of melanic-lipidic type which accumulate during aging. They are involved in protective and degenerative mechanisms depending on the cellular context, however their structures are still poorly understood. NMs from nine human brain areas were analyzed in detail. Elemental analysis led to identification of three types of NM, while infrared spectroscopy showed that NMs from neurons of substantia nigra and locus coeruleus, which selectively degenerate in Parkinson’s disease, have similar structure but different from NMs from brain regions not targeted by the disease. Synthetic melanins containing Fe and bovine serum albumin were prepared to model the natural product and help clarifying the structure of NMs. Extensive nuclear magnetic resonance spectroscopy studies showed the presence of dolichols both in the soluble and insoluble parts of NM. Diffusion measurements demonstrated that the dimethyl sulfoxide soluble components consist of oligomeric precursors with MWs in the range 1.4–52 kDa, while the insoluble part contains polymers of larger size but with a similar composition. These data suggest that the selective vulnerability of neurons of substantia nigra and locus coeruleus in Parkinson’s disease might depend on the structure of the pigment. Moreover, they allow to propose a pathway for NM biosynthesis in human brain.

Breast microcalcifications are a common mammographic finding. Microcalcifications are considered suspicious signs of breast cancer and a breast biopsy is required, however, cancer is diagnosed in only a few patients. Reducing unnecessary biopsies and rapid characterization of breast microcalcifications are unmet clinical needs. In this study, 473 microcalcifications detected on breast biopsy specimens from 56 patients were characterized entirely by Raman mapping and confirmed by X-ray scattering. Microcalcifications from malignant samples were generally more homogeneous, more crystalline, and characterized by a less substituted crystal lattice compared with benign samples. There were significant differences in Raman features corresponding to the phosphate and carbonate bands between the benign and malignant groups. In addition to the heterogeneous composition, the presence of whitlockite specifically emerged as marker of benignity in benign microcalcifications. The whole Raman signature of each microcalcification was then used to build a classification model that distinguishes microcalcifications according to their overall biochemical composition. After validation, microcalcifications found in benign and malignant samples were correctly recognized with 93.5% sensitivity and 80.6% specificity. Finally, microcalcifications identified in malignant biopsies, but located outside the lesion, reported malignant features in 65% of in situ and 98% of invasive cancer cases, respectively, suggesting that the local microenvironment influences microcalcification features. This study confirms that the composition and structural features of microcalcifications correlate with breast pathology and indicates new diagnostic potentialities based on microcalcifications assessment. Significance: Raman spectroscopy could be a quick and accurate diagnostic tool to precisely characterize and distinguish benign from malignant breast microcalcifications detected on mammography.