Every biomedical imaging technique exploitsdifferent physical principles and can provide peculiarinformation, which is often unachievable with differenttechniques and can be further enhanced by the employment ofsuitable contrast agents (CAs). However, each imaging techniquetypically requires its own specific CAs, with correspondingincrements of procedure duration, costs and invasiveness for thepatients, who should undergo two injections. In the last years,great effort has been addressed toward the development ofmultimodal CAs that can be real-time detected by differenttechniques. In this context, we developed a new type of bimodalnanoparticles (NPs), consisting of silica nanospheres (NSs)covered by an outer shell of smaller superparamagnetic NPs, tobe used as dual-mode imaging CAs for ultrasound and magneticresonance imaging techniques. Aim of the present study was toevaluate the echographic detectability of these bimodal NPsthrough a recently developed algorithm that was originallyimplemented to detect pure silica NSs. In particular, weperformed a series of "in vitro" experiments on custom-designedtissue-mimicking phantoms, focused on a specific objective ofdirect clinical interest: the detection of multimodal NPs with adiameter of about 330 nm at a low and biocompatible volumeconcentration (0.2%). The obtained results demonstrated thepossibility of deleting the US echoes coming from structuresother than NPs with high effectiveness, therefore enhancing thebrightness of nanosized contrast agents in the final diagnosticimages. The effectiveness of the proposed method shows verypromising perspectives for future clinical applications.

We investigated the possible clinical feasibility and accuracy of an innovative ultrasound (US) method for diagnosis of osteoporosis of the spine. A total of 342 female patients (aged 51-60 y) underwent spinal dual X-ray absorptiometry and abdominal echographic scanning of the lumbar spine. Recruited patients were subdivided into a reference database used for US spectral model construction and a study population for repeatability and accuracy evaluation. US images and radiofrequency signals were analyzed via a new fully automatic algorithm that performed a series of spectral and statistical analyses, providing a novel diagnostic parameter called the osteoporosis score (O.S.). If dual X-ray absorptiometry is assumed to be the gold standard reference, the accuracy of O.S.-based diagnoses was 91.1%, with k = 0.859 (p < 0.0001). Significant correlations were also found between O.S.-estimated bone mineral densities and corresponding dual X-ray absorptiometry values, with r(2) values up to 0.73 and a root mean square error of 6.3%-9.3%. The results obtained suggest that the proposed method has the potential for future routine application in US-based diagnosis of osteoporosis. (C) 2015 World Federation for Ultrasound in Medicine & Biology.

Aim of this work was to investigate the automatic echographic detection of an experimentaldrug delivery agent, halloysite clay nanotubes (HNTs), by employing an innovative methodbased on advanced spectral analysis of the corresponding "raw" radiofrequency backscattersignals. Different HNT concentrations in a low range (5.5-66  1010 part/mL, equivalent to0.25-3.00 mg/mL) were dispersed in custom-designed tissue-mimicking phantoms and imagedthrough a clinically-available echographic device at a conventional ultrasound diagnostic frequency(10 MHz). The most effective response (sensitivity = 60%, specificity = 95%), was found ata concentration of 33  1010 part/mL (1.5 mg/mL), representing a kind of best compromisebetween the need of enough particles to introduce detectable spectral modifications in thebackscattered signal and the necessity to avoid the losses of spectral peculiarity associated tohigher HNT concentrations. Based on theoretical considerations and quantitative comparisonswith literature-available results, this concentration could also represent an optimal concentrationlevel for the automatic echographic detection of different solid nanoparticles when employinga similar ultrasound frequency. Future dedicated studies will assess the actual clinical usefulness ofthe proposed approach and the potential of HNTs for effective theranostic applications.

Since the recognition of disease molecular basis, it has become clear that the keystone moments of medical practice, namely early diagnosis, appropriate therapeutic treatment and patient follow-up, must be approached at a molecular level. These objectives will be in the near future more effectively achievable thanks to the impressive developments in nanotechnologies and their applications to the biomedical field, starting-up the nanomedicine era. The continuous advances in the development of biocompatible smart nanomaterials, in particular, will be crucial in several aspects of medicine. In fact, the possibility of manufacturing nanoparticle contrast agents that can be selectively targeted to specific pathological cells has extended molecular imaging applications to non-ionizing techniques and, at the same time, has made reachable the perspective of combining highly accurate diagnoses and personalized therapies in a single theranostic intervention. Main developing applications of nanosized theranostic agents include targeted molecular imaging, controlled drug release, therapeutic monitoring, guidance of radiation-based treatments and surgical interventions. Here we will review the most recent findings in nanoparticles contrast agents and their applications in the field of cancer molecular imaging employing non-ionizing techniques and disease-specific contrast agents, with special focus on recent findings on those nanomaterials particularly promising for ultrasound molecular imaging and simultaneous treatment of cancer.

Mitochondrial dysfunction and oxidative stress occur in Parkinson's disease (PD), but the molecular mechanisms controlling these events are not completely understood. Peroxisome proliferator-activated receptor-gamma coactivator-1? (PGC-1?) is a transcriptional coactivator known as master regulator of mitochondrial functions and oxidative metabolism. Recent studies, including one from our group, have highlighted altered PGC-1? activity and transcriptional deregulation of its target genes in PD pathogenesis suggesting it as a new potential therapeutic target. Resveratrol, a natural polyphenolic compound proved to improve mitochondrial activity through the activation of several metabolic sensors resulting in PGC-1? activation. Here we have tested in vitro the effect of resveratrol treatment on primary fibroblast cultures from two patients with early-onset PD linked to different Park2 mutations. We show that resveratrol regulates energy homeostasis through activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) and raise of mRNA expression of a number of PGC-1?'s target genes resulting in enhanced mitochondrial oxidative function, likely related to a decrease of oxidative stress and to an increase of mitochondrial biogenesis. The functional impact of resveratrol treatment encompassed an increase of complex I and citrate synthase activities, basal oxygen consumption, and mitochondrial ATP production and a decrease in lactate content, thus supporting a switch from glycolytic to oxidative metabolism. Moreover, resveratrol treatment caused an enhanced macro-autophagic flux through activation of an LC3-independent pathway. Our results, obtained in early-onset PD fibroblasts, suggest that resveratrol may have potential clinical application in selected cases of PD-affected patients. © 2014 Elsevier B.V.

Among the various nanosized particles developed forinnovative biomedical applications, like selective molecularimaging and targeted drug delivery, silica nanoparticles (SiNPs)seem to be particularly attractive since of their low cost, lowtoxicity, ease of functionalization and acoustic properties. In fact,SiNPs have been demonstrated to effectively enhance ultrasoundcontrast at clinical diagnostic frequencies and, therefore, theymight be potentially employed in non-ionizing echographicmolecular imaging. Aim of this work was the development of asilica nanoparticle based system for in vitro molecular imaging ofhepatocellular carcinoma, using both ultrasound and laserscanningconfocal microscopy, by exploiting the particularfeature of these tumor cells to express on their surface high levelsof Glypican-3 protein (GPC-3). At this regard, we have designedand characterized novel GPC-3 ligand peptide-functionalizedfluorescent silica nanoparticles and tested them on GPC-3positive HepG2 cells, a human hepatocarcinoma cell line. Laserscanning confocal microscopy analysis showed that GPC-3-targeted fuorescent SiNP, in the concentration range used forexperimental ultrasound detection, did not exert significantcytotoxic effects and were effectively bound and taken up byHepG2 cells. These results suggest that silica nanoparticles mightbe a very promising contrast agents for non-ionizing ultrasoundmolecular imaging since of their high biocompatibility, targetingeffectiveness and ultrasound enhancement power.

Halloysite Clay nanotubes (HNTs) are naturallyoccurring nanomaterials composed of double layeredaluminosilicate minerals with a hollow tubular structure. Due totheir interesting structural characteristics, chemically activeexternal and internal surfaces, cheap and abundant availability,HNTs have recently become the subject of research attention as anew type of material for various biological applications, includingdrug and gene delivery vehicles, cancer cells isolation, boneimplants, ultrasound contrast agents, cancer and stem cellsisolation and cosmetics. Therefore, assessment of HNTbiocompatibility has gained importance to demonstrate itssuitability for clinical purposes. In this study, HNTs were denselycoated with poly(ethylene glycol) (PEG) and MTT measurementswere carried out on two different human cancer cell lines, namelyHeLa (cervical cancer) cells and HepG2 (hepatocarcinoma) cells,to quantify the biocompatibility of PEG-coated HNTs as afunction of nanotube dosage and incubation time. While noncoatednanotubes exhibited significant concentration- and timedependenttoxicity, PEG-coated HNTs resulted fullybiocompatible for concentrations up to 0.5 mg/mL and forincubation time up to 72 h, making them suitable candidates fornanomedicine applications.

Osteoporosis is a silent disease without any evidenceof disease until a fracture occurs. Approximately 200million people in the world are affected by osteoporosisand 8.9 million fractures occur each year worldwide.Fractures of the hip are a major public health burden,by means of both social cost and health condition ofthe elderly because these fractures are one of the maincauses of morbidity, impairment, decreased quality oflife and mortality in women and men. The aim of thisreview is to analyze the most important factors relatedto the enormous impact of osteoporotic fractures onpopulation. Among the most common risk factors, lowbody mass index; history of fragility fracture, environmentalrisk, early menopause, smoking, lack of vitaminD, endocrine disorders (for example insulin-dependentdiabetes mellitus), use of glucocorticoids, excessivealcohol intake, immobility and others represented themain clinical risk factors associated with augmentedrisk of fragility fracture. The increasing trend of osteoporosisis accompanied by an underutilization of theavailable preventive strategies and only a small numberof patients at high fracture risk are recognized andsuccessively referred for therapy. This report providesanalytic evidences to assess the best practices inosteoporosis management and indications for theadoption of a correct healthcare strategy to significantlyreduce the osteoporosis burden. Early diagnosis is thekey to resize the impact of osteoporosis on healthcaresystem. In this context, attention must be focused onthe identification of high fracture risk among osteoporoticpatients. It is necessary to increase nationalawareness campaigns across countries in order toreduce the osteoporotic fractures incidence.

Effective prevention and management of osteoporosis would require suitable methods for population screenings and early diagnosis. Current clinically-available diagnostic methods are mainly based on the use of either X-rays or ultrasound (US). All X-ray based methods provide a measure of bone mineral density (BMD), but it has been demonstrated that other structural aspects of the bone are important in determining fracture risk, such as mechanical features and elastic properties, which cannot be assessed using densitometric techniques. Among the most commonly used techniques, dual X-ray absorptiometry (DXA) is considered the current "gold standard" for osteoporosis diagnosis and fracture risk prediction. Unfortunately, as other X-ray based techniques, DXA has specific limitations (e.g., use of ionizing radiation, large size of the equipment, high costs, limited availability) that hinder its application for population screenings and primary care diagnosis. This has resulted in an increasing interest in developing reliable pre-screening tools for osteoporosis such as quantitative ultrasound (QUS) scanners, which do not involve ionizing radiation exposure and represent a cheaper solution exploiting portable and widely available devices. Furthermore, the usefulness of QUS techniques in fracture risk prediction has been proven and, with the last developments, they are also becoming a more and more reliable approach for assessing bone quality. However, the US assessment of osteoporosis is currently used only as a pre-screening tool, requiring a subsequent diagnosis confirmation by means of a DXA evaluation. Here we illustrate the state of art in the early diagnosis of this "silent disease" and show up recent advances for its prevention and improved management through early diagnosis.

Fetal malformations are very frequent in industrialized countries. Although advanced maternal age may affect pregnancy outcome adversely, 80%-90% of fetal malformations occur in the absence of a specific risk factor for parents. The only effective approach for prenatal screening is currently represented by an ultrasound scan. However, ultrasound methods present two important limitations: the substantial absence of quantitative parameters and the dependence on the sonographer experience. In recent years, together with the improvement in transducer technology, quantitative and objective sonographic markers highly predictive of fetal malformations have been developed. These markers can be detected at early gestation (11-14 wk) and generally are not pathological in themselves but have an increased incidence in abnormal fetuses. Thus, prenatal ultrasonography during the second trimester of gestation provides a "genetic sonogram", including, for instance, nuchal translucency, short humeral length, echogenic bowel, echogenic intracardiac focus and choroid plexus cyst, that is used to identify morphological features of fetal Down's syndrome with a potential sensitivity of more than 90%. Other specific and sensitive markers can be seen in the case of cardiac defects and skeletal anomalies. In the future, sonographic markers could limit even more the use of invasive and dangerous techniques of prenatal diagnosis (amniocentesis, etc.).

Halloysite clay nanotubes (HNTs) are natural materials with a characteristic hollow tubular structure in the nanometer range. Owing to this feature, they were found to be a suitable nanosized container for the loading of biologically active molecules like biocides and drugs. Also, HNTs have been reported to be of potential interest for other biological applications, such as gene delivery carriers, ultrasound contrast agents, cancer therapy and stem cells isolation. Therefore, biocompatibility of halloysite represents one the main requisites for the employment of HNTs for clinical purposes. Here we present a study aimed at assessing HNTs biocompatibility before and after their surface coating with poly(ethylene glycol) (PEG), a polymer which has been reported to increase biocompatibility, to prolong circulation time and to prevent protein adsorption and aggregation in biological environments. The dose- and time-dependent cytotoxicity of noncoated and PEG-coated HNTs obtained was evaluated in vitro by MTT cell viability assay carried out on both HeLa and HepG2 cells, two different human cancer cell lines. Binding and uptake of nanotubes were also analyzed at ultrastructural level by transmission electron microscopy (TEM). Interestingly, the results obtained showed that both the HNTs tested were actively taken up by the cells but, while noncoated nanotubes exhibited significant concentration- and time-dependent toxicity, PEG-coated HNTs were found to be highly biocompatible, being then suitable candidates for biomedical applications.

Aim of this work was to investigate the effect ofultrasound incident frequency on the echographic contrastenhancement power of an experimental drug delivery agent,halloysite clay nanotubes (HNTs), and to determine a suitableconfiguration in terms of both insonification frequency andparticle concentration for an effective employment as targetedcontrast agent. Various HNT concentrations (range 0.25-3.00mg/mL) were dispersed in custom-designed tissue-mimickingphantoms and exposed to different ultrasound frequencies (7-11MHz) through a conventional clinically-available echographicdevice. Off-line analysis included the evaluation of bothamplitude of backscattered ultrasound signals and imagebrightness. Amplitude of HNT-backscattered signals showed alinear increase with particle concentration, while imagebrightness enhancement was limited by logarithmic compressioneffects. On the other hand, backscatter amplitude showedsignificant increments with increasing ultrasound frequency upto 10 MHz, then showing a concentration-dependent behaviorwithout further enhancements. Overall, the most effectiveresponse was found when a 10-MHz ultrasound frequency wasemployed to insonify HNTs at a concentration of 1.5 mg/mL. Inconclusion, the present study optimized the combination ofincident ultrasound frequency and HNT concentration, in orderto obtain an echographic image enhancement suitable for medicalapplications. Future dedicated studies will assess the feasibility ofautomatic detection of HNTs within echographic images andtheir possible employment as theranostic agents.