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Glioblastoma multiforme (GBM) is the most common malignant brain tumor, associated with low long-term survival. Nanoparticles (NPs) developed against GBM are a promising strategy to improve current therapies, by enhancing the brain delivery of active molecules and reducing off-target effects. In particular, NPs hold high potential for the targeted delivery of chemotherapeutics both across the blood–brain barrier (BBB) and specifically to GBM cell receptors, pathways, or the tumor microenvironment (TME). In this review, the most recent strategies to deliver drugs to GBM are explored. The main focus is on how surface functionalizations are essential for BBB crossing and for tumor specific targeting. We give a critical analysis of the various ligand-based approaches that have been used to target specific cancer cell receptors and the TME, or to interfere with the signaling pathways of GBM. Despite the increasing application of NPs in the clinical setting, new methods for ligand and surface characterization are needed to optimize the synthesis, as well as to predict their in vivo behavior. An expert opinion is given on the future of this research and what is still missing to create and characterize a functional NP system for improved GBM targeting.

Extracellular Vesicles (EVs) are versatile carriers for biomarkers involved in the pathogenesis of multiple human disorders. Despite the increasing scientific and commercial interest in EV application in diagnostics, traditional biomolecular techniques usually require consistent sample amount, rely on operator-dependent and time- consuming procedures and cannot cope with the nano-size range of EVs, limiting both sensitivity and reproducibility of results. The application of biophotonics, i.e. light-based methods, for the diagnostic detection of EVs has brought to the development of innovative platforms with excellent sensitivity. In this review, we propose an overview of the most promising and emerging technologies used in the field of EV-related biomarker discovery. When tested on clinical samples, the reported biophotonic approaches in most cases have managed to discriminate between nanovesicles and contaminants, achieved much higher resolution compared to traditional procedures, and reached moderate to excellent diagnostic accuracy, thus demonstrating great potentialities for their clinical translation.

The pandemic of COVID-19 is continuously spreading, becoming a worldwide emergency. Early and fast identification of subjects with a current or past infection must be achieved to slow down the epidemiological widening. Here we report a Raman-based approach for the analysis of saliva, able to significantly discriminate the signal of patients with a current infection by COVID-19 from healthy subjects and/or subjects with a past infection. Our results demonstrated the differences in saliva biochemical composition of the three experimental groups, with modifications grouped in specific attributable spectral regions. The Raman-based classification model was able to discriminate the signal collected from COVID-19 patients with accuracy, precision, sensitivity and specificity of more than 95%. In order to translate this discrimination from the signal-level to the patient-level, we developed a Deep Learning model obtaining accuracy in the range 89–92%. These findings have implications for the creation of a potential Raman-based diagnostic tool, using saliva as minimal invasive and highly informative biofluid, demonstrating the efficacy of the classification model.

Heterochronic blood exchange (HBE) has demonstrated that circulating factors restore youthful features to aged tissues. However, the systemic mediators of those rejuvenating effects remain poorly defined. We show here that the beneficial effect of young blood on aged muscle regeneration was diminished when serum was depleted of extracellular vesicles (EVs). Whereas EVs from young animals rejuvenate aged cell bioenergetics and skeletal muscle regeneration, aging shifts EV subpopulation heterogeneity and compromises downstream benefits on recipient cells. Machine learning classifiers revealed that aging shifts the nucleic acid, but not protein, fingerprint of circulating EVs. Alterations in sub-population heterogeneity were accompanied by declines in transcript levels of the pro-longevity protein, α-Klotho, and injection of EVs improved muscle regeneration in a Klotho mRNA-dependent manner. These studies demonstrate that EVs play a key role in the rejuvenating effects of HBE and that Klotho transcripts within EVs phenocopy the effects of young serum on aged skeletal muscle.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease leading to progressive and irreversible muscle atrophy. The diagnosis of ALS is time-consuming and complex, with the clinical and neurophysiological evaluation accompanied by monitoring of progression and a long procedure for the discrimination of similar neurodegenerative diseases. The delayed diagnosis strongly slows the potential development of adequate therapies and the time frame for a prompt intervention. The discovery of new biomarkers could improve the disease diagnosis, as well as the therapeutic and rehabilitative effectiveness and monitoring of the pathological progression. In this work saliva collected from 19 patients with ALS, 10 affected by Parkinson’s disease, 10 affected by Alzheimer’s disease and 10 healthy subjects, was analysed using Raman spectroscopy, optimizing the parameters for detailed and reproducible spectra. The statistical multivariate analysis of the data revealed a significant difference between the groups, allowing the discrimination of the disease onset. Correlation of Raman data revealed a direct relationship with paraclinical scores, identifying multifactorial biochemical modifications related to the pathology. The proposed approach showed a promising accuracy in ALS onset discrimination, using a fast and sensitive procedure that can make more efficient the diagnostic procedure and the monitoring of therapeutic and rehabilitative processes in ALS.

Simple Summary Patients that survive after a stroke event may present disabilities that can persist for a long time or permanently after it. Clinical intervention with pharmacological and rehabilitation therapies must follow the correct timing and dosing to guarantee optimal recovery of the patients. Extracellular vesicles are nanometric cell products that can be detected in body fluids such as blood and urine; their use as biomarkers for the personalization of stroke therapy and rehabilitation (rehabilomics) might help clinicians and patients to reach the optimal recovery and ameliorate patient quality of life. Moreover, extracellular vesicles released by cells such as stem cells or other human cells are under investigation for their possible regenerative role that could be coupled to standard therapies to stimulate brain remodeling and ameliorate the recovery after stroke. In this review we describe some of the most recent advancements in the field and discuss the potentialities of extracellular vesicles in brain regeneration and rehabilitation after stroke. Abstract Patients that survive after a stroke event may present disabilities that can persist for a long time or permanently after it. If stroke prevention fails, the prompt and combinatorial intervention with pharmacological and rehabilitation therapy is pivotal for the optimal recovery of patients and the reduction of disabilities. In the present review, we summarize some key features of the complex events that occur in the brain during and after the stroke event, with a special focus on extracellular vesicles (EVs) and their role as both carriers of biomarkers and potential therapeutics. EVs have already demonstrated their ability to be used for diagnostic purposes for multiple brain disorders and could represent valuable tools to track the regenerative and inflammatory processes occurring in the injured brain after stroke. Last, but not least, the use of artificial or stem cell-derived EVs were proved to be effective in stimulating brain remodeling and ameliorating recovery after stroke. Still, effective biomarkers of recovery are needed to design robust trials for the validation of innovative therapeutic strategies, such as regenerative rehabilitation approaches.

Chronic Obstructive Pulmonary Disease (COPD) is a debilitating pathology characterized by reduced lung function, breathlessness and rapid and unrelenting decrease in quality of life. The severity rate and the therapy selection are strictly dependent on various parameters verifiable after years of clinical observations, missing a direct biomarker associated with COPD. In this work, we report the methodological application of Surface Enhanced Raman Spectroscopy combined with Multivariate statistics for the analysis of saliva samples collected from 15 patients affected by COPD and 15 related healthy subjects in a pilot study. The comparative Raman analysis allowed to determine a specific signature of the pathological saliva, highlighting differences in determined biological species, already studied and characterized in COPD onset, compared to the Raman signature of healthy samples. The unsupervised principal component analysis and hierarchical clustering revealed a sharp data dispersion between the two experimental groups. Using the linear discriminant analysis, we created a classification model able to discriminate the collected signals with accuracies, specificities, and sensitivities of more than 98%. The results of this preliminary study are promising for further applications of Raman spectroscopy in the COPD clinical field.

Mechanotransduction is the ability of cells to translate mechanical stimuli into biochemical signals that can ultimately influence gene expression, cell morphology and cell fate. Tenocytes are responsible for tendon mechanical adaptation converting mechanical stimuli imposed during mechanical loading, thus affecting extracellular matrix homeostasis. Since we previously demonstrated that MD-Tissue, an injectable collagen-based medical compound containing swine-derived collagen as the main component, is able to affect tenocyte properties, the aim of this study was to analyze whether the effects triggered by MD-Tissue were based on mechanotransduction-related mechanisms. For this purpose, MD-Tissue was used to coat Petri dishes and cytochalasin B was used to deprive tenocytes of mechanical stimulation mediated by the actin cytoskeleton. Cell morphology, migration, collagen turnover pathways and the expression of key mechanosensors were analyzed by morphological and molecular methods. Our findings confirm that MD-Tissue affects collagen turnover pathways and favors cell migration and show that the MD-Tissue-induced effect represents a mechanical input involving the mechanotransduction machinery. Overall, MD-Tissue, acting as a mechanical scaffold, could represent an effective medical device for a novel therapeutic, regenerative and rehabilitative approach to favor tendon healing in tendinopathies.

One of the main hurdles in the study of Alzheimer's Disease (AD) is the lack of easily accessible and sensitive biomarkers for the diagnosis, the prediction of the disease progression rate and the evaluation of rehabilitative and pharmacological treatments. Extracellular Vesicles (EVs) are nanoscale particles released by body cells, studied as promising biomarkers of AD as they are involved in the onset and progression of the disease. In the strive for a reliable and sensitive method to analyze EVs, we applied our recently developed biosensor based on Surface Plasmon Resonance imaging (SPRi) technology for the identification and profiling of neural EVs populations circulating in the plasma of 10 AD patients and 10 healthy subjects. The SPRi-array was designed to separate simultaneously EVs released by neurons, astrocytes, microglia and oligodendrocytes, and to evaluate the presence and the relative amount of specific surface molecules related to pathological processes including translocator protein (TSPO), β-Amyloid and ganglioside M1. As results, significant variations in the relative amount and cargoes of specific brain-derived populations of EVs were observed comparing EVs coming from AD patients and healthy subjects, finding the main differences in the activation phenotype of microglia EVs, in the lipid moieties on generic EVs and in the β-Amyloid expression on surfaces of neuronal EVs. Besides, the demonstrated correlation of SPRi data with Magnetic Resonance Imaging analysis, provided support for using the SPRi-based biosensor for the evaluation of neurodegeneration detecting and characterizing circulating EVs as peripheral biomarkers for the diagnosis and monitoring of progression and rehabilitation treatments in AD patients.

The growing field of regenerative rehabilitation has great potential to improve clinical outcomes for individuals with disabilities. However, the science to elucidate the specific biological underpinnings of regenerative rehabilitation-based approaches is still in its infancy and critical questions regarding clinical translation and implementation still exist. In a recent roundtable discussion from International Consortium for Regenerative Rehabilitation stakeholders, key challenges to progress in the field were identified. The goal of this article is to summarize those discussions and to initiate a broader discussion among clinicians and scientists across the fields of regenerative medicine and rehabilitation science to ultimately progress regenerative rehabilitation from an emerging field to an established interdisciplinary one. Strategies and case studies from consortium institutions-including interdisciplinary research centers, formalized courses, degree programs, international symposia, and collaborative grants-are presented. We propose that these strategic directions have the potential to engage and train clinical practitioners and basic scientists, transform clinical practice, and, ultimately, optimize patient outcomes.

Alzheimer disease (AD) is the most common form of dementia in the elderly, progressively affecting the cognitive functions with a complex diagnostic procedure that limits the time for a prompt intervention. In this study we optimized a reliable protocol for the analysis of AD patients and healthy subjects' serum using the Surface Enhanced Raman Spectroscopy (SERS), taking into consideration the effect of different variables on the final spectra, analyzed and compared through multivariate analysis and correlated with hippocampus volume. As results, we demonstrated a statistical difference between the spectra collected from the two investigated groups, with an accuracy, precision and specificity of respectively 83%, 86%, and 86%. The correlation of these data with those obtained from MRI, demonstrated a direct correlation between Raman spectra and hippocampus degeneration showing the Raman Spectroscopy (RS) as a potential tool for the monitoring of AD progression and rehabilitation treatments.

Children affected by orofacial disorders mix functional alterations with morphological problems, and suitable techniques should be devised for their analysis. Stereophotogrammetry and 3D-3D facial superimposition have already proven to reliably assess morphological differences even between twin siblings, separating the effect of genetic and environmental factors. However, little information is available about twin babies. We longitudinally analyzed a couple of healthy monozygotic twin sisters aged 6 months to 5 years (height time points). The entire 3D facial models of the two sisters were registered according to the least point-to-point distance, and the relevant RMS (root mean square) distance between the facial models was calculated at each time and compared with reference data recorded from adult twins (Mann-Whitney test, p < 0.05). RMS values in the twin sisters were on average 1.18 ± 0.21 mm, and 1.86 ± 0.53 mm in adults, with a significant difference (p < 0.01). Results showed that twins are more similar in early childhood when environmental factors are supposed to have not influenced facial morphology sufficiently. Additionally, the technique seems adequate to detect even small differences: the faces of the twin sisters were not fully identical. 3D-3D facial superimposition techniques can objectively quantify facial dissimilarity even in monozygotic twins. The method may be applied to the faces of twins discordant for some orofacial and maxillofacial pathology and potentially separate genetic and environmental factors.

Extracellular Vesicles (EVs) are implicated in the spread of pathogenic proteins in a growing number of neurological diseases. Given this, there is rising interest in developing inhibitors of Neutral Sphingomyelinase 2 (nSMase2), an enzyme critical in EV biogenesis. Our group recently discovered phenyl(R)‐(1‐(3‐(3,4‐dimethoxyphenyl)‐2,6‐dimethylimidazo[1,2‐b]pyridazin‐8‐yl)pyrrolidin‐3‐yl)carbamate (PDDC), the first potent, selective, orally-available, and brain-penetrable nSMase2 inhibitor, capable of dose-dependently reducing EVs release in vitro and in vivo. Herein, using multiplexed Surface Plasmon Resonance imaging (SPRi), we evaluated which brain cell-derived EVs were affected by PDDC following acute brain injury. Mice were fed PDDC-containing chow at doses which gave steady PDDC brain exposures exceeding its nSMase2 IC50. Mice were then administered an intra-striatal IL-1β injection and two hours later plasma and brain were collected. IL-1β injection significantly increased striatal nSMase2 activity which was completely normalized by PDDC. Using SPRi, we found that IL-1β-induced injury selectively increased plasma levels of CD171 + and PLP1 + EVs; this EV increase was normalized by PDDC. In contrast, GLAST1 + EVs were unchanged by IL-1β or PDDC. IL-1β injection selectively increased EVs released from activated versus non-activated microglia, indicated by the CD11b+/IB4 + ratio. The increase in EVs from CD11b + microglia was dramatically attenuated with PDDC. Taken together, our data demonstrate that following acute injury, brain nSMase2 activity is elevated. EVs released from neurons, oligodendrocytes, and activated microglial are increased in plasma and inhibition of nSMase2 with PDDC reduced these IL-1β-induced changes implicating nSMase2 inhibition as a therapeutic target for acute brain injury.

Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application.

The use of exosomes for diagnostic and disease monitoring purposes is becoming particularly appealing in biomedical research because of the possibility to study directly in biological fluids some of the features related to the organs from which exosomes originate. A paradigmatic example are brain-derived exosomes that can be found in plasma and used as a direct read-out of the status of the central nervous system (CNS). Inspired by recent remarkable development of plasmonic biosensors, we have designed a surface plasmon resonance imaging (SPRi) assay that, taking advantage of the fact that exosome size perfectly fits within the surface plasmon wave depth, allows the detection of multiple exosome subpopulations of neural origin directly in blood. By use of an array of antibodies, exosomes derived from neurons and oligodendrocytes were isolated and detected with good sensitivity. Subsequently, by injecting a second antibody on the immobilized vesicles, we were able to quantify the amount of CD81 and GM1, membrane components of exosomes, on each subpopulation. In this way, we have been able to demonstrate that they are not homogeneously expressed but exhibit a variable abundance according to the exosome cellular origin. These results confirm the extreme variability of exosome composition and demonstrate how SPRi can provide an effective tool for their characterization. Besides, our work paves the road toward more precise clinical studies on the use of exosomes as potential biomarkers of neurodegenerative diseases.

Here we describe a simple approach for the simultaneous detection of multiple microRNAs (miRNAs) using a single nanostructured reagent as surface plasmon resonance imaging (SPRi) enhancer and without using enzymatic reactions, sequence specific enhancers or multiple enhancing steps as normally reported in similar studies. The strategy involves the preparation and optimisation of neutravidin-coated gold nanospheres (nGNSs) functionalised with a previously biotinylated antibody (Ab) against DNA/RNA hybrids. The Ab guarantees the recognition of any miRNA sequence adsorbed on a surface properly functionalised with different DNA probes; at the same time, gold nanoparticles permit to detect this interaction, thus producing enough SPRi signal even at a low ligand concentration. After a careful optimisation of the nanoenhancer and after its characterisation, the final assay allowed the simultaneous detection of four miRNAs with a limit of detection (LOD) of up to 0.5 pM (equal to 275 attomoles in 500 μL) by performing a single enhancing injection. The proposed strategy shows good signal specificity and permits to discriminate wild-type, single- and triple-mutated sequences much better than non-enhanced SPRi. Finally, the method works properly in complex samples (total RNA extracted from blood) as demonstrated by the detection of four miRNAs potentially related to multiple sclerosis used as case study. This proof-of-concept study confirms that the approach provides the possibility to detect a theoretically unlimited number of miRNAs using a simple protocol and an easily prepared enhancing reagent, and may further facilitate the development of affordable multiplexing miRNA screening for clinical purposes., Research funding was provided by the Italian Ministry of Health within the framework of the European EuroNanoMedII Project (Call 2015) entitled “NanoPlasmiRNA”; DGA-FSE (Diputación General de Aragón–Fondo Social Europeo); Ministerio de Educación, Cultura y Deportes of Spanish Government, FPU grant (FPU014/06249).

Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves.

We present here the surface-enhanced Raman spectroscopic (SERS)-based detection of the Wilm’s tumor gene (WT1) sequence using dye-labeled reporter oligonucleotide and magnetic core @ gold shell nanoparticles. Thiolated single-stranded DNA (ssDNA) complementers of the WT1 sequence were used to functionalize the gold shell with capture oligonucleotides in a facile and fast two-step method. The signal amplification performance of the core @ shell colloidal SERS substrate was tested using malachite green as label dye. The Raman signal enhancing efficacy of the magnetic core @ gold shell nanomaterial was compared with the efficacy of spherical gold particles produced using the conventional citrate reduction method. The core @ shell particles were found to be superior both regarding robustness in SERS and facile separation in a heterogeneous reaction system. The core @ shell particles functionalized with target specific oligonucleotide were able to capture the WT1 target and worked as Raman signal amplifiers in our assay system. The good physicochemical characteristics of these particles and the sensitivity observed in SERS experiments allow us to expect good performance in the further development steps of a novel, fast and reliable spectroscopic method for WT1 detection in minimal residual disease patients.

Parkinson’s disease (PD) is a chronic neurodegenerative disorder, characterized by considerable clinical heterogeneity. Extracellular vesicles (EVs) were proposed as new biomarkers for PD because of their role as vehicles of multiple PD related molecules, but technical limitations exist in their detection and characterization in a clinical environment. We propose herein a Raman based protocol for the label-free analysis of circulating EVs as diagnostic and predictive tool for PD. After purification from serum of PD patients and healthy subjects, EVs were analyzed by Raman spectroscopy demonstrating the feasibility and reproducibility of the proposed biophotonic approach, its moderate accuracy in distinguishing PD patients from controls by their EV profile and the correlation between Raman data and clinical scales. Once validated, the Raman spectroscopy of circulating EVs could represent a reliable, automatable and sensitive method for the stratification of PD patients and for the evaluation of the effectiveness of rehabilitation and pharmacological treatments.

Paper-based SERS active substrates were prepared adsorbing spherical and star-shaped gold nanoparticles on a standard filter paper support. Besides the deposition conditions, morphological parameters of the particles were found to strongly affect the enhancer properties of the substrates. The developed substrate was tested regarding surface homogeneity as well as in the quantitative analysis of malachite green, - a well documented Raman reporter dye - and proved to be capable also to detect the oxidation products of apomorphine, a well-known drug molecule used in Parkinson's disease. This material is simple to prepare, easy to handle and dispose and as such it could be a perfect target for further development of a new family of mass-produced, cheap solid SERS substrates. © 2013 Elsevier B.V.

The detection of biomarkers by means of Surface Enhanced Raman Spectroscopy (SERS) is foreseen to became a very important tool in the clinical practice because of its excellent sensitivity and potential for the simultaneous detection of multiple biomarkers. In the present paper we describe how it was possible to build a sensor for the detection of genetic biomarkers involved in acute myeloid leukemia. The assay is based on the use of a specifically designed SERS substrate made of a 2D crystal structure of polymeric pillars embedded in a gold layer. This substrate is characterized by good enhancing properties coupled with an excellent homogeneity. The SERS substrate was conjugated with DNA probes complementary to a target sequence and used in a sandwich assay with gold nanoparticles labeled with a second DNA probe and a Raman reporter. The so developed assay allowed the detection of a leukemia biomarker (WT1 gene) and an housekeeping gene with low picomolar sensitivity. At last, we optimized the assay in order to tackle one of the main limitations of SERS based assay: the loss of signal that is observed when the Raman spectra are collected in liquid. Combining a preferential functionalization on the polymeric pillars with a different height of the polymer pillars from the gold layer the assay demonstrated its effectiveness even when measured in buffer.

The early radiation of epidermal reactions can lead to healing of the lesion or radiation necrosis. There is no general agreement for either the prevention and/or treatment of radiation skin response, also as little is known about the immediate phases of this phenomenon. We investigated the early effects exerted by Healing and Wound Emulsion (HWE) on human skin response after ionizing radiation. Epidermal morphology, Heat Shock Protein (HSP) 70, and Transforming Growth Factor-beta1 (TGF-beta1) gene expression were investigated in organotypic human skin cultures undergoing a double dose of gamma-rays (2 Gy). HSP70 gene expression tended to be induced in the HWE group 6 hours after cream administration and was significantly up-regulated after 48 hours, when epidermal morphological alterations were evident. TGF-beta1 seems not affected in cream treated samples. HWE may stimulate skin to mount an early defensive response against damage induced by gamma rays.

Toll-like receptors (TLRs) 4, 5, 7 and 9 belong to a family of proteins that recognize mainly conserved microbial motifs. Though each TLR has a highly specific ability to recognize a particular microbial pattern, recent papers suggest that some ligands are able to affect the expression of different TLRs. In this paper, we have investigated TLR4, 5, 7 and 9 expression, both at mRNA and protein level, following treatment of different intestinal epithelial cell lines with LPS, flagellin, loxiribine, CpG-oligodeoxynucleotide and peptidoglycan, to assess if the different TLR ligands may modulate the expression of the respective TLR and of the unrelated ones. Our results show that a cross-talk exists between TLRs and various ligands, indicating a cross-regulation among these pattern recognition receptors. In particular, TLR4 was generally down-regulated by treatment with ligands other than LPS, while flagellin and unrelated microbial-associated molecular patterns exerted a general stimulatory activity as regards TLR5 expression. Concerning TLR7 and 9, we have observed a more variable behaviour of the various cell lines with the different ligands. Together, our results demonstrate that the expression of TLRs in intestinal cells is highly dynamic and tightly regulated in response to encountered microbial stimuli.

Casein phosphopeptides (CPPs) form aggregated complexes with calcium phosphate and induce Ca2+ influx into HT-29 cells that have been shown to be differentiated in culture. The relationship between the aggregation of CPPs assessed by laser light scattering and their biological effect was studied using the CPPs beta-CN(1-25)4P and alpha(s1)-CN(59-79)5P, the commercial mixture CPP DMV, the 'cluster sequence' pentapeptide, typical of CPPs, and dephosphorylated beta-CN(1-25)4P, [beta-CN(1-25)0P]. The biological effect was found to be: (a) maximal with beta-CN(1-25)4P and null with the 'cluster sequence'; (b) independent of the presence of inorganic phosphate; and (c) maximal at 4 mmol.L(-1) Ca2+. The aggregation of CPP had the following features: (a) rapid occurrence; (b) maximal aggregation by beta-CN(1-25)4P with aggregates of 60 nm hydrodynamic radius; (c) need for the concomitant presence of Ca2+ and CPP for optimal aggregation; (d) lower aggregation in Ca2+-free Krebs/Ringer/Hepes; (e) formation of bigger aggregates (150 nm radius) with beta-CN(1-25)0P. With both beta-CN(1-25)4P and CPP DMV, the maximum biological activity and degree of aggregation were reached at 4 mmol.L(-1) Ca2+.

In this article, we describe how it is possible to tune the size and the aspect ratio of gold nanorods obtained using a highly efficient protocol based on the use of hydroquinone as a reducing agent by varying the amounts of CTAB and silver ions present in the “seed-growth” solution. Our approach not only allows us to prepare nanorods with a four times increased Au3+ reduction yield, when compared with the commonly used protocol based on ascorbic acid, but also allows a remarkable reduction of 50–60 % of the amount of CTAB needed. In fact, according to our findings, the concentration of CTAB present in the seed-growth solution do not linearly influence the final aspect ratio of the obtained nanorods, and an optimal concentration range between 30 and 50 mM has been identified as the one that is able to generate particles with more elongated shapes. On the optimized protocol, the effect of the concentration of Ag+ ions in the seed-growth solution and the stability of the obtained particles has also been investigated.

Surface-enhanced Raman spectroscopy (SERS) widely improves the sensitivity of traditional Raman analysis, thus allowing this technique to be exploited for the development of new bio-analytical tests. In this work, 3D substrates made of zinc oxide tetrapods (ZnOTP) are decorated with branched gold nanoparticles by means of a new photochemical approach. The SERS enhancing properties of the obtained substrate are tested using different Raman dyes and apomorphine, a drug used for the management of Parkinson disease. The results prove that the enhancing properties depend on the shape of the gold nanoparticles grown on the branches of ZnO tetrapods. The optimized substrate here developed is characterized by an enhancing factor up to 7 x 10(6) and a detection limit for apomorphine of 1 mM. Finally, the new substrates are tested to study single cancer cells showing enhanced Raman signals related to the portion of the cell interacting with the 3D structure of the substrate.

Summary Background Skin reaction is the most common side-effect of radiation therapy. Radiation-induced dermal fibrosis has been characterized histologically, but little is known about the epidermis overlying fibronecrotic lesions. Objectives To characterize the epidermal response 24 h after a single clinically relevant dose of γ-rays in cultured human breast skin. Methods Biopsies obtained from cosmetic surgery (n = 7) were placed epidermis upwards in a Transwell system, and were exposed to a single dose of γ-irradiation (2 Gy). A parallel set of nonirradiated skin fragments was incubated under the same conditions. Both irradiated and nonirradiated fragments were harvested 24 h after irradiation and processed for light microscopy and molecular biology analysis. A quantitative analysis of cell proliferation was performed after 5-bromo-2′-deoxyuridine incorporation. Cytokeratin 10 (CK10) and desmocollin 1 (Dsc1) expression was evaluated by immunofluorescence. Dsc1 and transforming growth factor (TGF)-β1 gene expression was measured by reverse transcriptase–polymerase chain reaction analysis. Results The mean percentage inhibition of epidermal proliferation in irradiated samples was 53·7% (P

Tumor Necrosis Factor-α (TNF-α) plays a pivotal role in psoriasis, an immuno-mediated and genetic skin disease. Anti-TNF-α inhibitors, such as etanercept, are widely used in clinical practice. By immunofluorescence, we investigated the expression of junctional transmembrane proteins in desmosomes (desmocollin-1, Dsc1; desmoglein-1, Dsg1), adherens junctions (E-cadherin), tight junctions (occludin), biomarkers of keratinocyte differentiation (keratin-10, K10; keratin-14, K14; keratin-16, K16; involucrin), epithelial proliferation and apoptosis in psoriatic skin before/after etanercept treatment (n = 5) and in control skin samples (n = 5). Occludin, K14, K16 and involucrin expressions were altered in psoriatic epidermis, while Dsc1, Dsg1, E-cadherin and K10 localisations were comparable to controls. Etanercept promoted the restoration of the physiological condition as suggested by a more differentiated keratinocyte phenotype and a reduced epidermal proliferation rate.

Surface Enhanced Raman Spectroscopy is a powerful analytical technique that combines the excellent chemical specificity of Raman spectroscopy with the good sensitivity provided by the enhancement of the signal observed when a molecule is located on (or very close to) the surface of suitable nanostructured metallic materials. The availability of cheap, reliable and easy to use SERS substrates would pave the road to the development of bioanalytical tests that can be used in clinical practice. SERS, in fact, is expected to provide not only higher sensitivity and specificity, but also the simultaneous and markedly improved detection of several targets at the same time with higher speed compared to the conventional analytical methods. Here, we present the SERS activity of 2-D plasmonic crystals made by polymeric pillars embedded in a gold matrix obtained through the combination of soft-lithography and plasma deposition techniques on a transparent substrates. The use of a transparent support material allowed us to perform SERS detection from support side opening the possibility to use these substrates in combination with microfluidic devices. In order to demonstrate the potentialities for bioanalytical applications, we used our SERS active gold surface to detect the oxidation product of apomorphine, a well-known drug molecule used in Parkinson’s disease which has been demonstrated being difficult to study by traditional HPLC based approaches.

Surface Enhanced Raman Spectroscopy (SERS) is a popular method in bio-analytical chemistry and a potentially powerful enabling technology for in vitro diagnostics. SERS combines the excellent chemical specificity of Raman spectroscopy with the good sensitivity provided by enhancement of the signal that is observed when a molecule is located on (or very close to) the surface of nanostructured metallic materials. Star-like gold nanoparticles (SGN) are a new class of multibranched nanoparticles that in the last few years have attracted the attention of SERS community for their plasmonic properties. In this work we present a new method to prepare star-like gold nanoparticles with a simple one step protocol at room temperature using hydroquinone as reducing agent. Besides we compare the enhancement of Raman signal of malachite green, a dye commonly employed as label in biological studies, by star-like gold nanoparticles having different size, directly in liquid. This study shows that SGN provide good enhancement of Raman signal and that the effect of their dimension is strongly dependent on the wavelength used. Moreover preliminary results suggest that SGN produced using this method are characterized by good physical-chemical properties and they can be functionalized using the standard thiol chemistry. Overall, these results suggest that star-like gold nanoparticles produced through this method could be used for the further development of highly specific and sensitive SERS-based bio-analytical tests. © 2013 Copyright SPIE.

Monitoring biosignals, such as in electrocardiography (ECG), electromiography (EMG) and electroencephalography (EEG), is important for a better understanding of the pathological and physiological conditions of human subjects. In clinical practice the recording of biopotentials is carried out in general with wet electrodes, considered as the golden standard, although they have shown some limits: (i) the susceptibility to motions artifacts, critical aspect in ECG and EMG monitoring, (ii) the time-consuming procedure to be applied in EEG examinations, and (iii) the inability to record biosignals in long-term applications. In the past years innovative solutions have been suggested, among which microstructured dry electrodes showed very promising features. This study represents a first technological assessment of a novel type of microneedles-based dry electrodes. In this paper it has been proved that these electrodes: (i) seem to allow a better electro-mechanical interface with human skin, (ii) have performance comparable to wet electrodes in recording EEG, EMG and static ECG signals, showing an improvement in the monitoring of ECG signal in dynamic conditions, and (iii) do not need the long-lasting skin preparation as wet electrodes for EEG applications, appearing easy to use and to apply. In conclusion, our microneedles based dry electrodes seem to be a promising alternative to standard wet electrodes for the recording of biosignals in clinical examinations.

The present work reports on Raman and Surface Enhanced Raman Scattering (SERS) vibrational fingerprints of β-carotene and haemoglobin in fresh whole blood (i.e. right after blood test) with different laser excitations, i.e. visible (514 nm) and near-infrared (NIR, 785 nm). The use of colloidal silver nanoparticles significantly increases the Raman signal, thus providing a clear SERS spectrum of blood. The collected spectra have been examined and marker bands of β-carotene and of the haem prosthetic group of haemoglobin have been found. In particular, the fundamental features of β-carotene (514 nm excitation), blood proteins and haem molecules (785 nm excitation) were recognized and assigned. Moreover haemoglobin SERS signals can be identified and related with its oxygenation state (oxy-haemoglobin). The data reported show the prospects of Raman and SERS techniques to detect important bio-molecules in a whole blood sample with no pre-treatment.