Rapid and specific detection of wheat spindle streak mosaic virus using RT-LAMP in durum wheat crude leaf extract.

To accurately determine the spread of any pathogen, including plant viruses, a quick, sensitive, cost-effective, point-of-care diagnostic assay is necessary. Wheat spindle streak mosaic virus (WSSMV) is a Bymovirus, transmitted by the plasmodiophorid Polymyxa graminis Led, which causes yellow mosaic and reduces the grain yield in wheat. Currently, detection protocols for WSSMV use ELISA or more sensitive PCR-based approaches requiring specialized laboratory and personnel. A protocol for reverse transcription loop mediated isothermal amplification (RT-LAMP) has been developed and optimized for the rapid detection of viruses using crude extracts from wheat leaves. The protocol was specific for WSSMV detection, while no reaction was observed with SBCMV or SBWMV, the non-target viruses transmitted by the same vector. The RT-LAMP assay was shown to be as sensitive as the one-step WSSMV specific RT-PCR. The RT-LAMP assay can be performed under field conditions using a portable instrument, and can help the actual spread of WSSMV, an aspect of this virus not yet well understood, to be explored.

Fast and Sensitive Detection of Soil-Borne Cereal Mosaic Virus in Leaf Crude Extract of Durum Wheat.

Soil-borne cereal mosaic virus (SBCMV) is a furovirus with rigid rod-shaped particles containing an ssRNA genome, transmitted by Polymyxa graminis Led., a plasmodiophorid that can persist in soil for up to 20 years. SBCMV was reported on common and durum wheat and it can cause yield losses of up to 70%. Detection protocols currently available are costly and time-consuming (real-time PCR) or have limited sensitivity (ELISA). To facilitate an efficient investigation of the real dispersal of SBCMV, it is necessary to develop a new detection tool with the following characteristics: no extraction steps, very fast results, and high sensitivity to allow pooling of a large number of samples. In the present work, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) protocol with such characteristics, and we have compared it with real-time PCR. Our results show that the sensitivity of LAMP and real-time PCR on cDNA and RT-LAMP on crude extracts are comparable, with the obvious advantage that RT-LAMP produces results in minutes rather than hours. This paves the way for extensive field surveys, leading to a better knowledge of the impact of this virus on wheat health and yield.

Cross-sectional and longitudinal reliability of semiquantitative osteoarthritis assessment at 1.0T extremity MRI: Multi-reader data from the MOST study.

Objective: To determine reliability among four experienced and calibrated readers in cross-sectional and longitudinal semi-quantitative MRI assessments of knee osteoarthritis (OA) in the Multicenter Osteoarthritis (MOST) study. Design: From all MOST participants with at least one knee with readable 60-month and 84-month paired knee MRIs (1.0 â€‹T extremity systems), we selected 10 subjects having a spectrum of baseline disease severity of cartilage, bone marrow lesions, and meniscal damage and a spectrum of longitudinal changes in severity at 24 months follow-up. MRIs were independently assessed using the WORMS grading system by four musculoskeletal radiologists with the chronological sequence known to the readers. Kappa statistics were used to determine agreement between each pair of readers and Kendall's coefficient of concordance to determine average agreement across readers. Results: For most features, cross-sectional reliability was substantial to almost perfect. Regarding longitudinal reliability (detection of longitudinal change), inter-reader reliability as weighted kappa values ranged from 0.62 to 0.78 for cartilage damage, 0.75-0.88 for bone marrow lesions, 0.75-0.92 for meniscal tears, 0.67-0.95 for meniscal extrusion, 0.51-0.77 for bone attrition, 0.43-0.76 for osteophytes, 0.31-0.70 for Hoffa-synovitis, and 0.47-0.85 for effusion-synovitis. Kendall's coefficient ranged from 0.65 to 0.98. Conclusion: High levels of cross-sectional reliability and moderate to high longitudinal reliability was achieved using four experienced readers in semiquantitative MRI-assessment of most knee OA features.

Structural effects of sprifermin in knee osteoarthritis: a post-hoc analysis on cartilage and non-cartilaginous tissue alterations in a randomized controlled trial.

BACKGROUND: A recent publication on efficacy of Sprifermin for knee osteoarthritis (OA) using quantitatively MRI-defined central medial tibio-femoral compartment cartilage thickness as the structural primary endpoint reported no statistically significant dose response. However, Sprifermin was associated with statistically significant, dose-dependent reductions in loss of total and lateral tibio-femoral cartilage thickness. Based on these preliminary promising data a post-hoc analysis of secondary assessment and endpoints was performed to evaluate potential effects of Sprifermin on semi-quantitatively evaluated structural MRI parameters. Aim of the present analysis was to determine effects of sprifermin on several knee joint tissues over a 12 month period. METHODS: 1.5 T or 3 T MRIs were acquired at baseline and 12 months follow-up using a standard protocol. MRIs were read according to the Whole-Organ Magnetic Resonance Imaging Score (WORMS) scoring system (in 14 articular subregions) by four muskuloskeletal radiologists independently. Analyses focused on semiquantitative changes in the 100 µg subgroup and matching placebo of multiple MRI-defined structural alterations. Analyses included a delta-subregional and delta-sum approach for the whole knee and the medial and lateral tibio-femoral (MTFJ, LTFJ), and patello-femoral (PFJ) compartments, taking into account number of subregions showing no change, improvement or worsening and changes in the sum of subregional scores. Mann-Whitney - Wilcoxon tests assessed differences between groups. RESULTS: Fifty-seven and 18 patients were included in the treatment and matched placebo subgroups. Less worsening of cartilage damage was observed from baseline to 12 months in the PFJ (0.02, 95 % confidence interval (CI) (-0.04, 0.08) vs. placebo 0.22, 95 % CI (-0.05, 0.49), p = 0.046). For bone marrow lesions (BMLs), more improvement was observed from 6 to 12 months for whole knee analyses (-0.14, 95 % CI (-0.48, 0.19) vs. placebo 0.44, 95 % CI (-0.15, 1.04), p = 0.042) although no significant effects were seen from the baseline visit, or in Hoffa-synovitis, effusion-synovitis, menisci and osteophytes. CONCLUSIONS: In this post-hoc analysis cartilage showed less worsening from baseline to 12 months in the PFJ, and BMLs showed more improvement from 6 to 12 months for the whole knee. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01033994 .

A mechanistic study on the cardiotoxicity of 5-fluorouracil in vitro and clinical and occupational perspectives.

Fluoropyrimidines are key agents for the treatment of gastrointestinal tract adenocarcinomas. The possible cardiotoxic effects in patients and occupationally exposed workers are multifactorial and remain a puzzle to solve for investigators. In the present study, we study what cell death pathways and what doses can determine direct cardiotoxic effects of 5-fluorouracil (5-FU) and doxorubicin (DOXO) on rat cardiocytes (H9c2) and a human colon adenocarcinoma (HT-29) cell line, already reported to be sensitive to 5-FU. We have found that 5-FU induced 50% growth inhibition (IC:50) at 72 h with concentrations of 400 µM and 4 µM on H9c2 and HT-29, respectively. Moreover, we have found that the addition of Levofolinic Acid (LF) to 5-FU potentiated the growth inhibition induced by 5-FU. The growth inhibition induced by 5-FU alone or in combination with LF in cardiocytes was paralleled by an increase of thiobarbituric acid-reactive species (Tbars) and end products of nitric oxide (NO) suggesting the increase of the oxidative stress status in cardiocytes. Interestingly, these effects were strongly potentiated by the addition of LF, a biochemical modulator of 5-FU activity. Our data suggest that agents such as 5-FU different from anthracyclines, conventionally related to the induction of cardiotoxic effects, can also induce cardiocyte damage paralleled by oxidative stress. The strategies based upon the use of scavengers could be used in order to prevent this effect.

Delayed gadolinium-enhanced magnetic resonance imaging of medial tibiofemoral cartilage and its relationship with meniscal pathology: a longitudinal study using 3.0T magnetic resonance imaging.

OBJECTIVE: To evaluate the relationship between medial meniscal pathology and cartilage matrix status using delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) in medial tibiofemoral cartilage in a sample of middle-aged women. METHODS: A total of 148 women ages ≥40 years were included, and 3.0T MRI of the knee was performed at baseline and at 1 year. T2-weighted, fat-suppressed and 3-dimensional inversion recovery-prepared spoiled gradient-recalled echo sequences were acquired 90 minutes after gadolinium injection. Baseline medial meniscal pathology was scored on a scale of 0-3, where 0 = normal, 1 = intrasubstance meniscal signal change, 2 = single tears, and 3 = complex tears/maceration. The central medial femur, the medial tibial plateau, and the posterior medial femur were subjected to dGEMRIC at baseline and at 1 year. Analysis of covariance was used to examine whether baseline and 1-year dGEMRIC indices in the same regions were related to the severity of meniscal damage at baseline, using normal medial menisci (grade 0) as the reference. RESULTS: Medial compartments with grade 3 lesions showed significantly lower dGEMRIC indices (less proteoglycan content) at the central medial femur region compared with compartments with normal menisci. Mean ± SEM differences in dGEMRIC indices between grade 3 and grade 0 menisci at the central medial femur were -119.1 ± 34.2 msec at baseline (P = 0.03) and -120.3 ± 35.2 msec at followup (P = 0.04). CONCLUSION: High-grade damage of the medial meniscus showed significant associations with lower dGEMRIC indices. The dGEMRIC technique may be a useful tool in detecting early degenerative changes of cartilage when meniscal function is lost.

Synovitis in knee osteoarthritis assessed by contrast-enhanced magnetic resonance imaging (MRI) is associated with radiographic tibiofemoral osteoarthritis and MRI-detected widespread cartilage damage: the MOST study.

OBJECTIVE: To examine the cross-sectional association of whole-knee synovitis assessed by contrast-enhanced magnetic resonance imaging (CEMRI) with radiographic tibiofemoral osteoarthritis (OA), non-CEMRI-assessed cartilage damage, and meniscal status. METHODS: Multicenter Osteoarthritis Study (MOST) is a cohort study of people with or at high risk of knee OA. Subjects are a subset of MOST who volunteered for both CEMRI and non-CEMRI. Using CEMRI, synovitis was assessed at 11 sites and graded 0-2 at each site. Presence of "whole-knee synovitis" was defined as the synovitis score of ≥ 1 at any site from each knee. Cartilage and meniscal damage was evaluated using non-CEMRI based on the Whole Organ MRI Score. Logistic regression was used to assess associations of synovitis with radiographic OA (Kellgren-Lawrence grade ≥ 2), widespread cartilage damage, and meniscal damage, adjusting for age, sex, and body mass index (BMI). Additional analyses were performed excluding subjects who had chondrocalcinosis on radiography and those taking antiinflammatory medications. RESULTS: Four hundred four subjects were included (mean age 58.8 ± 7.0 yrs, BMI 29.6 ± 4.9 kg/m(2), 45.5% women). On CEMRI, the maximum synovitis score across 11 sites in each knee was 0 in 106 knees (26.2%), 1 in 135 (33.4%), and 2 in 163 (40.4%). Synovitis was associated with radiographic OA [adjusted OR (aOR) 3.25, 95% CI 1.98-5.35] and widespread cartilage damage (aOR 1.91, 95% CI 1.24-2.92). Severe meniscal damage showed a borderline significant association with synovitis (aOR 1.74, 95% CI 0.99-3.04). Additional analyses as described did not notably change the results. CONCLUSION: CEMRI-detected synovitis is strongly associated with tibiofemoral radiographic OA and MRI-detected widespread cartilage damage.

Urotensin-II receptor is over-expressed in colon cancer cell lines and in colon carcinoma in humans.

BACKGROUND: Urotensin (U)-II receptor (UTR) has been previously reported to be over-expressed in a number of tumours. Whether UTR-related pathway plays a role in colon carcinogenesis is unknown. METHODS: We evaluated UTR protein and mRNA expression in human epithelial colon cancer cell lines and in normal colon tissue, adenomatous polyps and colon cancer. U-II protein expression was assessed in cancer cell lines. Moreover, we evaluated the effects of U-II(4-11) (an UTR agonist), antagonists and knockdown of UTR protein expression through a specific shRNA, on proliferation, invasion and motility of human colon cancer cells. RESULTS: Cancer cell lines expressed U-II protein and UTR protein and mRNA. By immunohistochemistry, UTR was expressed in 5-30% of epithelial cells in 45 normal controls, in 30-48% in 21 adenomatous polyps and in 65-90% in 48 colon adenocarcinomas. UTR mRNA expression was increased by threefold in adenomatous polyps and eightfold in colon cancer, compared with normal colon. U-II(4-11) induced a 20-40% increase in cell growth while the blockade of the receptor with specific antagonists caused growth inhibition of 20-40%. Moreover, the knock down of UTR with a shRNA or the inhibition of UTR with the antagonist urantide induced an approximately 50% inhibition of both motility and invasion. CONCLUSIONS: UTR appears to be involved in the regulation of colon cancer cell invasion and motility. These data suggest that UTR-related pathway may play a role in colon carcinogenesis and that UTR may function as a target for therapeutic intervention in colon cancer.

Radiographically occult and subtle fractures: a pictorial review.

Radiographically occult and subtle fractures are a diagnostic challenge. They may be divided into (1) "high energy trauma fracture," (2) "fatigue fracture" from cyclical and sustained mechanical stress, and (3) "insufficiency fracture" occurring in weakened bone (e.g., in osteoporosis and postradiotherapy). Independently of the cause, the initial radiographic examination can be negative either because the findings seem normal or are too subtle. Early detection of these fractures is crucial to explain the patient's symptoms and prevent further complications. Advanced imaging tools such as computed tomography, magnetic resonance imaging, and scintigraphy are highly valuable in this context. Our aim is to raise the awareness of radiologists and clinicians in these cases by presenting illustrative cases and a discussion of the relevant literature.

Stealth liposomes encapsulating zoledronic acid: a new opportunity to treat neuropathic pain.

In the pathogenesis of neuropathic pain, the conversion of astrocytes in the reactive state and the ras-dependent Erk-mediated pathway play an important role. Zoledronic acid (ZOL) is a potent inhibitor of the latter pathway, but its activity in neurological diseases is hampered by its biodistribution that is almost exclusively limited to the bone. We have developed nanotechnological devices able to increase the accumulation of ZOL in extra bone sites. In this work, we have evaluated the effects of ZOL-encapsulating PEGylated liposomes (LipoZOL) on an animal model of neuropathic pain. We have found that 2 iv administrations (10 µg of ZOL, either as free or encapsulated into liposomes) at days 2 and 4 after the injury markedly reduced mechanical hypersensitivity at 3 and 7 days after nerve injury. On the other hand, free ZOL did not exert any significant alteration of the mechanical threshold. Immunohistochemical analysis of spinal cord revealed that GFAP-labeled astrocytes appeared hypertrophic activated cells in the ispilateral dorsal horn of spinal cord 7 days after SNI. LipoZOL significantly changed astrocyte morphology, by inducing a protective phenotype, without changing the total cell number. Moreover, the astrocytes of the spinal cord of LipoZOL-treated mice were positive for interleukin-10. Delivery of ZOL into the CNS was confirmed by biodistribution of fluorescently labeled liposomes. In particular, liposomes accumulated in the liver and kidney in both groups of normal and neuropathic animals; on the other hand, only in the case of neuropathic animals, a fluorescence increase in the brain and spinal cord occurred only in neuropathic animals at 30 min and 1 h. These data demonstrate that ZOL, only by using a delivery system able to cross the altered BBB, could be a new opportunity to treat neuropathic pain.

Co-localisation of non-cartilaginous articular pathology increases risk of cartilage loss in the tibiofemoral joint--the MOST study.

PURPOSE: To assess risk of cartilage loss in the tibiofemoral joint in relation to baseline damage severity, and to analyse the association of nearby pathologic findings on the risk of subsequent cartilage loss. METHODS: The Multicenter Osteoarthritis Study is a longitudinal study of individuals with or at high risk for knee osteoarthritis. MRI examinations were assessed according to the Whole Organ MRI Score. Included were all knees with available baseline and 30 months MRIs. Ordinal logistic regression was used to estimate risk of cartilage loss in each subregion in relation to the number of associated articular features including bone marrow lesions, meniscal damage and extrusion and also in regard to baseline damage severity, respectively. RESULTS: 13 524 subregions of 1365 knees were included. 3777 (27.9%) subregions exhibited prevalent cartilage damage at baseline and 1119 (8.3%) subregions showed cartilage loss at 30-month follow-up. Risk of cartilage loss was increased for subregions with associated features (OR 2.53, 95% CI 2.03 to 3.15 for one, 4.32 95% CI 3.42 to 5.47 for two and 5.30 95% CI 3.95 to 7.12 for three associated features; p for trend<0.0001). Subregions with prevalent cartilage damage showed increased risk for further cartilage loss compared to subregions with intact cartilage at baseline with small superficial defects exhibiting highest risk. CONCLUSIONS: Risk of cartilage loss is increased for subregions with associated pathology and further increased when more than one type of associated feature is present. In addition, prevalent cartilage damage increases risk for subsequent cartilage loss.

Type I interferons: ancient peptides with still under-discovered anti-cancer properties.

Type I interferons (IFNs) represent a group of cytokines that act through a common receptor composed by two chains (IFNAR-1 and IFNAR-2). Several in vitro and in vivo studies showed a potent antitumor activity induced by these cytokines. IFN-α, the first cytokine to be produced by recombinant DNA technology, has emerged as an important regulator of cancer cell growth and differentiation, affecting cellular communication and signal transduction pathways. IFN-α, is currently the most used cytokine in the treatment of cancer. However, the potential anti-tumour activity of IFN-α is limited by the activation of tumour resistance mechanisms. This article reviews the current knowledge about the antitumor activity of type I IFNs, focusing on new potential strategies able to strengthen the antitumor activity of these cytokines.

Scapholunate advanced collapse and scaphoid nonunion advanced collapse: MDCT arthrography features.

OBJECTIVE: The aim of this article is to present the imaging features of scapholunate advanced collapse (SLAC) and scaphoid nonunion advanced collapse (SNAC) on MDCT arthrography. CONCLUSION: MDCT arthrography is an excellent tool for patients with clinically suspected SLAC or SNAC wrist because it allows identification of the spectrum of findings for diagnosis and proper classification, which directly impact management.

5-Fluorouracil induces apoptosis in rat cardiocytes through intracellular oxidative stress.

BACKGROUND: Cardiotoxicity is a major complication of anticancer drugs, including anthracyclines and 5-fluorouracil(5-FU) and it can have detrimental effects both in patients and workers involved in the preparation of chemotherapy. METHODS: Specifically, we have assessed the effects of increasing concentrations of 5-FU and doxorubicin (DOXO) on proliferation of H9c2 rat cardiocytes and HT-29 human colon adenocarcinoma cells by MTT assay. Cells were treated for 24, 48 and 72 h with different concentrations of the two drugs alone or with 5-FU in combination with 10(-4) M of levofolene (LF). RESULTS: 5-FU induced a time- and dose-dependent growth inhibition in both cell lines. The 50% growth inhibition (IC:50) was reached at 72 h with concentrations of 4 µM and 400 µM on HT-29 and H9c2, respectively. The addition of LF to 5-FU enhanced this effect. On the other hand, the IC:50 of DOXO was reached at 72 h with concentrations of 0.118 µM on H9c2 and of 0.31 µM for HT-29. We have evaluated the cell death mechanism induced by 50% growth inhibitory concentrations of 5-FU or DOXO in cardiocytes and colon cancer cells. We have found that the treatment with 400 µM 5-FU induced apoptosis in 32% of H9c2 cells. This effect was increased by the addition of LF to 5-FU (38% of apoptotic cells). Apoptosis occurred in only about 10% of HT-29 cells treated with either 5-FU or 5-FU and LF in combination. DOXO induced poor effects on apoptosis of both H9c2 and HT-29 cells (5-7% apoptotic cells, respectively). The apoptosis induced by 5-FU and LF in cardiocytes was paralleled by the activation of caspases 3, 9 and 7 and by the intracellular increase of O(2-) levels. CONCLUSIONS: These results suggest that cardiotoxic mechanism of chemotherapy agents are different and this disclose a new scenario for prevention of this complication.

Factors associated with meniscal extrusion in knees with or at risk for osteoarthritis: the Multicenter Osteoarthritis study.

PURPOSE: To assess the associations of meniscal tears, knee mal-alignment, cartilage damage, knee effusion, and body mass index with meniscal extrusion. MATERIALS AND METHODS: The Multicenter Osteoarthritis study is an observational study of individuals who have or are at risk for knee osteoarthritis (OA). The HIPAA-compliant protocol was approved by the institutional review boards of all participating centers, and written informed consent was obtained from all patients. All subjects with available baseline knee radiographs and magnetic resonance (MR) images were included. MR imaging assessment of meniscal morphologic characteristics, meniscal position, and cartilage morphologic characteristics with use of the Whole-Organ Magnetic Resonance Imaging Score system was performed by two musculoskeletal radiologists. Cross-sectional associations of severity of meniscal tears, knee malalignment, tibiofemoral cartilage damage, knee effusion, and body mass index with meniscal extrusion were assessed by using logistic regression, with multiadjustments when testing each predictor. RESULTS: A total of 1527 subjects (2131 knees; 2116 medial and 2106 lateral menisci) were included. Medially, meniscal tears, varus malalignment, and cartilage damage were associated with meniscal extrusion, with odds ratios (ORs) of 6.3 (95% confidence interval [CI]: 5.0, 8.0), 1.3 (95% CI: 1.1, 1.7), and 1.8 (95% CI: 1.4, 2.2), respectively. Laterally, meniscal tears, valgus malalignment, and cartilage damage were associated with meniscal extrusion, with ORs of 10.3 (95% CI: 7.1, 14.9), 2.2 (95% CI: 1.5, 3.2), and 2.0 (95% CI: 1.3, 2.9), respectively. CONCLUSION: Meniscal tears are not the only factors associated with meniscal extrusion; other factors include knee malalignment and cartilage damage. Meniscal extrusion is probably an effect of the complex interactions among joint tissues and mechanical stresses involved in the OA process.

Tumour-specific uptake of anti-cancer drugs: the future is here.

A challenge of anti-cancer treatment is the specific delivery of the drugs in order to avoid deleterious effects on normal cells. In fact, anti-cancer drugs have potent effects also on normal cells due to the strong similarity of the mechanisms of growth regulation of normal cells if compared to their transformed counterparts. The recent developments in nanotechnology allow the old Ehrlich's dream to deliver anti-cancer drugs in tumour tissue through their encapsulation in drug delivery systems (DDS). In the present review we analyze the different reasons to encapsulate an anti-tumour drug in DDS including eventual damages induced by their extravasation or by eccipients used to their solubilisation, the rapid break-down of the drug in vivo and the specific bio-distribution of the drug in tumour tissues. The delivery strategies of anti-cancer drugs are based upon the particular structure of tumour neo-angiogenic vessels that allow the passive targeting or enhanced permeability and retention (EPR). In order to avoid the entrapping of DDS in reticulo-endothelial system the nanoparticles can be modified with the addition on their surface of inert polyetilenglicole (PEG) molecules that inhibit the opsonisation of DDS by macrophages. The addition of targeting moieties, antibodies or Fab fragments or small peptides and aptamers, on the surface of DDS can allow the active targeting of DDS to tumour cells. In conclusion, a new avenue in anti-cancer treatment has been disclosed with the use of DDS.

New self-assembly nanoparticles and stealth liposomes for the delivery of zoledronic acid: a comparative study.

Zoledronic acid (ZOL) is a drug whose potent anti-cancer activity is limited by its short plasma half-life and rapid uptake and accumulation within bone. We have recently proposed new delivery systems to avoid ZOL accumulation into the bone, thus improving extra-skeletal bioavailability. In this work, we have compared the technological and anti-cancer features of either ZOL-containing self-assembly PEGylated nanoparticles (NPs) or ZOL-encapsulating PEGylated liposomes (LIPO-ZOL). ZOL-containing NPs showed superior technological characteristics in terms of mean diameter, size distribution, and ZOL encapsulation efficiency, compared to LIPO-ZOL. Moreover, the anti-cancer activity of NPs in nude mice xenografted with prostate cancer PC3 cells was higher than that one induced by LIPO-ZOL. In addition, NPs induced the complete remission of tumour xenografts and an increase of survival time higher than that one observed with LIPO-ZOL. It has also to be considered that PC3 tumour xenografts were almost completely resistant to the anti-cancer effects induced by free ZOL. Both nanotechnological products did not induce toxic effects not affecting the mice weight nor inducing deaths. Moreover, the histological examination of some vital organs such as liver, kidney and spleen did not find any changes in terms of necrotic effects or modifications in the inflammatory infiltrate. On the other hand, NPs but not LIPO-ZOL caused a statistically significant reduction of the tumour associated macrophages (TAM) in tumour xenografts. This effect was paralleled by a significant increase of both necrotic and apoptotic indexes. The effects of the NPs were also higher in terms of neo-angiogenesis inhibition. These results suggest the future preclinical development of ZOL-encapsulating NPs in the treatment of human cancer.

The PPAR-γ agonist troglitazone antagonizes survival pathways induced by STAT-3 in recombinant interferon-ß treated pancreatic cancer cells.

We have previously shown that cancer cells can protect themselves from apoptosis induced by type I interferons (IFNs) through a ras→MAPK-mediated pathway. In addition, since IFN-mediated signalling components STATs are controlled by PPAR gamma we studied the pharmacological interaction between recombinant IFN-ß and the PPAR-γ agonist troglitazone (TGZ). This combination induced a synergistic effect on the growth inhibition of BxPC-3, a pancreatic cancer cell line, through the counteraction of the IFN-ß-induced activation of STAT-3, MAPK and AKT and the increase in the binding of both STAT-1 related complexes and PPAR-γ with specific DNA responsive elements. The synergism on cell growth inhibition correlated with a cell cycle arrest in G0/G1 phase, secondary to a long-lasting increase of both p21 and p27 expressions. Blockade of MAPK activation and the effect on p21 and p27 expressions, induced by IFN-ß and TGZ combination, were due to the decreased activation of STAT-3 secondary to TGZ. IFN-ß alone also increased p21 and p27 expression through STAT-1 phosphorylation and this effect was attenuated by the concomitant activation of IFNbeta-induced STAT-3-activation. The combination induced also an increase in autophagy and a decrease in anti-autophagic bcl-2/beclin-1 complex formation. This effect was mediated by the inactivation of the AKT→mTOR-dependent pathway. To the best of our knowledge this is the first evidence that PPAR-γ activation can counteract STAT-3-dependent escape pathways to IFN-ß-induced growth inhibition through cell cycle perturbation and increased autophagic death in pancreatic cancer cells.

Molecular targets and oxidative stress biomarkers in hepatocellular carcinoma: an overview.

Hepatocellular carcinoma (HCC) is a complex and heterogeneous tumor with multiple genetic aberrations. Several molecular pathways involved in the regulation of proliferation and cell death are implicated in the hepatocarcinogenesis. The major etiological factors for HCC are both hepatitis B virus (HBV) and hepatitis C virus infection (HCV). Continuous oxidative stress, which results from the generation of reactive oxygen species (ROS) by environmental factors or cellular mitochondrial dysfunction, has recently been associated with hepatocarcinogenesis. On the other hand, a distinctive pathological hallmark of HCC is a dramatic down-regulation of oxido-reductive enzymes that constitute the most important free radical scavenger systems represented by catalase, superoxide dismutase and glutathione peroxidase. The multikinase inhibitor sorafenib represents the most promising target agent that has undergone extensive investigation up to phase III clinical trials in patients with advanced HCC. The combination with other target-based agents could potentiate the clinical benefits obtained by sorafenib alone. In fact, a phase II multicenter study has demonstrated that the combination between sorafenib and octreotide LAR (So.LAR protocol) was active and well tolerated in advanced HCC patients. The detection of molecular factors predictive of response to anti-cancer agents such as sorafenib and the identification of mechanisms of resistance to anti-cancer agents may probably represent the direction to improve the treatment of HCC.

Analysis of interaction partners for eukaryotic translation elongation factor 1A M-domain by functional proteomics.

The eukaryotic translation elongation factor 1A (eEF1A), besides to its canonical role in protein synthesis, is also involved in several other cellular processes, depending on changes in cellular location, cell type, concentration of ligands, substrates or cofactors. Therefore eEF1A is a moonlighting protein that participates to a network of molecular interactions involving its structural domains. Since the identification of novel protein-protein interactions represents important tasks in post-genomic era, the interactome of eEF1A1 M-domain was investigated by using a proteomic approach. To this purpose, the eEF1A1 M-domain was fused with glutathione-S-transferase (GST) and Strep-tag (ST) at it's N- and C-terminal, respectively. The recombinant protein (GST-M-ST) was purified and incubated with a mouse embryo lysate by applying an affinity chromatography strategy. The interacting proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Besides the known partners, the pool of interacting proteins contained sorbin, a polypeptide of 153 amino acids present in SH3 domain-containing adaptor proteins, such as SORBS2. This interaction was also assessed by Western blot on immunoprecipitate from mouse embryo or H1355 cell lysates with anti-eEF1A or anti-SORBS2 antibodies and on eEF1A1-His pull-down from H1355 cell lysate with antibody anti-SORBS2. Furthermore, the interaction between eEF1A and SORBS2 was also confirmed by confocal microscopy and FRET analysis. Interestingly, a co-localization of SORBS2 and eEF1A was evidenced at level of plasma membrane, thus suggesting the involvement of eEF1A1 in novel key signal transduction complexes.