Identification and characterization of a new potent inhibitor targeting CtBP1/BARS in melanoma cells.

BACKGROUND: The C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with "cancer hallmarks" and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT). Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions. Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities. METHODS: Structured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model. RESULTS: We identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models.  CONCLUSIONS: This study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.

Doppler ultrasonographic measurements of the lateral digital palmar artery in pregnant mares.

Cardiovascular changes have been reported in late pregnancy in mares. However, there are no data on changes in peripheral blood flow. Doppler ultrasound represents a sensitive method for assessing the blood flow directed to the hoof. The aims of this study were to evaluate the blood flow parameters of the lateral palmar digital artery (LPDA) in pregnant mares and to assess intra- and interrater agreement between two observers with different levels of experience. The LPDAs of pregnant Italian Standardbred mares were examined. The vessels were located with B-mode ultrasound and analyzed with color and pulsed wave Doppler. The following parameters were recorded by the operators: heart rate (HR), peak systolic velocity (PSV), end-diastolic velocity (EDV), and resistive index (RI). Measurements were performed between 2 and 3 months of gestation (T1), in the last month of pregnancy (T2) and a week after delivery (T3). Seventeen mares aged 3-18 years met the inclusion criteria. Ultrasound examinations of the LPDA were subjectively easy to perform and well tolerated by the mares. Interrater and intrarater agreement were good and moderate, respectively. The HR was higher at T2 than at T1 and T3. The PSV and RI changed significantly during pregnancy, with higher values at T2 and T3, whereas the EDV remained unchanged throughout the examination. Doppler examination showed that peripheral flow changes were present in mares in late pregnancy. However, the persistence of higher values after delivery invites further investigation to assess the correlation between metabolic/endocrine changes related to pregnancy and Doppler parameters.

Computed Tomographic Assessment of Pituitary Gland Dimensions in Domestic Short-Haired Cats.

The detection of subtle changes in the pituitary dimensions has relevant clinical implications. In cats, a few studies have established the cut-off values of the pituitary gland's dimensions using small and inhomogeneous samples. The aims of this study were: to determine by computed tomography (CT) the pituitary linear dimensions and the pituitary-to-brain (P:B) ratio in a sample of domestic short-haired (DSH) cats; to assess the effects of sex, age, and weight on pituitary dimensions; and to evaluate the inter- and intra-observer agreement for such measurements. All skull CTs of DSH cats performed over four years using a multidetector CT and a standardized protocol were retrospectively reviewed. The exclusion criteria were: clinical, laboratory, or CT alterations of the pituitary gland, brain diseases, fractures of the neurocranium, and diabetes. The pituitary dimensions and brain area were assessed by two different observers using multiplanar reconstructions and automated segmentation tools. Fifty-one cats were included in the final sample. The intraclass correlation coefficients for intra- and inter-observer reliability were good/excellent, and moderate/good, respectively. No differences between sexes were detected, and negligible correlations were found between age and weight. According to this study, a pituitary gland with a height > 4 mm or a P:B ratio > 0.49 mm should be considered enlarged.

Retrobulbar Filling for Enophthalmos Treatment in Dogs: Technique, Description and Computed-Tomographic Evaluation. Preliminary Cadaveric Study.

A new therapeutic approach for enophthalmos may be retrobulbar lipofilling. This study aims to standardize the intraconal filling technique and to evaluate the degree of eyeball displacement by computed tomography (CT). Skull CT was performed on six dog cadavers before and after intraconal injection of two 5% iodinated, viscoelastic solutions, one per eye, using an ultrasound-guided supratemporal approach. The volume to be injected was calculated using formulas for retrobulbar cone anesthesia. After CT, the dogs underwent necropsy and histopathology to evaluate damages that eventually occurred to retrobulbar structures. Eyeball displacement was estimated using two CT-based methods, named M1 and M2. The Wilcoxon signed-rank test revealed no significant difference between the two injected materials in both M1 (p > 0.99), and M2 (lateral p = 0.84 and rostral p = 0.84 displacement). A statistically significant difference was found between the pre- and post-injection group M1 (p = 0.002), M2 (p = 0.004) for the lateral and (p = 0.003) for rostral displacement. Although the slight eyeball displacement, the retrobulbar filling can lead to enophthalmos resolution. Compared to M1, the M2 method has better-defined anatomical landmarks. Further, preclinical in vivo studies are necessary to assess retrobulbar filling efficacy and safety.

Imaging techniques in veterinary medicine. Part II: Computed tomography, magnetic resonance imaging, nuclear medicine.

Radiography and ultrasonography are the most used techniques in veterinary clinical practice, due to organizational, managerial and, mostly, economic reasons. However, in the last decades, Computed tomography (CT), Magnetic Resonance Imaging (MRI) and, to a lesser extent, Nuclear Medicine (MN) are increasingly used. As we said in the previous article, all the Diagnostic Imaging techniques are actually "indispensable" in Veterinary Medicine, where many patients do not show any symptoms.This second part describes Computed Tomography (CT), Magnetic Resonance (MRI) and Nuclear Medicine techniques in Veterinary Medicine are described.

The heart to single vertebra ratio: A new objective method for radiographic assessment of cardiac silhouette size in dogs.

Vertebral heart size (VHS) is widely determined in clinical practice as an objective method to assess the cardiac silhouette dimensions. However, a key limitation is that it is difficult to determine VHS in dogs with vertebral alterations. This retrospective, method comparison, observer agreement study sought to overcome this limitation by using the heart-to-single vertebra ratio (HSVR), by evaluating the level of agreement between VHS and HSVR, as well as the intra- and inter-observer agreement for HSVR. Three independent observers retrospectively evaluated thoracic radiographs obtained over a set time period. Exclusion criteria were the presence of alterations of the thoracic spine and the inability to clearly outline the cardiac silhouette. The lengths of the vertebral bodies, from the fourth to eighth thoracic vertebra, and VHS were measured on each radiograph. The HSVR was calculated by dividing the sum of the cardiac long and short axes by the length of each vertebral body. Eighty dogs of different breeds were included in the final analysis. Lin's concordance correlation coefficients revealed strong correlations between VHS and HSVR (0.91-0.96), and the Bland-Altman plots showed low bias (0.01-0.2) between the methods. The mean absolute errors indicated low average magnitudes of error (0.11-0.28). The intraclass correlation coefficients showed good to excellent inter-observer (0.87-0.92; P = 0.000) and intra-observer (0.87-0.99; P < .001) agreement. In the authors' opinion, this new method, which is less time consuming and more objective, could offer a valuable alternative to VHS.

Description of a low-cost picture archiving and communication system based on network-attached storage.

High costs for installing, maintaining, and updating a standard picture archiving and communication system (PACS) can be prohibitive for small/medium-sized veterinary facilities. The aims of this prospective, exploratory study were to describe the design, implementation, and author experiences for 1 year's use of a low-cost PACS based on network-attached storage. The system described here was easily installed and resiliently stored redundant copies of data. It excellently balanced data recovery, system speed, security, and available memory for storage. A virtual private network also allowed off-site data review. This system can also be used for future off-site backup of data in the cloud.

Preclinical Imaging Evaluation of miRNAs' Delivery and Effects in Breast Cancer Mouse Models: A Systematic Review.

BACKGROUND: We have conducted a systematic review focusing on the advancements in preclinical molecular imaging to study the delivery and therapeutic efficacy of miRNAs in mouse models of breast cancer. METHODS: A systematic review of English articles published in peer-reviewed journals using PubMed, EMBASE, BIOSIS™ and Scopus was performed. Search terms included breast cancer, mouse, mice, microRNA(s) and miRNA(s). RESULTS: From a total of 2073 records, our final data extraction was from 114 manuscripts. The most frequently used murine genetic background was Balb/C (46.7%). The most frequently used model was the IV metastatic model (46.8%), which was obtained via intravenous injection (68.9%) in the tail vein. Bioluminescence was the most used frequently used tool (64%), and was used as a surrogate for tumor growth for efficacy treatment or for the evaluation of tumorigenicity in miRNA-transfected cells (29.9%); for tracking, evaluation of engraftment and for response to therapy in metastatic models (50.6%). CONCLUSIONS: This review provides a systematic and focused analysis of all the information available and related to the imaging protocols with which to test miRNA therapy in an in vivo mice model of breast cancer, and has the purpose of providing an important tool to suggest the best preclinical imaging protocol based on available evidence.

Effects of Oral Administration of Lepidium meyenii on Morphology of Mice Testis and Motility of Epididymal Sperm Cells After Tetrahydrocannabinol Exposure.

Background: Tetrahydrocannabinol (THC) administration is associated with testicular damage and reduced semen quality. Oral administration of Lepidium Meyenii (maca) improves spermatogenesis and sperm motility and count and reduces spermatogenic damage. Objectives: The aim of this study was to evaluate the effect of administration of THC, maca, and their combination on testicular tissue and semen parameters. Materials and Methods: Thirty-six-week-old male mice were classified into control, THC, Maca, and THC + Maca groups. The mice were subjected to Eco Color Doppler ultrasound examination of the testicles before and after treatment. After euthanasia, the epididymis, testes, liver, and kidney were collected for histological examination. For morphometry of the testis, tubular diameters and seminiferous epithelium height were measured. Sperm concentration and sperm motilities were assessed. Differences among the groups were assessed using the Kruskal-Wallis and Dunn's post-hoc test. Results: In all the groups, there were no significant changes in testicular morphology before and after treatment. Histological assessment of the testes showed no alterations in control, no significant alterations in Maca, mild to moderate alterations in THC, and mild alterations in THC + Maca groups. Histological examination of the other organs showed no significant differences among the groups. Tubular diameter showed significantly increased thickening for THC and THC + Maca compared with that for Maca and control. Moreover, seminiferous epithelium height decreased for THC compared with that in the control, Maca, and THC + Maca groups. No statistically significant reduction in the spermatogenic index was observed for THC compared with that for Maca and THC + Maca. Epididymal cross-sections of the groups showed no significant alterations. Sperm concentration and motility were higher for control and THC + Maca groups than in group THC and Maca. Conclusion: In vivo maca administration reduced the deleterious effect of THC on testicular parenchyma and semen production.

Imaging techniques in Veterinary Medicine. Part I: Radiography and Ultrasonography.

In Veterinary Medicine all the Imaging techniques are used and described but, due to organizational, managerial and, mostly, economical reasons, Radiography and Ultrasonography are the most used. Veterinary Radiology teaching has a relatively small number of educational credits in the degree courses but, nowday, educational opportunities are remarkably increased thanks to a number of post-degree courses and masters, organized both by the universities and private veterinary associations. The relevance of Diagnostic Imaging is particularly true in Veterinary Medicine, in which Radiology could be considered "indispensable" for diagnosis, prognosis and follow up. Furthermore, it should not be forgotten that the diagnostic image represents a "document" and, has a relevant role in legal medical debats. In this first part, Radiography and Ultrasonography are described.

A computed tomography-based method for the assessment of canine retrobulbar cone volume for ophthalmic anaesthesia.

OBJECTIVE: To develop a comprehensive formula for calculating the volume of local anaesthetic solution used for retrobulbar anaesthesia in dogs with different skull morphologies. STUDY DESIGN: Retrospective cohort imaging study. ANIMALS: Skull computed tomography (CT) images of 188 dogs of different breeds collected between January 2009 and December 2017. METHODS: Anatomical integrity of the orbit and adjacent structures, presenting complaint, clinical signs and CT findings were verified to exclude ocular abnormalities. The volume of the retrobulbar cone of 376 eyes was calculated using CT scans of the dogs' skulls. Additional data recorded included morphology of the skull, body weight, sex and size of the dogs, all of which were matched for possible association to the retrobulbar cone volume through univariable and multivariable linear regression models. Results of linear regression models were expressed as estimated beta coefficients with the corresponding 95% confidence intervals (95% CIs). RESULTS: Using univariate analysis, the retrobulbar cone volume was positively associated with weight and male sex. In addition, brachycephalic and dolichocephalic dogs showed a larger retrobulbar cone volume than mesocephalic dogs, while sex was no longer significantly associated with the retrobulbar cone volume. In multivariate analysis, when considering all variables in the model, weight emerged as the strongest predictor (beta coefficient: 0.062 mL kg-1, 95% CI: 0.056-0.067 mL kg-1, p < 0.001). CONCLUSIONS: and clinical relevance In the veterinary literature, there is no agreement on the precise volume of local anaesthetic solution that should be used to achieve intraconal retrobulbar anaesthesia in dogs. Here we suggest a formula to calculate the retrobulbar cone volume and, accordingly, the injection volume of local anaesthetic solution for effective retrobulbar anaesthesia.

The phosphatase Shp1 interacts with and dephosphorylates cortactin to inhibit invadopodia function.

BACKGROUND: Invadopodia are actin-based cell-membrane protrusions associated with the extracellular matrix degradation accompanying cancer invasion. The elucidation of the molecular mechanisms leading to invadopodia formation and activity is central for the prevention of tumor spreading and growth. Protein tyrosine kinases such as Src are known to regulate invadopodia assembly, little is however known on the role of protein tyrosine phosphatases in this process. Among these enzymes, we have selected the tyrosine phosphatase Shp1 to investigate its potential role in invadopodia assembly, due to its involvement in cancer development. METHODS: Co-immunoprecipitation and immunofluorescence studies were employed to identify novel substrate/s of Shp1AQ controlling invadopodia activity. The phosphorylation level of cortactin, the Shp1 substrate identified in this study, was assessed by immunoprecipitation, in vitro phosphatase and western blot assays. Short interference RNA and a catalytically-dead mutant of Shp1 expressed in A375MM melanoma cells were used to evaluate the role of the specific Shp1-mediated dephosphorylation of cortactin. The anti-invasive proprieties of glycerophosphoinositol, that directly binds and regulates Shp1, were investigated by extracellular matrix degradation assays and in vivo mouse model of metastasis. RESULTS: The data show that Shp1 was recruited to invadopodia and promoted the dephosphorylation of cortactin at tyrosine 421, leading to an attenuated capacity of melanoma cancer cells to degrade the extracellular matrix. Controls included the use of short interference RNA and catalytically-dead mutant that prevented the dephosphorylation of cortactin and hence the decrease the extracellular matrix degradation by melanoma cells. In addition, the phosphoinositide metabolite glycerophosphoinositol facilitated the localization of Shp1 at invadopodia hence promoting cortactin dephosphorylation. This impaired invadopodia function and tumor dissemination both in vitro and in an in vivo model of melanomas. CONCLUSION: The main finding here reported is that cortactin is a specific substrate of the tyrosine phosphatase Shp1 and that its phosphorylation/dephosphorylation affects invadopodia formation and, as a consequence, the ability of melanoma cells to invade the extracellular matrix. Shp1 can thus be considered as a regulator of melanoma cell invasiveness and a potential target for antimetastatic drugs. Video abstract.

The Use of Platelet-Rich Plasma for Treatment of Tenodesmic Lesions in Horses: A Systematic Review and Meta-Analysis of Clinical and Experimental Data.

The use of platelet-rich plasma (PRP) to enhance tenodesmic lesion healing has been questioned over the years. The aim of this study was to evaluate current literature to establish the effectiveness of PRP for treating tenodesmic lesions through a systematic review, in accordance with the PRISMA guidelines, and a meta-analysis. Studies comparing PRP with placebo or other treatments for horses with tenodesmic injuries or evaluated PRP effect on tendon and ligament explants were included. Outcomes were clinical, ultrasound, histologic, molecular evaluation, and adverse effects. Two authors independently extracted data and assessed each study's risk of bias. Treatment effects were evaluated using risk ratios for dichotomous data, together with 95% CI. Data were pooled using the random-effects model. The quality of the evidence for each outcome was assessed using GRADE criteria. Twenty-four trials met inclusion criteria for systematic review, while fifteen studies were included in the meta-analysis. Results showed no significant differences in the outcomes between PRP and control groups. Finally, there is no definitive evidence that PRP enhances tendons and ligaments healing. Therefore, there is a need for more controlled trials to draw a firmer conclusion about the efficacy of PRP as a treatment for tenodesmic lesions in the horse.

Ultrasonographic measurement of kidney-to-aorta parameters in Whippets.

In a previous study, an ultrasonographic method to assess kidney size in dogs as a ratio of kidney length to aortic luminal diameter (KL/AoD ratio) was proposed. The main limitation of this method was the wide range of normal values (5.5-9.1), which resulted in poor sensitivity and specificity. The aim of this prospective, observational, reference interval study was to determine whether the KL/AoD normal cut-off values in a single breed (Whippets) would have a narrower range than the previously reported normal reference ranges. The influence of sex, age, weight, and side on kidney length (KL) and of sex, age, weight, and scanning plane (longitudinal vs transversal) on aortic luminal diameter (AoD) were also investigated. Thirty-six clinically healthy Whippets (16 males, 20 females) without ultrasonographic renal lesions were included in this study. The 95% confidence interval of mean KL/AoD was found to be narrower than the previously reported range (ie, 6.3-6.9 versus 5.5-9.1). This was considered to be especially notable in that the KL in this breed exhibits marked sexual dimorphism. The KL/AoD ratio did not differ between right versus left sides or male versus female sexes in Whippets (P > .05). Findings from the current study provided KL/AoD ratio normal reference range cut-off values for future use in Whippets and supported the use of breed-specific KL/AoD ratio values for characterizing abnormal renal size in other canine breeds.

FOXE1 Gene Dosage Affects Thyroid Cancer Histology and Differentiation In Vivo.

The transcription factor Forkhead box E1 (FOXE1) is a key player in thyroid development and function and has been identified by genome-wide association studies as a susceptibility gene for papillary thyroid cancer. Several cancer-associated polymorphisms fall into gene regulatory regions and are likely to affect FOXE1 expression levels. However, the possibility that changes in FOXE1 expression modulate thyroid cancer development has not been investigated. Here, we describe the effects of FOXE1 gene dosage reduction on cancer phenotype in vivo. Mice heterozygous for FOXE1 null allele (FOXE1+/-) were crossed with a BRAFV600E-inducible cancer model to develop thyroid cancer in either a FOXE1+/+ or FOXE1+/- genetic background. In FOXE1+/+ mice, cancer histological features are quite similar to that of human high-grade papillary thyroid carcinomas, while cancers developed with reduced FOXE1 gene dosage maintain morphological features resembling less malignant thyroid cancers, showing reduced proliferation index and increased apoptosis as well. Such cancers, however, appear severely undifferentiated, indicating that FOXE1 levels affect thyroid differentiation during neoplastic transformation. These results show that FOXE1 dosage exerts pleiotropic effects on thyroid cancer phenotype by affecting histology and regulating key markers of tumor differentiation and progression, thus suggesting the possibility that FOXE1 could behave as lineage-specific oncogene in follicular cell-derived thyroid cancer.

New CXCR4 Antagonist Peptide R (Pep R) Improves Standard Therapy in Colorectal Cancer.

The chemokine receptor CXCR4 is overexpressed and functional in colorectal cancer. To investigate the role of CXCR4 antagonism in potentiating colon cancer standard therapy, the new peptide CXCR4 antagonist Peptide R (Pep R) was employed. Human colon cancer HCT116 xenograft-bearing mice were treated with chemotherapeutic agents (CT) 5-Fluorouracil (5FU) and oxaliplatin (OX) or 5FU and radio chemotherapy (RT-CT) in the presence of Pep R. After two weeks, CT plus Pep R reduced by 4-fold the relative tumor volume (RTV) as compared to 2- and 1.6-fold reductions induced, respectively, by CT and Pep R. In vitro Pep R addition to CT/RT-CT impaired HCT116 cell growth and further reduced HCT116 and HT29 clonal capability. Thus, the hypothesis that Pep R could target the epithelial mesenchyme transition (EMT) process was evaluated. While CT decreased ECAD and increased ZEB-1 and CD90 expression, the addition of Pep R restored the pretreatment expression. In HCT116 and HT29 cells, CT/RT-CT induced a population of CD133+CXCR4+ cells, supposedly a stem-resistant cancer cell population, while Pep R reduced it. Taken together, the results showed that targeting CXCR4 ameliorates the effect of treatment in colon cancer through inhibition of cell growth and reversal of EMT treatment-induced markers, supporting further clinical studies.

Ultrasound-guided percutaneous antegrade pyelography for suspected ureteral obstruction in 6 pet guinea pigs (Cavia porcellus).

OBJECTIVES: To describe the feasibility and safety of ultrasound-guided percutaneous antegrade pyelography (US-PAP) in pet guinea pigs (Cavia porcellus) with suspected ureteral obstruction. MATERIALS AND METHODS: Six adult pet guinea pigs (4 females and 2 males, all intact) were evaluated for suspected ureteral obstruction. The mean weight of the guinea pigs was 0.8 ± 0.25 kg (range 0.4-1.1 kg), and mean age was 4.07 ± 1.63 years (range 2-7 years). All animals were free from comorbid diseases, had clinical signs of urologic disease and were referred based on either strong clinical suspicion of, or diagnostic imaging of ureteral obstruction. Data on signalment and clinical examination findings, response to anaesthesia and imaging findings were recorded. RESULTS: Partial ureteral obstruction was confirmed in all guinea pigs but one, in which a complete ureteral obstruction occurred. Uroliths were in both ureters of 5 cases and in both the left renal pelvis and ureters in 1 case. All guinea pigs showed a normal appetite and regular defaecation within 2 h following the procedure. No intraoperative or immediate postoperative complications were encountered after the procedure. The only complication was contrast medium leakages in the subcapsular perinephric, retroperitoneal and, in one case, peritoneal space, which caused no overt clinical consequences afterwards. In one male patient, mobilisation of the ureteral calculus occurred and the urolith was found in the urinary bladder on the radiograph taken after contrast medium injection. CLINICAL SIGNIFICANCE: The US-PAP technique is a useful, safe and easy-to-perform diagnostic tool in guinea pigs with hydronephrosis and hydroureter.

Junctional adhesion molecule-A is down-regulated in anaplastic thyroid carcinomas and reduces cancer cell aggressiveness by modulating p53 and GSK3 α/ß pathways.

Junctional adhesion molecule A (JAM-A) is a transmembrane protein that contributes to different biological process, including the epithelial to mesenchymal transition (EMT). Through an EMT profiler array, we explored the molecular players associated with human thyroid cancer progression and identified JAM-A as one of the genes mostly deregulated. The quantitative real-time polymerase chain reaction and immunohistochemistry analyses showed that downregulation of JAM-A occurred in anaplastic thyroid carcinoma (ATC) compared with normal thyroid (NT) and papillary thyroid carcinoma (PTC) tissues and correlated with extrathyroid infiltration, tumor size, and ATC histotype. In ATC cell lines, JAM-A restoration suppressed malignant hallmarks of transformation including cell proliferation, motility, and transendothelial migration. Accordingly, knockdown of JAM-A enhanced thyroid cancer cell proliferation and motility in PTC cells. Through the proteome profiler human phospho-kinase array, we demonstrated that higher expression of JAM-A was associated with a significant increased level of phosphorylation of p53 and GSK3 α/ß proteins. In conclusion, our findings highlight a novel role of JAM-A in thyroid cancer progression and suggest that JAM-A restoration could have potential clinical relevance in thyroid cancer treatment.

Helper-dependent adenovirus-mediated gene transfer of a secreted LDL receptor/transferrin chimeric protein reduces aortic atherosclerosis in LDL receptor-deficient mice.

Familial hypercholesterolemia (FH) is a genetic hyperlipidemia characterized by elevated concentrations of plasma LDL cholesterol. Statins are not always effective for the treatment of FH patients; unresponsive patients have poor prognosis and rely on LDL apheresis. In the past, we developed safe and effective gene therapy strategies for the expression of anti-atherogenic proteins using PEGylated helper-dependent adenoviral (HD-Ad) vectors. We recently developed a HD-Ad vector for the expression of the soluble form of the extracellular portion of the human LDL receptor (LDLR) fused with a rabbit transferrin dimer (LDLR-TF). We evaluated the efficacy of the LDLR-TF chimeric protein  in CHOLDLA7, a cell line lacking LDLR expression, restoring the ability to uptake LDL. Subsequently, we administered intravenously 1 × 10E13 vp/kg of this vector in LDLR-deficient mice and observed amelioration of lipid profile and reduction of aortic atherosclerosis. Finally, we studied LDL distribution after HD-Ad vector-mediated expression of LDLR-TF in LDLR-deficient mice and found LDL accumulation in liver, and in heart and intestine. These results support the possibility of lowering LDL-C levels and reducing aortic atherosclerosis using a secreted therapeutic transgene; the present strategy potentially can be modified and adapted to non-systemic gene transfer with expression of the secreted chimeric protein in muscle or other tissues. Intramuscular or local administration strategies could improve the safety profile of this strategy and facilitate applicability.

Measurement of [123I]FP-CIT binding to the dopamine transporter (DAT) in healthy mouse striatum using dedicated small animal SPECT imaging: feasibility, optimization and validation.

BACKGROUND: In-vivo imaging of dopamine transporter (DAT), a reliable marker of degeneration of nigrostriatal dopaminergic innervation, has gained increasing interest in preclinical neurodegenerative research for studying disease mechanisms and testing new therapeutic strategies. We assessed the feasibility and the reliability of in vivo and ex vivo quantification of Methyl (3S,4S,5R)-8-(3-fluoropropyl)-3-(4-iodophenyl)-8-azabicyclo[3.2.1]octane-4-carboxylate ([123I]FP-CIT) binding to striatal DAT sites in mouse brain. METHODS: Dedicated small animal single-photon emission computed tomography (SPECT) images of [123I]FP-CIT binding were obtained in 3 groups of healthy mice: untreated (N.=6), pre-treated with lugol solution (N.=4), and pre-treated with selective dopamine transporter uptake inhibitor GBR12909 (N.=4). Ex-vivo autoradiography studies were performed at the end of SPECT studies with phosphor image system in 4 out of the 6 untreated mice and in all mice pre-treated with lugol. Regions of interest were defined over the striatum. The specific binding (SB) was calculated using the cerebral cortex as reference region. RESULTS: SPECT images in untreated mice showed high [123I]FP-CIT uptake in the striatum and extracerebral regions. Lugol pretreatment improved striatal images quality decreasing salivary and thyroid glands uptake. SB was higher (P<0.0001) in mice pre-treated with lugol (5.97±0.60) than in untreated mice (2.25±0.28). Autoradiography showed similar SB findings in untreated (2.27±0.33) and lugol-treated (4.27±0.57) mice (P<0.0001). In-vivo striatal 123I-FP-CIT SB and ex-vivo striatal 123I-FP-CIT SB were significantly correlated (r=0.87; P<0.0001). SPECT competition studies showed a significant (P<0.0001) reduction of [123I]FP-CIT SB in the striatum after GBR12909. CONCLUSIONS: We demonstrated the feasibility of [123I]FP-CIT imaging of the normal mouse brain using small-animal SPECT without pinhole collimators. The reliability of quantitative measurement of striatal [123I]FP-CIT SB is supported by competition studies showing measurable inhibition of uptake induced by GBR12909 and by the strong correlation between in vivo and ex vivo striatal [123I]FP-CIT SB. Our data also demonstrate that pre-treatment with lugol might improve striatal [123I]FP-CIT SB in mice.