Validation of an Engineered Cell Model for In Vitro and In Vivo HIF-1 alpha Evaluation by Different Imaging Modalities

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po 259 inhibition of the hexosamine biosynthetic pathway by targeting pgm3 causes breast cancer growth arrest and apoptosis

Introduction Cancer aberrant N - and O -linked protein glycosylation, frequently resulting from an augmented flux through the Hexosamine Biosynthetic Pathway (HBP), play different roles in tumour progression. Recent studies reported an association between the tumorigenic potential, metastasis and chemoresistance of several type of breast cancer cells and tumours, among which the Triple Negative Breast Cancer (TNBC), and the alteration of their membrane glycans composition and ramification as well as of their level of protein O -Glc N Ac. However, the low specificity and toxicity of the existing HBP inhibitors prevented their use for cancer treatment. Material and methods In order to identify a novel inhibitor of HBP pathway and in particular of the PGM3 enzyme, we performed a virtual screening by using computational approaches. These approaches lead us to the identification of a lead compound. This compound, named FR054, has been synthetized and in vitro and in vivo tested by using several biophysical methods (NMR, LC/MS, HPLC) and biochemical assay (CETSA, ITDRF, FACS analysis) as well as tested in TNBC xenograft mice model. Results and discussions Here we report the preclinical evaluation of FR054, a novel inhibitor of the HBP enzyme PGM3, with a remarkable anti-breast cancer effect. In fact, FR054 induces in different breast cancer cells a dramatic decrease in cell proliferation and survival. In particular, in a model of Triple Negative Breast Cancer (TNBC) cells, MDA-MB-231, we show that these effects are correlated to FR054-dependent reduction of both N - and O -glycosylation level that cause also to a strong reduction of cancer cell adhesion and migration. Moreover we show that impaired survival of cancer cells upon FR054 treatment is associated with activation of the Unfolded Protein Response (UPR) and accumulation of intracellular ROS. Finally, we show that FR054 suppresses cancer growth in MDA-MB-231 xenograft mice. Conclusion Our data support the advantage of targeting HBP for therapeutic purpose and encourage further investigation about the use of this small-molecule as promising compound for breast cancer therapy

Predictors of 1-year compliance with adaptive servoventilation in patients with heart failure and sleep disordered breathing: preliminary data from the ADVENT-HF trial

Despite its effectiveness in suppressing sleep disordered breathing (SDB), positive airway pressure therapy (PAP) is not always well tolerated by patients and long-term adherence can be problematic. Recently, two multicentre, randomised clinical trials (RCTs) tested the effects of PAP for patients with cardiovascular disease and co-existing SDB on morbidity and mortality with negative outcomes [1, 2]. Relatively poor adherence to PAP therapy (mean 3.7 and 3.3 h·day-1, respectively) in these two trials might have contributed to their poor results. Indeed, higher PAP use per day is associated with better clinical outcomes than lower use [3]

Quality and fertilizing ability of electro-ejaculated cat spermatozoa frozen with or no Equex STM Paste

An optimal protocol for cat semen cryopreservation has not yet been defined. Addition of Equex STM Paste has been tested for epididymal cat spermatozoa but not for ejaculated cat spermatozoa. Furthermore, the effect of Equex STM Paste on fertilizing ability of cryopreserved semen has never been evaluated in that species. Therefore, the aims of the current study were to investigate if addition of Equex STM Paste to a freezing extender for electroejaculated cat (Felis catus) semen would improve postthaw sperm quality and if sperm fertilizing ability after cryopreservation with or without Equex STM Paste was preserved. Semen was collected by electroejaculation and frozen in a Tris-glucose-citrate egg yolk extender supplemented with (0.5% vol/vol) or without Equex STM Paste. In Experiment 1, sperm motility, membrane integrity, and acrosomal status were determined immediately after collection and at 0, 3, and 6 h postthaw. In Experiment 2, frozen semen from the two groups was used for in vitro fertilization (IVF) of in vitro-matured cat oocytes. Cleavage rate was recorded 30 h after IVF, and embryo development was evaluated on Days 6 and 7 of culture. In Experiment 1, the rate of motile spermatozoa after freezing-thawing was higher when Equex STM Paste was added to the freezing extender, but progressive motility score was not influenced (P > 0.05). Sperm membrane integrity was positively affected (P 0.05) between groups. Even if the decreasing rates of motility and membrane integrity were more rapid in presence of Equex than those in controls, total motility and sperm viability were similar at 3 and 6 h after thawing (P > 0.05). In Experiment 2, there was no difference in fertilizing ability and embryo development between the two groups (P > 0.05). The results of this study demonstrate that the addition of Equex STM Paste in the freezing extender avoids the loss of motile spermatozoa and maintains fertilizing ability of frozen-thawed spermatozo

Ovarioisterectomia e vulvovaginectomia per il trattamento di recidive di neoplasie vaginali in una cagna

Un’ovarioisterectomia associata ad una vulvovaginectomia radicale, per il trattamento di recidive di neoplasie vaginali, è stata eseguita in una Labrador di 14 anni, già sottoposta in tre occasioni ad interventi chirurgici d’asportazione di neoformazioni. L’intervento ha avuto esito positivo e la cagna non ha evidenziato alcun problema nel postoperatorio

Sperm quality after collection with three different UrCaPI (Urethral Catheterization after Pharmacological Induction) protocols

The urethral catheterization after the medetomidine pharmacological effect (UrCaPI) is the most practical technique to obtain a good cat ejaculate because not requires a specific equipment, as the electroejaculation (EE), or a trained tomcat, as the sperm collection with artificial vagina. As reported in literature the sperm collected by UrCaPI or by EE could be used for cryopreservation or for in vitro fertilization with the same results. The aim of this study is to evaluate if different protocols used for UrCaPI collection permits to obtain significative differences in sperm quality.In this experiment were enrolled male cats referred, between January and May 2009, to Animal Reproduction Unit of Veterinary Clinical Department \u2013 University of Bologna, in order to be submitted to orchiectomy. A total of 59 adult (1-4 years), mixed-breed male cats, privately owned or belonging to a cat pound, clinically healthy with two palpably normal descended testicles were used for this experiment. Before starting the study, the cats were random assigned to one of three groups for sperm collection using different protocols of UrCaPI. The UrCaPI was performed as described by Zambelli et al., using a 3F by 11 cm long urinary tomcat catheter with the tip cutted. All the animals were intramuscularly injected with medetomidine (120 μg/kg of body weight) in order to perform sperm collection and then the anaesthesia was maintained with 1.5-2% isoflurane in oxygen in order to perform the orchiectomy. In 11/59 cats (group 1) the collection was performed immediately once medetomidine pharmacological effect was obtained, 23/59 cats (group 2) were collected three times every 5 minutes after the pharmacological effect, finally in 25/59 (group 3) UrCaPI was performed 20 minutes after pharmacological effect was reached. Semen was collected into a pre-warmed plastic 1,5 mL eppendorf tube. The volume was measured using a calibrated pipette. Successively, 2 \ub5l of sperm was diluted with 18 \ub5L of Tris-glucose-citrate in order to be correctly evaluated. The percent motility (0-100%) and progressive motility (scale 0-5) were immediately estimated using a phase-contrast microscope (X400) equipped with a warming plate. A B\ufcrker chamber was used for measuring sperm concentration (x106/ml). Data from semen evaluation have been expressed as mean \ub1 S.D. and analysed using a t-test for independent samples or a Wald-Wolfowitz runs test, depending on data distribution (Statistica for Windows, Stat Soft Inc., Tulsa, Oklahoma, USA). A value of p<0.01 was considered significant. Sperm quality did not significantly differ between the three method of collection (p>0.01). Conclusions. Quality of sperm collected using a single catheterization (Group 1) immediately after pharmacological medetomidine effect was statistically not different (p>0.01) in comparison with sperm quality obtained with the other two protocols. Moreover this method avoids the urethra traumatism that a multiple catheterization could induce