Nutraceutics is a growing research field in which researchers study and attempt to improve the biological properties of metabolites in food. Wine is one of the most consumed products in the world and contains a plethora of molecules biologically relevant to human health. In this article, several polyphenols with potential antioxidant activity were measured in wines from Apulia, in Southeast Italy. Hydroxytyrosol, gallic and syringic acids, luteolin, quercetin, and trans-resveratrol were identified and quantified by HPLC. The amount of the analyzed metabolites in wines were largely dependent on their color, with red ones being the richest compared to white and rose wines. Gallic acid was the most abundant polyphenol, followed by syringic acid and luteolin. Nevertheless, significant amounts of hydroxytyrosol, quercetin, and trans-resveratrol were also found. The average concentration of polyphenols found in these wines could have potential health-promoting effects, especially if consumed in moderate quantities on a regular basis.

Purpose: The aim of this study was to investigate the angiogenic role of the hepatocyte growth factor (HGF)/cMET pathway and its inhibition in bone marrow endothelial cells (EC) from patients with multiple myeloma versus from patients with monoclonal gammopathy of undetermined significance (MGUS) or benign anemia (control group). Experimental Design: The HGF/cMET pathway was evaluated in ECs from patients with multiple myeloma (multiple myeloma ECs) at diagnosis, at relapse after bortezomib- or lenalidomide-based therapies, or on refractory phase to these drugs; in ECs from patients with MGUS (MGECs); and in those patients from the control group. The effects of a selective cMET tyrosine kinase inhibitor (SU11274) on multiple myeloma ECs’ angiogenic activities were studied in vitro and in vivo. Results: Multiple myeloma ECs express more HGF, cMET, and activated cMET (phospho (p)-cMET) at both RNAand protein levels versus MGECs and control ECs. Multiple myeloma ECs are able to maintain the HGF/cMET pathway activation in absence of external stimulation, whereas treatment with anti-HGF and anti-cMET neutralizing antibodies (Ab) is able to inhibit cMET activation. The cMET pathway regulates several multiple myeloma EC activities, including chemotaxis, motility, adhesion, spreading, and whole angiogenesis. Its inhibition by SU11274 impairs these activities in a statistically significant fashion when combined with bortezomib or lenalidomide, both in vitro and in vivo. Conclusions: An autocrine HGF/cMET loop sustains multiple myeloma angiogenesis and represents an appealing new target to potentiate the antiangiogenic management of patients with multiple myeloma.

In the near future, the wireless technologies will play a very important role in healthcare applications, especially for tracing and tracking systems of objects and people. Among these, the Radio Frequency Identification (RFID) solutions promise to substantially improve both many processes in healthcare environments and the patient safety connected to the reduction of errors in drug prescriptions and administration. Unfortunately, there are still some barriers limiting the large-scale deployment of these innovative technologies in the healthcare sector. In order to face these challenges, multidisciplinary skills are required. A recent research project has attempted to coordinate heterogeneous activities focused on the tracing and tracking of drugs at item level on the pharmaceutical supply chain. One of these is related to the evaluation of potential effects of exposure to electromagnetic fields, generated by RFID devices in UHF band, on drugs. This paper aims to introduce some investigative techniques useful to carry out an experimental protocol able to evaluate the potential alterations of the molecular structure of some biological drugs exposed to electromagnetic fields. The work highlighted that the experimental analysis is complex because it depends strongly on particular molecules. Some experimental results on ActrapidTM, a human insulin preparation, are reported. They showed the absence of substantial alterations both in molecular structure and biological activity of ActrapidTM.

Uterine leiomyoma is a benign smooth muscle tumor characterized by a high incidence in women of reproductive age. The aetiology of this tumor is still unknown but established risk factors include high levels of female hormones, family history, African ancestry, early age of menarche and obesity. Here, to identify proteomic features associated with this tumor type, we performed a liquid cromatography-mass spectrometry (LC-MS/MS) analysis of uterine myomas. The identified proteins were subjected to a gene ontology analysis to generate biological functions, molecular processes, and protein networks that were relevant to the uploaded dataset. Pathway-based analysis was an effective approach to investigate the molecular mechanisms underlying the disease and to create biological hypotheses about regulation of our proteins including the identification of upstream regulators and main protein nodes. Moreover, proteomic and in silico data were combined with immunohistochemistry and western blotting to identify a group of proteins representative of some selected pathways, with a dysregulated expression in in myoma, pseudocapsule, and normal myometrium samples. Based on these results, we confirmed the over-expression of extracellular matrix components, and estrogen and progesterone receptors in uterine myomas, and proposed biological networks, canonical pathways and functions that may be relevant to the pathophysiology of this tumor.

Fibroids or myomas involve large proportion of women of reproductive age. The myoma formation starts from the transformation of the myometrium, causing the progressive formation of a pseudocapsule, which is made of compressed muscle fibers. Numerous studies investigated on myoma pseudocapsule anatomy, discovering many neurotransmitters and neuropeptides, as a neurovascular bundle, influencing myometrial physiology. These substances have a positive impact on wound healing and muscular restoring, also playing a role in sexual and reproductive function. Based on investigations, a distinct surgical technique evolved, called "intracapsular myomectomy", meaning myoma removal from its pseudocapsule, which enables protection of the myoma pseudocapsule, containing neuropeptides and neurofibers involved in physiological myometrial healing. This technique, performed by a gentle myoma enucleating by stretching from myometrium and sparing pseudocapsule, reduces surgical trauma caused by iatrogenic myoma pseudocapsule damage. Intracapsular myomectomy meets the basic surgical anatomy principle: myoma is removed by a bloodless, precise and careful dissection sparing myometrium, as much as possible. The rationale of intracapsular myomectomy should be applied to all myoma removals; therefore, it has been used for both laparoscopic and laparotomic myomectomy, as well as for cesarean myomectomy. Scientific research is still seeks to clarify some reports of myomas with infertility, especially in the case of intramural myomas, but it is clear that in the case of performing myomectomy, it must do by the described intracapsular technique. This enables myometrial preservation, especially peripherally to myoma bed, promoting myometrial healing after myoma removal.

Dietary phytochemicals found in vegetables and fruits consist of a wide variety of biologically active compounds with anti-carcinogenic activity. The aim of this study was to evaluate the antigrowth activity of carnosol, a dietary diterpene, as a single agent or in combination with other dietary phytochemicals or chemotherapeutic drugs against a panel of tumor cell lines. Carnosol decreased cell viability in human breast, ovarian, and intestinal tumor cell lines, and inhibited cancer cell adhesion on fibronectin and growth of cancer cells in suspension. Carnosol also inhibited EGF-induced epithelial mesenchymal transition in ovarian cancer cells. The combination treatment with other dietary phytochemicals increased the anti-proliferative activity of carnosol. The combination with curcumin resulted in a synergistic reduction of vitality in SKOV-3 and MDA-231 cells and potently inhibited viability of primary cancer cells isolated from the pleural fluid or ascites of patients with metastatic cancers. These results provide additional evidence about the anticancer role of carnosol and its potential in blocking the growth of tumor cells.

Angiogenesis critically sustains the progression of both physiological and pathological processes. Copper behaves as an obligatory co-factor throughout the angiogenic signalling cascades, so much so that a deficiency causes neovascularization to abate. Moreover, the progress of several angiogenic pathologies (e.g. diabetes, cardiac hypertrophy and ischaemia) can be tracked by measuring serum copper levels, which are being increasingly investigated as a useful prognostic marker. Accordingly, the therapeutic modulation of body copper has been proven effective in rescuing the pathological angiogenic dysfunctions underlying several disease states. Vascular copper transport systems profoundly influence the activation and execution of angiogenesis, acting as multi-functional regulators of apparently discrete pro-angiogenic pathways. This review concerns the complex relationship among copper-dependent angiogenic factors, copper transporters and common pathological conditions, with an unusual accent on the multi-faceted involvement of the proteins handling vascular copper. Functions regulated by the major copper transport proteins (CTR1 importer, ATP7A efflux pump and metallo-chaperones) include the modulation of endothelial migration and vascular superoxide, known to activate angiogenesis within a narrow concentration range. The potential contribution of prion protein, a controversial regulator of copper homeostasis, is discussed, even though its angiogenic involvement seems to be mainly associated with the modulation of endothelial motility and permeability.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune demyelinating disease of the central nervous system. There are four clinical forms of MS, the most common of which is characterized by a relapsing remitting course (RRMS). The etiology of MS is unknown, but many studies suggested that genetic, environmental and infectious agents may contribute to the development of this disease. In experimental autoimmune encephalomyelitis (EAE), the animal model for MS, it has been shown that CD4+ T cells play a key role in MS pathogenesis. In fact, these cells are able to cross the blood-brain barrier and cause axonal damage with neuronal death. T cell activation critically depends on mitochondrial ATP synthesis and reactive oxygen species (ROS) production. Interestingly, lots of studies linked the oxidative damage arising from mitochondrial changes to neurodegenerative disorders, such as MS. Based on these evidences, this work focused on the metabolic reprogramming of CD4+ T cells in MS subjects, being this cell population directly implicated in pathogenesis of disease, paying attention to mitochondrial function and response to oxidative stress. Such aspects, once clarified, may open new opportunities for a therapeutic metabolic modulation of MS disorder.

Classical Hodgkin lymphoma (cHL) is a malignancy with complex pathogenesis. The hallmark of the disease is the presence of large mononucleated Hodgkin and bi- or multinucleated Reed/Sternberg (H/RS) cells. The origin of HRS cells in cHL is controversial as these cells show the coexpression of markers of several lineages. Using a proteomic approach, we compared the protein expression profile of cHL models of T- and B-cell derivation to find proteins differentially expressed in these cell lines. A total of 67 proteins were found differentially expressed between the two cell lines including metabolic proteins and proteins involved in the regulation of the cytoskeleton and/or cell migration, which were further validated by western blotting. Additionally, the expression of selected B- and T-cell antigens was also assessed by flow cytometry to reveal significant differences in the expression of different surface markers. Bioinformatics analysis was then applied to our dataset to find enriched pathways and networks, and to identify possible key regulators. In the present study, a proteomic approach was used to compare the protein expression profiles of two cHL cell lines. The identified proteins and/or networks, many of which not previously related to cHL, may be important to better define the pathogenesis of the disease, to identify novel diagnostic markers, and to design new therapeutic strategies.

The epithelial to mesenchymal transition (EMT) is a cellular program associated with the organ morphogenesis but also with the disease progression. EMT in the cancer field fuels neoplastic progression promoting the resistance to cell death, the resistance to chemotherapy and the acquisition of stem cell properties. Considering the crucial role of EMT in breast cancer metastasis, a better understanding of this process may provide new therapeutic options. Here, by using a proteomic approach we identified a set of proteins differentially expressed between an epithelial and a mesenchymal breast cancer cell line. The protein-protein network of these identified proteins was determined by an in silico analysis highlighting, in the EMT program, the role of proteins involved in cell adhesion, migration, and invasion, together with protein kinases involved in proliferation and survival, with many of these emerging as possible targets of novel biological agents. Finally, the pharmacological inhibition of some of these kinases was able to reverse the mesenchymal phenotype to an epithelial phenotype.

Extra-virgin olive oil (EVOO) is among the basic constituents of the Mediterranean diet. Its nutraceutical properties are due mainly, but not only, to a plethora of molecules with antioxidant activity known as biophenols. In this article, several biophenols were measured in EVOOs from South Apulia, Italy. Hydroxytyrosol, tyrosol and their conjugated structures to elenolic acid in different forms were identified and quantified by high performance liquid chromatography (HPLC) together with lignans, luteolin and α-tocopherol. The concentration of the analyzed metabolites was quite high in all thecultivarsstudied, but it was still possible to discriminate them through multivariate statistical analysis (MVA). Furthermore, principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) were also exploited for determining variances among samples depending on the interval time between harvesting and milling, on the age of the olive trees, and on the area where the olive trees were grown.

Cuprizone is used to obtain demyelination in mice. Cuprizone-treated mice show symptoms similar to several neurodegenerative disorders such as severe status spongiosus. Although it has a simple chemical formula, its neurotoxic mechanism is still unknown. In this work, we examined both physico–chemical properties and biological effects of cuprizone. Our results indicate that cuprizone has very complicated and misunderstood solution chemistry. Moreover, we show here the inability of cuprizone to cross neither the intestinal epithelial barrier nor the neuronal cell membrane, as well its high tolerability by cultured neurons. If added to mice diet, cuprizone does not accumulate in liver or in brain. Therefore, its neurotoxic effect is explainable only in terms of its capability to chelate copper, leading to chronic copper deficiency.

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. Genes which have been implicated in autosomal-recessive PD include PARK2 which codes for parkin, an E3 ubiquitin ligase that participates in a variety of cellular activities. In this study, we compared parkin-mutant primary fibroblasts, from a patient with parkin compound heterozygous mutations, to healthy control cells. Western blot analysis of proteins obtained from patient’s fibroblasts showed quantitative differences of many proteins involved in the cytoskeleton organization with respect to control cells. These molecular alterations are accompanied by changes in the organization of actin stress fibers and biomechanical properties, as revealed by confocal laser scanning microscopy and atomic force microscopy. In particular, parkin deficiency is associated with a significant increase of Young’s modulus of null-cells in comparison to normal fibroblasts. The current study proposes that parkin influences the spatial organization of actin filaments, the shape of human fibroblasts, and their elastic response to an external applied force.

In this review we will overview novel nanotechnological nanocarrier systems for cancer therapy focusing on recent development in polyelectrolyte capsules for targeted delivery of antineoplastic drugs against cancer cells. Biodegradable polyelectrolyte microcapsules (PMCs) are supramolecular assemblies of particular interest for therapeutic purposes, as they can be enzymatically degraded into viable cells, under physiological conditions. Incorporation of small bioactive molecules into nano-to-microscale delivery systems may increase drug's bioavailability and therapeutic efficacy at single cell level giving desirable targeted therapy. Layer-by- layer (LbL) self-assembled PMCs are efficient microcarriers that maximize drug's exposure enhancing antitumor activity of neoplastic drug in cancer cells. They can be envisaged as novel multifunctional carriers for resistant or relapsed patients or for reducing dose escalation in clinical settings

The growing counterfeiting problem and the significant fragmentation of the pharmaceutical market are resulting on an increase of difficulty to trace medicines. In these scenarios, where an item-level traceability is crucial, the Radio Frequency Identification (RFID) technology holds the promise to eliminate many of the previous problems. Unfortunately, there are still some technical and economic barriers that are retarding the deployment of these innovative technologies in large-scale application scenarios. For the pharmaceutical supply chain, there have been concerns raised regarding the potential effects on the quality of drugs due to exposure to electromagnetic fields. In this paper, some results, obtained by a recent experimental study focused on the evaluation of potential effects on biological drugs, have been reported. This work aimed to evaluate potential effects of tracing RFID systems on the molecular structure of biological drugs. In particular, some samples of a commercial human insulin preparation have been exposed for different periods to electromagnetic fields generated by RFID devices. In order to evaluate possible alterations on the molecular structure, the following diagnostic techniques have been used: High Pressure Liquid Chromatography (HPLC) and Nuclear Magnetic Resonance (NMR). The experimental results have shown that the electromagnetic field generated by UHF RFID readers does not cause any damage on the structure of the insulin molecule.

Curcumin is a natural hydrophobic polyphenol found in the powdered rhizomes of Curcuma longa. Due to its capacity to interfere with many signalling pathways, it has been shown that curcumin has potential beneficial pharmacological effects including antioxidant, anti-inflammatory, anticarcinogenic properties. However, the use of curcumin is fairly restricted because of its poor water solubility, low bioavailability, inadequate tissue absorption and degradation at alkaline pH. In the present contribution, we first verified the anti-proliferative effects of natural curcuminoids towards two different cell lines derived from an ovarian and a breast adenocarcinoma cancer. Later, curcuminoids were successfully encapsulated into reconstituted oil bodies. Once encapsulated into the triacylglycerol cores of the reconstituted oil bodies, curcumin, the most hydrophobic and active of the three curcuminoids, was better stabilized in comparison with albumin stabilization. Oil body encapsulated curcuminoids showed the same effects on cancer cell viability as the free drug, confirming the great potential of natural oil bodies as micro/nano-capsules in drug delivery applications.

The application of RFID technologies along the pharmaceutical supply chain represents a guarantee of transparency in the drug flow, against the growing counterfeiting problem. Unfortunately, the large-scale deployment of these solutions is still limited because of concerns over potential deteriorating effects of RF emissions on traced pharmaceuticals. This work aims at delineating an experimental framework to evaluate potential exposure risks and get new insight into poorly explored safety issues. Heterogeneous skills (engineering, chemistry and physiology) have been recruited to draw up a suitable experimental protocol, consisting of three main steps: (i) simulation of drug exposure to electromagnetic fields generated by RFID devices in a test environment reproducing the pharmaceutical supply chain; (ii) investigation of potential drug structural changes by High Pressure Liquid Chromatography (HPLC) techniques; (iii) analysis of performance by in vitro functional assays. An example of application of this protocol on a commercial FSH (Follicle Stimulating Hormone) preparation has been reported, showing no alterations of the hormone integrity following RF exposure. Conclusively, a wide adoption of "trace and track" RFID technologies passes through the assessment of related safety issues. The varying nature of drugs as well as their different biological targets make it a hard challenge, that can be successfully overcome by multidisciplinary approaches.

The Radio Frequency Identification (RFID) technology promises to improve several processes in the healthcare scenario, especially those related to traceability of people and things. Unfortunately, there are still some barriers limiting the large-scale deployment of these innovative technologies in the healthcare field. Among these, the evaluation of potential thermal and non-thermal effects due to the exposure of biopharmaceutical products to electromagnetic fields is very challenging, but still slightly investigated. This paper aims to setup a controlled RF exposure environment, in order to reproduce a worst-case exposure of pharmaceutical products to the electromagnetic fields generated by the UHF RFID devices placed along the supply chain. Radiated powers several times higher than recommended by current normative limits were applied (10 W and 20 W). The electric field strength at the exposed sample location, used in tests, was as high as 100 V/m. Non-thermal effects were evaluated by chromatography techniques and in vitro assays. The results obtained for a particular case study, the ActrapidTM human insulin preparation, showed temperature increases lower than 0.5°C and no significant changes in the structure and performance of the considered drug.

The item-level tracing and tracking requirements are growing more and more in the pharmaceutical sector, where the counterfeiting problem and the significant fragmentation of the market contribute significantly to complicate the scenario. The Radio Frequency Identification (RFID) technology holds the promise to eliminate many of the previous problems. Unfortunately, there are still some barriers limiting the largescale deployment of these innovative technologies. Currently, it is not easy finding an exhaustive analysis about potential effects of exposure to electromagnetic fields of the RFID systems on drugs. This work aimed to evaluate the effects of RFID systems on the molecular structure and potency of a biological drug. In particular, some samples of a commercial human insulin preparation (ActrapidTM) were exposed for different periods to electromagnetic fields generated by RFID devices in UHF band. In order to evaluate both possible alterations of the molecular structure and possible adverse effects on drug performance, the following techniques have been used: Reverse Phase-High Pressure Liquid Chromatography and in vitro cell proliferation assays. The experimental results have shown that the electromagnetic field generated by UHF RFID devices does not cause significant biological effects on ActrapidTM insulin.

Bortezomib (bort) has improved overall survival in patients with multiple myeloma (MM), but the majority of them develop drug resistance. In this study, we demonstrate that bone marrow (BM) fibroblasts (cancer-associated fibroblasts; CAFs) from bort-resistant patients are insensitive to bort and protect the RPMI8226 and patients' plasma cells against bort-induced apoptosis. Bort triggers CAFs to produce high levels of interleukin (IL)-6, IL-8, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF) β. Proteomic studies on CAFs demonstrate that bort resistance parallels activation of oxidative stress and pro-survival autophagy. Indeed, bort induces reactive oxygen species in bort-resistant CAFs and activates autophagy by increasing light chain 3 protein (LC3)-II and inhibiting p62 and phospho-mammalian target of rapamycin. The small-interfering RNA knockdown of Atg7, and treatment with 3-methyladenine, restores bort sensitivity in bort-resistant CAFs and produces cytotoxicity in plasma cells co-cultured with CAFs. In the syngeneic 5T33 MM model, bort-treatment induces the expansion of LC3-II(+) CAFs. TGFβ mediates bort-induced autophagy, and its blockade by LY2109761, a selective TβRI/II inhibitor, reduces the expression of p-Smad2/3 and LC3-II and induces apoptosis in bort-resistant CAFs. A combination of bort and LY2109761 synergistically induces apoptosis of RPMI8226 co-cultured with bort-resistant CAFs. These data define a key role for CAFs in bort resistance of plasma cells and provide the basis for a novel targeted therapeutic approach.

Astrocytes have a key role in the pathogenesis of several diseases, including multiple sclerosis, and are proposed as a possible target for immunotherapy. Our earlier study reported that astrocytes treated with IFN-β modified their biomechanical properties possibly due to changes in the expression of the proteins involved in cytoskeleton organization and other important physiological processes. To gain insight into the mechanism underlying IFN-β action during inflammation, we stimulated astrocytes with LPS, a bacterial wall component used as a model for both in vitro and in vivo immunological stimulation of microglia and astrocytes. We showed that IFN-β reverses the effects of LPS on the proteome of astrocytes. To better examine this result, we performed a proteomic analysis of astrocytes treated with LPS or LPS plus IFN-β. Treatment with LPS caused increases both in a series of proteins mainly involved in cytoskeletal changes and in protein degradation, as well as protective enzymes like superoxide dismutase. IFN-β reverses LPS effects on astrocyte proteome, supporting its protective role during inflammatory insults.

AIM: The lack of sensitivity of chronic myeloid leukemia (CML) stem cells to imatinib mesylate (IM) commonly leads to drug dose escalation or early disease relapses when therapy is stopped. Here, we report that packaging of IM into a biodegradable carrier based on polyelectrolyte microcapsules increases drug retention and antitumor activity in CML stem cells, also improving the ex vivo purging of malignant progenitors from patient autografts. MATERIALS & METHODS: Microparticles/capsules were obtained by layer-by-layer (LbL) self-assembly of oppositely charged polyelectrolyte multilayers on removable calcium carbonate (CaCO(3)) templates and loaded with or without IM. A leukemic cell line (KU812) and CD34(+) cells freshly isolated from healthy donors or CML patients were tested. RESULTS & DISCUSSION: Polyelectrolyte microcapsules (PMCs) with an average diameter of 3 microm, fluorescently labelled multilayers sensitive to the action of intracellular proteases and 95-99% encapsulation efficiency of IM, were prepared. Cell uptake efficiency of such biodegradable carriers was quantified in KU812, leukemic and normal CD34(+) stem cells (range: 70-85%), and empty PMCs did not impact cell viability. IM-loaded PMCs selectively targeted CML cells, by promoting apoptosis at doses that exert only cytostatic effects by IM alone. More importantly, residual CML cells from patient leukapheresis products were reduced or eliminated more efficiently by using IM-loaded PMCs compared with freely soluble IM, with a purging efficiency of several logs. No adverse effects on normal CD34(+) stem-cell survival and their clonogenic potential was noticed in long-term cultures of hematopoietic progenitors in vitro. CONCLUSION: This pilot study provides the proof-of-principle for the clinical application of biodegradable IM-loaded PMC as feasible, safe and effective ex vivo purging agents to target CML stem cells, in order to improve transplant outcome of resistant/relapsed patients or reduce IM dose escalation.

Proprotein convertases are a family of kexin-like serine proteases that process proteins at single and multiple basic residues. Among the predicted and identified PC substrates, an increasing number of proteins having functions in cancer progression indicate that PCs may be potential targets for antineoplastic drugs. In support of this notion, we identified PACE4 as a vital PC involved in prostate cancer proliferation and progression, contrasting with the other co-expressed PCs. The aim of the present study was to test the importance of PCs in ovarian cancer cell proliferation and tumor progression. Based on tissue-expression profiles, furin, PACE4, PC5/6 and PC7 all displayed increased expression in primary tumor, ascites cells and metastases. These PCs were also expressed in variable levels in three model ovarian cell lines tested, namely SKOV3, CAOV3 and OVCAR3 cells. Since SKOV3 cells closely represented the PC expression profile of ovarian cancer cells, we chose them to test the effects of PC silencing using stable gene-silencing shRNA strategy to generate knockdown SKOV3 cells for each expressed PC. In vitro and in vivo assays confirmed the role of PACE4 in the sustainment of SKOV3 cell proliferation, which was not observed with the other three PCs. We also tested PACE4 peptide inhibitors on all three cell lines and observed consequent reduced cell proliferation which was correlated with PACE4 expression. Overall, these data support a role of PACE4 in promoting cell proliferation in ovarian cancer and provides further evidence for PACE4 as a potential therapeutic target.

The Radio Frequency Identification (RFID) is a wireless technology that is becoming more and more important as auto-identification solution for many application scenarios. The adoption of this innovative technology in the pharmaceutical sector promises to solve several problems related to tracing and tracking systems at item level. Unfortunately, there are still some barriers limiting the large-scale deployment of RFID technologies. One of these is related to very interesting research topics on the evaluation of potential effects of electromagnetic fields on drugs. In detail, this work aimed to analyze the impact of UHF RFID devices, used in tracing systems, on the molecular structure and potency of a commercial human insulin preparation, ActrapidTM. In order to investigate possible induced alterations of molecular structure, the Reverse Phase-High Pressure Liquid Chromatography and the Nuclear Magnetic Resonance spectroscopy have been mainly used in the experimental protocol. To obtain some indications about drug performance, in vitro cell proliferation assays have been also conducted. The experimental results, achieved by a protocol combining an accurate structural analysis on 5 min to 24 h irradiated drug samples with functional in vitro assays, have shown that the electromagnetic field generated by UHF RFID devices does not cause significant effects on ActrapidTM insulin. These findings are strongly encouraging the use of RFID-based technologies for item-level tracing systems in the pharmaceutical supply chain.

Chronic Myelogenous Leukemia (CML)-initiating cells (CICs) express the hybrid oncoprotein BCR-ABL at the highest levels compared to their differentiated progeny but fail to expand at the same rate as downstream leukemic myeloid cells. Moreover, the primitive stem cell clone that originates the indolent CML chronic phase (CP) remains almost invariant as the disease evolves to a fatal blast crisis (BC). Compared to their healthy counterpart, the most dormant BCR-ABL+ CICs show the tendency to remain in a somewhat unusual 'proliferative quiescence', i.e. a prolonged low-energy viable state that restrains the frequency of symmetrical (self-renewing) cell divisions while enforcing cell cycle entry and myeloid commitment under cytokine support. Over the past few years, we and others have proposed the nutrient-sensing protein serine/threonine kinase GSK3β (glycogen synthase kinase 3β) as an attractive target to eradicate leukemia-initiating cells while sparing normal haematopoiesis. Beyond its natural negative effects on self-renewal, through the inhibitory phosphorylation of β-Catenin (Wnt signalling) and c-MYC (Hedgehog signalling), hyperactive GSK3β is reportedly crucial to link energy metabolism and nutrient availability to stem cell homeostasis processes. This review will integrate current evidence pertaining to the biological relevance of GSK3β in normal and malignant haematopoiesis, with particular emphasis on its role(s) at the CML CP stage and BC transformation. Preclinical evidence earmarking the use of novel small-molecule inhibitors of GSK3β as effective anti-leukemia agents are also discussed.

The sonication-assisted layer-by-layer (SLBL) technology was developed to combine necessary factors for an efficient drug-delivery system: (i) control of nanocolloid size within 100 - 300 nm, (ii) high drug content (70% wt), (iii) shell biocompatibility and biodegradability, (iv) sustained controlled release, and (v) multidrug-loaded system. Stable nanocolloids of Paclitaxel (PTX) and lapatinib were prepared by the SLBL method. In a multidrug-resistant (MDR) ovarian cancer cell line, OVCAR-3, lapatinib/PTX nanocolloids mediated an enhanced cell growth inhibition in comparison with the PTX-only treatment. A series of in vitro cell assays were used to test the efficacy of these formulations. The small size and functional versatility of these nanoparticles, combined with their ability to incorporate various drugs, indicates that lapatinib/PTX nanocolloids may have in vivo therapeutic applications.

Purpose: To determine the in vivo and in vitro antiangiogenic power of lenalidomide, a "lead compound" of IMiD immunomodulatory drugs in bone marrow (BM) endothelial cells (EC) of patients with multiple myeloma (MM) in active phase (MMEC). Experimental Design: The antiangiogenic effect in vivo was studied using the chorioallantoic membrane (CAM) assay. Functional studies in vitro (angiogenesis, "wound" healing and chemotaxis, cell viability, adhesion, and apoptosis) were conducted in both primary MMECs and ECs of patients with monoclonal gammopathies (MGUS) of undetermined significance (MGEC) or healthy human umbilical vein endothelial cells (HUVEC). Real-time reverse transcriptase PCR, Western blotting, and differential proteomic analysis were used to correlate morphologic and biological EC features with the lenalidomide effects at the gene and protein levels. Results: Lenalidomide exerted a relevant antiangiogenic effect in vivo at 1.75 mu mol/L, a dose reached in interstitial fluids of patients treated with 25 mg/d. In vitro, lenalidomide inhibited angiogenesis and migration of MMECs, but not of MGECs or control HUVECs, and had no effect on MMEC viability, apoptosis, or fibronectin-and vitronectin-mediated adhesion. Lenalidomide-treated MMECs showed changes in VEGF/VEGFR2 signaling pathway and several proteins controlling EC motility, cytoskeleton remodeling, and energy metabolism pathways. Conclusions: This study provides information on the molecular mechanisms associated with the antimigratory and antiangiogenic effects of lenalidomide in primary MMECs, thus giving new avenues for effective endothelium-targeted therapies

Lipid and fatty acid compositions of Mytilus galloprovincialis cultured in the Mar Grande of Taranto (southern Italy): feeding strategies and trophic relationships. Zoological Studies 49(2): 211-219. Lipid and fatty acid (FA) compositions were determined in the mussel Mytilus galloprovincialis collected from June 2006 to May 2007 in the Mar Grande of Taranto, southern Italy. Total lipids significantly differed throughout the study period (ANOVA, p < 0.05), with higher values in summer (24.7% dry weight (DW)) and the lowest values in winter (3.5% DW). Triacylglycerols (TAGs) were the dominant lipid class in spring and summer accounting for 55.28% and 60.3% of total lipids, respectively, while in the autumn and winter phospholipids (PLs) were considerably greater than TAGs, comprising 55.16% and 47.5% of total lipids, respectively. Cholesterol did not show large variations over the seasons. Predominant FAs were saturated FAs (SAFAs) followed by monounsaturated FAs (MUFAs). The amount of polyunsaturated FAs (PUFAs) was low. The 14:0, 16:0, 18:0, and 22:0 SAFAs, together with 14:1, 16:1 omega 7, 18:1 omega 9, 18:1 omega 7, 20:1 omega 9, and 24:1 omega 9 MUFAs, and the PUFA, non-methylene interrupted dienoic (NMID), were the most abundant FAs. FA biomarkers are frequently used to identify trophic relationships among marine invertebrates. In order to obtain indications on food sources of M. galloprovincialis, a variety of FA ratios and the sum of some FAs were determined. The sum of 18:1 omega 7 + odd-branched FAs indicated a moderate bacterial contribution to the mussel diet. A high 18:1 omega 9/18:1 omega 7 ratio together with a high level of 20:1w9 indicated an animal dietary input. Trophic markers suggested lows contribution of diatoms and dinoflagellates to the diet of M. galloprovincialis.

Parkin mutations are a major cause of early-onset Parkinson's disease (PD). The impairment of protein quality control system together with defects in mitochondria and autophagy process are consequences of the lack of parkin, which leads to neurodegeneration. Little is known about the role of lipids in these alterations of cell functions. In the present study, parkin-mutant human skin primary fibroblasts have been considered as cellular model of PD to investigate on possible lipid alterations associated with the lack of parkin protein. Dermal fibroblasts were obtained from two unrelated PD patients with different parkin mutations and their lipid compositions were compared with that of two control fibroblasts. The lipid extracts of fibroblasts have been analyzed by combined matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) and thin-layer chromatography (TLC). In parallel, we have performed direct MALDI-TOF/MS lipid analyses of intact fibroblasts by skipping lipid extraction steps. Results show that the proportions of some phospholipids and glycosphingolipids were altered in the lipid profiles of parkin-mutant fibroblasts. The detected higher level of gangliosides, phosphatidylinositol, and phosphatidylserine could be linked to dysfunction of autophagy and mitochondrial turnover; in addition, the lysophosphatidylcholine increase could represent the marker of neuroinflammatory state, a well-known component of PD.

Lipid contents and fatty acid compositions of Idotea baltica and Sphaeroma serratum (Crustacea: Isopoda) as indicators of food sources. Zoological Studies 51(1): 38-50. The lipid and fatty acid (FA) compositions of Idotea baltica and Sphaeroma serratum, from Mar Piccolo basin at Taranto (Ionian Sea), Italy, were analyzed during winter and summer to assess their feeding habits. The 2 isopods showed strong similarities in total lipid contents. Phospholipids (PLs) were the major lipid class in both species, followed by triacylglycerols (TAGs). A low proportion of energystorage lipids suggested a regular food supply. Twenty-seven fatty acids were identified in the species studied. Unsaturated FAs (UFAs) represented the predominant proportion in both species in the seasons studied. Among them, monounsaturated FAs (MUFAs) showed higher levels. Regarding FAs corresponding to the potential food of the 2 isopods studied, I. baltica and S. serratum displayed different FA profiles. Large amounts of 18:2n-6 and18:3n-3 were found, especially in S. serratum suggesting a specific selection of phytodetritus from green algae or terrestrial material of neighboring vegetation. The FA marker for diatoms of I. baltica differed from that of S. serratum, although both species showed major consumption of diatoms during summer. Idotea baltica showed higher levels of 22:6n-3 and 20:4n-6 in winter suggesting a preference for dinoflagellates and macroalgae in this period. High levels of the carnivorous marker (the 18:1n-9/18:1n-7 ratio) reflected consumption of animal materials, especially in winter. Examination of trophic markers indicated that I. baltica and S. serratum consumed a mixed diet, showing that they have the ability to choose among available food sources.

Glatiramer acetate (GA; Copaxone) is a random copolymer of glutamic acid, lysine, alanine, and tyrosine used for the treatment of patients with multiple sclerosis (MS). Its mechanism of action has not been already fully elucidated, but it seems that GA has an immune-modulatory effect and neuro-protective properties. Lymphocyte mitochondrial dysfunction underlines the onset of several autoimmune disorders. In MS first diagnosis patients, CD4+, the main T cell subset involved in the pathogenesis of MS, undergo a metabolic reprogramming that consist in the up-regulation of glycolysis and in the down-regulation of oxidative phosphorylation. Currently, no works exist about CD4+ T cell metabolism in response to GA treatment. In order to provide novel insight into the potential use of GA in MS treatment, blood samples were collected from 20 healthy controls (HCs) and from 20 RR MS patients prior and every six months during the 12 months of GA administration. GA treated patients' CD4+ T cells were compared with those from HCs analysing their mitochondrial activity through polarographic and enzymatic methods in association with their antioxidant status, through the analysis of SOD, GPx and CAT activities. Altogether, our findings suggest that GA is able to reduce CD4+ T lymphocytes' dysfunctions by increasing mitochondrial activity and their response to oxidative stress.

Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrPC. In addition, SERS has also been used to reveal the prion protein–Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa). A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.

The new goal of anticancer agent research is the screening of natural origin drugs with lower systemic adverse effects than synthetic compounds. Here, we focus on curcumin, an important polyphenolic pigment classically used as spice in the Indian cuisine. The molecule has high pleiotropic activities including strong antioxidant and anti-inflammatory properties. However, its clinical potential is limited due its low solubility and bioavailability. We have developed a layer by layer functionalization of Fe3 O4 nanoparticles (nano-Fe3 O4 ) by coating biodegradable polyelectrolyte multilayers such as Dextran (DXS) and Poly(l-lysine) (PLL). Physico-chemical studies were performed to obtain a high upload of curcumin in Fe3 O4 nanoparticles. Nano-Fe3 O4 were then tested against an ovarian cancer cell line, SKOV-3, to demonstrate their therapeutic efficacy.

Radio Frequency Identification (RFID) is going to play a crucial role as auto-identification technology in a wide range of applications such as healthcare, logistics, supply chain management, ticketing, et cetera. The use of electromagnetic waves to identify, trace, and track people or goods allows solving many problems related to auto-identification devices based on optical reading (i.e. bar code). Currently, high interest is concentrated on the use of Radio Frequency (RF) solutions in healthcare and pharmaceutical supply chain, in order to improve drugs flow transparency and patients’ safety. Unfortunately, there is a possibility that drug interaction with electromagnetic fields (EMFs) generated by RF devices, such as RFID readers, deteriorate the potency of bioactive compounds. This chapter proposes an experimental multidisciplinary approach to investigate potential alterations induced by EMFs on drug molecular structure and performance. To show the versatility of this approach, some experimental results obtained on two biological pharmaceuticals (peptide hormone-based) are discussed.

The item-level traceability is a very important requirement for many practical application scenarios, where it needs to guarantee perfect transparency for products flow along the whole supply chain. Among these, the pharmaceutical distribution is a very interesting scenario, characterized by many challenges, where, the Radio Frequency Identification (RFID) technology will play a very important role. Unfortunately, there are still some technical barriers that are retarding the deployment of these innovative technologies in large-scale. For the pharmaceutical supply chain, there have been concerns raised regarding the potential effects on the quality of drugs due to electromagnetic fields exposure. This work aimed to evaluate potential effects of tracing RFID systems on the molecular structure of biological drugs. In particular, some samples of a commercial human insulin preparation have been exposed for different periods to electromagnetic fields generated by RFID devices. In order to evaluate possible alterations on the molecular structure, the following diagnostic techniques were used: High Pressure Liquid Chromatography (HPLC) and Nuclear Magnetic Resonance (NMR). HPLC analysis demonstrated that there is are no differences between the RFID exposed samples and the control. On the contrary, a first and partial NMR analysis detected some changes on the insulin molecule spectra after one hour of exposition to the electromagnetic field. Unfortunately, this approach did not allow us to verify possible damages on the protein because of presence of expicients and low drug concentration. Further investigations, e.g. in vitro functional analysis, are required.

Adaptation of organisms to extreme environments requires proteins to work at thermodynamically unfavorable conditions. To adapt to subzero temperatures, proteins increase the flexibility of parts of, or even the whole, 3D structure to compensate for the lower thermal kinetic energy available at low temperatures. This may be achieved through single-site amino acid substitutions in regions of the protein that undergo large movements during the catalytic cycle, such as in enzymes or transporter proteins. Other strategies of cold adaptation involving changes in the primary amino acid sequence have not been documented yet. In Antarctic icefish (Chionodraco hamatus) peptide transporter 1 (PEPT1), the first transporter cloned from a vertebrate living at subzero temperatures, we came upon a unique principle of cold adaptation. A de novo domain composed of one to six repeats of seven amino acids (VDMSRKS), placed as an extra stretch in the cytosolic COOH-terminal region, contributed per se to cold adaptation. VDMSRKS was in a protein region uninvolved in transport activity and, notably, when transferred to the COOH terminus of a warm-adapted (rabbit) PEPT1, it conferred cold adaptation to the receiving protein. Overall, we provide a paradigm for protein cold adaptation that relies on insertion of a unique domain that confers greater affinity and maximal transport rates at low temperatures. Due to its ability to transfer a thermal trait, the VDMSRKS domain represents a useful tool for future cell biology or biotechnological applications.

E-cadherin is the core protein of the epithelial adherens junction. Through its cytoplasmic domain, E-cadherin interacts with several signaling proteins; among them, - and -catenins mediate the linkof E-cadherin to the actin cytoskeleton. Loss of E-cadherin expression is a crucial step of epithelial-mesenchymal transition (EMT) and is involved in cancer invasion and metastatization. In human tumors,down-regulation of E-cadherin is frequently associated with poor prognosis. Despite the critical roleof E-cadherin in cancer progression, little is known about proteome alterations linked with its down-regulation. To address this point, we investigated proteomics, biophysical and functional changes ofepithelial breast cancer cell lines upon shRNA-mediated stable knockdown of E-cadherin expression(shEcad). shEcad cells showed a distinct proteomic signature including altered expression of enzymes andproteins involved in cytoskeletal dynamic and migration. Moreover, these results suggest that, besidestheir role in mechanical adhesion, loss of E-cadherin expression may contribute to cancer progressionby modifying a complex network of pathways that tightly regulate fundamental processes as oxidativestress, immune evasion and cell metabolism. Altogether, these results extend our knowledge on thecellular modifications associated with E-cadherin down-regulation in breast cancer cells.

An important goal of biomedical research is the development of tools for high throughput evaluation of drug effects and cytotoxicity tests. In this respect, electrochemical impedance spectroscopy (EIS) is an emerging technique for on-chip cell-based assays. For cell layers, RET (electron transfer resistence) and C (capacitance) are correlated to cell viability, adhesion and cytoskeleton organization and this approach has been successfully exploited to gain real-time information on cell behaviour. Here, for the first time, impedance based biochips in combination with complementary methodologies (including state-of-the-art AFM, viability test and western blot) are employed to perform a complete characterization of cell morphology and changes induced by copper ions on two cell lines (B104 and HeLa cells). Our results reveal a strong correlation between EIS data and both MTT test and AFM characterization. As a consequence, we expect that such on-chip assays can replace in vitro drug tests based on conventional biochemical methods, being very cheap and reusable and allowing to perform cytotoxicity tests without using any expensive reagent or equipment.

Carcinoma progression is associated with the loss of epithelial features, and the acquisition of a mesenchymal phenotype by tumour cells. Herein we show that exposure of MCF-7 cells to epidermal growth factor (EGF) resulted in morphological alterations characteristic of epithelial-to-mesenchymal transition (EMT). EGF treatment resulted in increased motility along with an up-regulation of transcription factors Slug, Zeb1, Zeb2, and mesenchymal markers Vimentin and N-cadherin. Treatment of MCF-7 cells with a combined stimulation of EGF and resveratrol, a naturally occurring stilbene with antitumor properties, failed to alter cell morphology, motility and overexpression of EMT markers induced by EGF. Using specific chemical inhibitors, we demonstrated that EGF-induced EMT is mediated by extracellular signal-regulated kinase 1/2 (ERK 1/2) signalling pathway and that resveratrol is able to repress EGF-induced ERK activation. In summary, these data provide new evidence of the inhibitory effect of resveratrol on EGF-induced EMT cell transformation.

Phytochemicals constitute a heterogeneous group of substances with an evident role in human health. Their properties on cancer initiation, promotion and progression are well documented. Particular attention is now devoted to better understand the molecular basis of their anticancer action. In the present work, we studied the effect of resveratrol on the ovarian cancer cell line OVCAR-3 by a proteomic approach. Our findings demonstrate that resveratrol down-regulates the protein cyclin D1 and, in a concentration dependent manner, the phosphorylation levels of protein kinase B (Akt) and glycogen synthase kinase-3β (GSK-3β). The dephosphorylation of these kinases could be responsible for the decreased cyclin D1 levels observed after treatment. We also showed that resveratrol reduces phosphorylation levels of the extracellular signal-regulated kinase (ERK) 1/2. Chemical inhibitors of phosphatidylinositol 3-kinase (PI3K) and ERK both increased the in vitro therapeutic efficacy of resveratrol. Moreover, resveratrol had an inhibitory effect on the AKT phosphorylation in cultured cells derived from the ascites of ovarian cancer patients and in a panel of human cancer cell lines. Thus, resveratrol shows antitumor activity in human ovarian cancer cell lines targeting signalling pathway involved in cell proliferation and drug-resistance.

Radio Frequency Identification (RFID) is a very promising wireless technology able to trace and track individual objects. The pharmaceutical supply chain is a challenging scenario, where an item-level traceability is crucial to guarantee transparency and safety in the drug flow. Unfortunately, there are still some barriers limiting the large-scale deployment of these innovative technologies. In order to face these challenges, multidisciplinary skills are required. A recent research project has attempted to coordinate heterogeneous activities focused on drug traceability. One of these is related to the evaluation of potential effects of exposure to electromagnetic fields on drugs. This paper aims to briefly describe both the main features of the defined framework for the item-level tracing of drugs on the whole supply chain and the most interesting results obtained by the evaluation of the potential effects of RFID systems on drugs. In particular, the potential alterations of the molecular structure of a commercial human insulin preparation have been analyzed by using investigative techniques such as Reverse Phase-High Pressure Liquid Chromatography and in vitro cell proliferation assays. The experimental results are strongly encouraging the use of RFID-based technologies for item-level tracing systems in the pharmaceutical supply chain.

CTR1 (Copper Transporter 1), PrPC (Cellular Prion Protein), NRAMP2 (Natural Resistance - Associated Macrophage Protein 2) and ATP7A proteins control the cell absorption and efflux of copper (Cu) ions in nervous tissues upon physiological conditions. Little is known about their regulation under reduced Cu availability, a condition underlying the onset of diffused neurodegenerative disorders. In the current study, rat neuron-like cells were exposed to Cu starvation for 48 h. The activation of Caspase-3 enzymes and the impairment of Cu,Zn Superoxide Dismutase (Cu,Zn SOD) activity depicted the initiation of a pro-apoptotic program, preliminary to the appearance of the morphological signs of apoptosis. The transcriptional response related to Cu transport proteins has been investigated. Notably, PrPC transcript and protein levels were consistently elevated upon Cu deficiency. The CTR1 protein amount was stable, despite a two-fold increase in the transcript amount, meaning the activation of post-translational regulatory mechanisms. NRAMP2 and ATP7A expression was unvaried. The up-regulated PrPC has been demonstrated to enhance the cell Cu uptake ability by about 50% with respect to the basal transport, and so sustain the Cu delivery to the Cu,Zn SOD cuproenzymes. Conclusively, the study suggests a pivotal role for PrPC in the cell adaptation to Cu limitation through a direct activity of ion uptake. In this view, the PrPC accumulation observed in several cancer cell lines could be interpreted as a molecular marker of cell Cu deficiency and a potential target of therapeutic interventions against disorders caused by metal imbalances.

La Sclerosi Multipla (SM) è una malattia infiammatoria cronica demielinizzante, a carattere autoimmune, che colpisce il Sistema Nervoso Centrale (SNC). Nonostante siano stati effettuati una serie di studi allo scopo di chiarire le cause della patologia, alcuni meccanismi patogenetici restano ancora oscuri e l’eziologia rimane sconosciuta. Il modello murino della malattia, l'encefalopatia autoimmune, ha tuttavia permesso di ricondurre il processo di demielinizzazione all'azione dei linfociti T CD4+ che, una volta attivati, sarebbero in grado di penetrare attraverso la barriera ematoencefalica e di causare danno assonale con conseguente morte di cellule neuronali. L’attivazione delle cellule T è accompagnata da uno shift metabolico che prevede il passaggio da un metabolismo ossidativo energeticamente efficiente ad un programma prettamente glicolitico; nelle malattie autoimmuni i dati in letteratura concernenti il fenotipo metabolico dei linfociti T sono piuttosto esigui e, tuttora, non vi sono lavori condotti a tal riguardo nella SM.

Objective: Mutations in Mediator Complex Subunit 12 (MED12) gene are typical genomic aberrations, commonly detected in a high percentage of uterine leiomyomas (ULs). The aim of this investigation was to define the fibroid or non-tumor origin of uterine leiomyoma pseudocapsule (PC) surrounding fibroids and its possible therapeutic targets in uterine fibroid management. Research design and methods: A non-randomized observational study was performed on 36 women, not subjected to any previous drug treatment, undergoing laparoscopic intracapsular myomectomy. Specimens of myometrium (UM), ULs and corresponding PCs were sampled to analyze MED12 gene status, by direct sequencing of exon 2. Main outcome measures: Defining the status of MED12 gene in PCs associated to ULs harboring mutations. Results: PCs always showed a wild type MED12 gene status, even when associated to a UL harboring a specific MED12 aberration. Conclusion: The wild-type status of MED12 gene in the PCs indicates the non-tumoral origin of this structure: it appears as a protective structure for the healthy tissue that could enhance regenerative mechanisms. The limitations of this study, as the restrained number of patients, will be solved in the future extending the analysis to a larger cohort of women, as tester of such pharmacological treatments on PC.

The prion proteins and their interaction with copper ion represent a suitable marker in neurodegenerative disorders. A SERS based optical sensor has been developed in order to detect and quantify the prion proteins (PrPC) onto the cell membrane using the higher binding affinity of such proteins for copper ions. A combined method of hydrothermal “green” synthesis and thermal treatment allows us to obtain impurity-free surfaces for SERS measurement, suitable for cell growth. The plasmon absorption of the gold nanostructures was monitored by UV–vis spectrometry. The most significant red shift in the longitudinal plasmon resonance absorption of gold nanostructures was maximized in order to achieve the highest electromagnetic enhancement in Raman measurements. Our SERS based optical sensor has been used to detect and quantify the PrPC –Cu2+ interaction in vitro as a function of copper concentration and time in a rat neuroblastoma cell line (B104) and in three other cell models (SH-SY5Y, GN11, HeLa), expressing PrPC at different levels. The proposed methodology can be engineered in order to obtain an extremely fast and low-cost diagnostic tool to evaluate the subject’s proneness to incur neurodegenerative processes.

The targeting of BCR-ABL, a hybrid oncogenic tyrosine (Y) kinase, does not eradicate chronic myeloid leukemia (CML)-initiating cells. Activation of β-catenin was linked to CML leukemogenesis and drug resistance through its BCR-ABL-dependent Y phosphorylation and impaired binding to GSK3β (glycogen synthase kinase 3β). Herein, we show that GSK3β is constitutively Y(216) phospho-activated and predominantly relocated to the cytoplasm in primary CML stem/progenitor cells compared with its balanced active/inactive levels and cytosolic/nuclear distribution in normal cells. Under cytokine support, persistent GSK3β activity and its altered subcellular localization were correlated with BCR-ABL-dependent and -independent activation of MAPK and p60-SRC/GSK3β complex formation. Specifically, GSK3β activity and nuclear import were increased by imatinib mesylate (IM), a selective ABL inhibitor, but prevented by dasatinib that targets both BCR-ABL- and cytokine-dependent MAPK/p60-SRC activity. SB216763, a specific GSK3 inhibitor, promoted an almost complete suppression of primary CML stem/progenitor cells when combined with IM, but not dasatinib, while sparing bcr-abl-negative cells. Our data indicate that GSK3 inhibition acts to prime a pro-differentiative/apoptotic transcription program in the nucleus of IM-treated CML cells by affecting the β-catenin, cyclinD1, C-EBPα, ATF5, mTOR, and p27 levels. In conclusion, our data gain new insight in CML biology, indicating that GSK3 inhibitors may be of therapeutic value in selectively targeting leukemia-initiating cells in combination with IM but not dasatinib.

The current therapy for ovarian cancer has advanced from alkylating agents, to a combination of carboplatinum and paclitaxel offering increased survival. Although most patients respond to this first-line therapy, initially, the majority of these patients relapse within 2 years. The mechanisms responsible for acquired drug resistance in ovarian cancer have been elucidated only in part. They include i) enhanced drug export, ii) activation/inhibition of intracellular signalling pathways, iii) molecular alterations in tubulin isotype composition. A better understanding of these mechanisms is needed, in order to develop new approaches, aimed at overcoming resistance to anticancer agents, and to reveal the complexity of causes, which contribute to drug resistance. In this review we offer an updated overview of proteomic studies on the molecular mechanisms of paclitaxel resistance. These proteomic studies also identify potential targets for modulating drug resistance, that could be predictive of response to chemotherapy in ovarian carcinomas.

In this work we explored the possibility of using genetically modified Arabidopsis thaliana plants as a rapid and low-cost screening tool for evaluating human anticancer drugs action and efficacy. Here, four different inhibitors with a validated anticancer effect in humans and distinct mechanism of action were screened in the plant model for their ability to interfere with the cytoskeletal and endomembrane networks. We used plants expressing a green fluorescent protein (GFP) tagged microtubule-protein (TUA6-GFP), and three soluble GFPs differently sorted to reside in the endoplasmic reticulum (GFPKDEL) or to accumulate in the vacuole through a COPII dependent (AleuGFP) or independent (GFPChi) mechanism. Our results demonstrated that drugs tested alone or in combination differentially influenced the monitored cellular processes including cytoskeletal organization and endomembrane trafficking. In conclusion, we demonstrated that A. thaliana plants are sensitive to the action of human chemotherapeutics and can be used for preliminary screening of drugs efficacy. The cost-effective subcellular imaging in plant cell may contribute to better clarify drugs subcellular targets and their anticancer effects.

The growing understanding of the molecular mechanisms underlying epithelial-to-mesenchymal transition (EMT) may represent a potential source of clinical markers. Despite EMT drivers have not yet emerged as candidate markers in the clinical setting, their association with established clinical markers may improve their specificity and sensitivity. Mass spectrometry-based platforms allow analyzing multiple samples for the expression of EMT candidate markers, and may help to diagnose diseases or monitor treatment efficiently. This review highlights proteomic approaches applied to elucidate the differences between epithelial and mesenchymal tumors and describes how these can be used for target discovery and validation.

Physical and mechanical properties of extracellularmatrix (ECM) have been proved to be crucial in the metastatic process. However, currently available studies on the interplay between ECM stiffness and cancer cell invasive behaviour are performed on planar assays, while the in vivo interaction takes place in three-dimensions. To take into consideration the ECM structural and mechanical complexity in the cell/structure interactions, we fabricated 3D microscaffolds through two-photon lithography (2PL) and tested how they are invaded by human colorectal adenocarcinoma (LS-174T) tumor cells, showing that it is possible to detect significant differences in cells/structure interactionwhen structural parameters are modified. In particular, both scaffold geometry and 2PL fabrication parameters were optimized to obtain 3D polymeric cylindrical structures with controlled Young's modulus and with linear stiffness gradients. The ability of LS-174T to migrate in the scaffolds was tested in different experimental conditions, including scaffolds functionalization and under β-catenin downregulation. It was observed that high Young's modulus scaffolds are always less invaded than softer ones, confirming the role of the 3D micro-environmental stiffness in mediating cells migration, including when specific functionalization or pharmacological treatments are performed.