Transgenic analysis of Smad4

Mammary gland involution proceeds through massive, highly controlled epithelial cell apoptosis and tissue remodelling. Thus, mammary gland represents an ideal physiological environment in which to study apoptosis. Recently, the Smad gene family has been identified as mediators of TGF-P superfamily signaling and has been implicated in mediating epithelial cell apoptosis. The Smad family of signal transduction proteins focuses around a central mediator, Smad4, and this report presents a number of analyses which have been undertaken to investigate the potential apoptotic role of Smad4 in the mammary gland. To investigate the role of Smad4 I have utilised an over-expressing Smad4 transgenic mouse. This transgenic mouse has been designed to increase Smad4 protein in the secretory epithelial cells of the mammary gland during pregnancy and lactation by using the biological properties of the BLG promoter. Histological investigation of Smad4 slides indicated accelerated involution and significant increase of apoptosis at day 2 and 3 of involution. Molecular analysis through Western blots for STAT3 and STAT5a levels showed a deregulation of both proteins at the same time points. The mechanism with which transgenic mice regulated apoptosis was prove by Western blots to be via the p27Kipl molecular pathway and independent of p21wa or Box. Microarray analysis for day 3 involuting mice showed an over expression of Vitamin D Receptor. A result which was confirmed by both semi quantitative RT-PCR and Western blot analysis. Raw microarray data showed a down regulation of Methyl Binding Domain 2 (MBD2) in Smad4 transgenic mice indicating that MBD2 could play a role in mammary gland involution. However, analysis of conditional mammary knockout MBD2 mice at day 3 involution did not show any involvement of MBD2 in the regulation mammary gland. Another way of investigating Smad4 was by passing the Smad4 construct to Stat3 mammary knockout animals with the hypothesis that Smad4 could restore STAT3's delayed involution. Based on the deregulation of STAT3 protein levels in the smad4 transgenics the above hypothesis was investigated at day 3 of involution. Phenotypic investigation showed that over expression of smad4 is not able to restore the delayed phenotype of STAT3 knockouts. My final investigation was to identify molecules whose expression is altered by a conditional deletion of STAT3 in the mammary gland. This investigation was approached through microarrays which demonstrated an over expression of the Bone Morphogenic Protein receptor, a result which demonstrates once more the synergistic cooperation of Smads and STATS.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Transgenic analysis of Smad4

Mammary gland involution proceeds through massive, highly controlled epithelial cell apoptosis and tissue remodelling. Thus, mammary gland represents an ideal physiological environment in which to study apoptosis. Recently, the Smad gene family has been identified as mediators of TGF-P superfamily signaling and has been implicated in mediating epithelial cell apoptosis. The Smad family of signal transduction proteins focuses around a central mediator, Smad4, and this report presents a number of analyses which have been undertaken to investigate the potential apoptotic role of Smad4 in the mammary gland. To investigate the role of Smad4 I have utilised an over-expressing Smad4 transgenic mouse. This transgenic mouse has been designed to increase Smad4 protein in the secretory epithelial cells of the mammary gland during pregnancy and lactation by using the biological properties of the BLG promoter. Histological investigation of Smad4 slides indicated accelerated involution and significant increase of apoptosis at day 2 and 3 of involution. Molecular analysis through Western blots for STAT3 and STAT5a levels showed a deregulation of both proteins at the same time points. The mechanism with which transgenic mice regulated apoptosis was prove by Western blots to be via the p27Kipl molecular pathway and independent of p21wa or Box. Microarray analysis for day 3 involuting mice showed an over expression of Vitamin D Receptor. A result which was confirmed by both semi quantitative RT-PCR and Western blot analysis. Raw microarray data showed a down regulation of Methyl Binding Domain 2 (MBD2) in Smad4 transgenic mice indicating that MBD2 could play a role in mammary gland involution. However, analysis of conditional mammary knockout MBD2 mice at day 3 involution did not show any involvement of MBD2 in the regulation mammary gland. Another way of investigating Smad4 was by passing the Smad4 construct to Stat3 mammary knockout animals with the hypothesis that Smad4 could restore STAT3's delayed involution. Based on the deregulation of STAT3 protein levels in the smad4 transgenics the above hypothesis was investigated at day 3 of involution. Phenotypic investigation showed that over expression of smad4 is not able to restore the delayed phenotype of STAT3 knockouts. My final investigation was to identify molecules whose expression is altered by a conditional deletion of STAT3 in the mammary gland. This investigation was approached through microarrays which demonstrated an over expression of the Bone Morphogenic Protein receptor, a result which demonstrates once more the synergistic cooperation of Smads and STATS

Guanylyl cyclase activation reverses resistive breathing–induced lung injury and inflammation

Inspiratory resistive breathing (RB), encountered in obstructive lung diseases, induces lung injury. The soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway is down-regulated in chronic and acute animal models of RB, such as asthma, chronic obstructive pulmonary disease, and in endotoxin-induced acute lung injury. Our objectives were to: (1) characterize the effects of increased concurrent inspiratory and expiratory resistance in mice via tracheal banding; and (2) investigate the contribution of the sGC/cGMP pathway in RB-induced lung injury. Anesthetized C57BL/6 mice underwent RB achieved by restricting tracheal surface area to 50% (tracheal banding). RB for 24 hours resulted in increased bronchoalveolar lavage fluid cellularity and protein content, marked leukocyte infiltration in the lungs, and perturbed respiratory mechanics (increased tissue resistance and elasticity, shifted static pressure–volume curve right and downwards, decreased static compliance), consistent with the presence of acute lung injury. RB down-regulated sGC expression in the lung. All manifestations of lung injury caused by RB were exacerbated by the administration of the sGC inhibitor, 1H-[1,2,4]oxodiazolo[4,3-]quinoxalin-l-one, or when RB was performed using sGCα1 knockout mice. Conversely, restoration of sGC signaling by prior administration of the sGC activator BAY 58-2667 (Bayer, Leverkusen, Germany) prevented RB-induced lung injury. Strikingly, direct pharmacological activation of sGC with BAY 58-2667 24 hours after RB reversed, within 6 hours, the established lung injury. These findings raise the possibility that pharmacological targeting of the sGC–cGMP axis could be used to ameliorate lung dysfunction in obstructive lung diseases

Combined Endovascular/Surgical Management of a Ruptured Para-Anastomotic Aneurysm of the Left Common Iliac Artery#

A 75-year old man presented with signs and symptoms of acute abdomen and a clinical picture of hypovolemic shock. An emergency CT scan revealed a ruptured para-anastomotic left common iliac artery aneurysm. The patient had undergone an elective abdominal aortic aneurysm repair operation and placement of an aortoiliac bifurcated graft 10 years before. Para-anastomotic aneurysms had developed in all 3 (aortic and the 2 iliac) anastomosis. As the patient was highrisk, a combined endovascular/surgical approach was undertaken. The patient was discharged 4 days later

Characteristics of patients presenting to the vascular emergency department of a tertiary care hospital: a 2-year study

<p>Abstract</p> <p>Background</p> <p>The structure of health care in Greece is receiving increased attention to improve its cost-effectiveness. We sought to examine the epidemiological characteristics of patients presenting to the vascular emergency department of a Greek tertiary care hospital during a 2-year period. We studied all patients presenting to the emergency department of vascular surgery at Red Cross Hospital, Athens, Greece between 1^stJanuary 2009 and 31st December 2010.</p> <p>Results</p> <p>Overall, 2452 (49.4%) out of 4961 patients suffered from pathologies that should have been treated in primary health care. Only 2509 (50.6%) needed vascular surgical intervention.</p> <p>Conclusions</p> <p>The emergency department of vascular surgery in a Greek tertiary care hospital has to treat a remarkably high percentage of patients suitable for the primary health care level. These results suggest that an improvement in the structure of health care is needed in Greece.</p

Carotid disease and periodontal disease

Aim: The aim of this pilot study was to investigate whether periodontal disease is associated with the instability of carotid plaques as indicated by ultrasound and histopathologic markers. Material and method: The study consisted of 70 patients that underwent surgery for carotid artery stenosis at “Red Cross” Hospital in Athens. Prior to surgery symptomatic plaques were recorded and patients were examined with ultrasound for plaque stability and histopathologic bay CD-68 and a-SMA markers. Additionally, patients received full mouth periodontal examination including measurements of attachment loss (AL), probing pocket depth (PPD) and periodontal inamed surface area (PISA). Statistical analysis was performed using the X2 test. Result: Instable carotid plaque, as diagnosed based to GSM, CD-68 and α-SMA markers, is related to increased values of probing pocket depth - PPD, attachment loss - AL, bleeding during control - BoP, periodontal inamed surface area - PISA and the presence of periodontitis (PERIO1). Conclusion: Periodontitis is related to instable carotid plaque and can be considered as an indepented factor to symptomatic carotid disease.Σκοπός: Σκοπός της παρούσης ερευνητικής εργασίας ήταν η μελέτη της σχέσης μεταξύ αθηρωματικής νόσου του καρωτιδικού διχασμού και της περιοδοντικής νόσου. Ειδικότερα διερευνήθηκε κατά πόσο η περιοδοντική νόσος σχετίζεται με την αστάθεια των αθηρωματικών πλακών, όπως υποδεικνύεται από το υπερηχογράφημα και τα παθολογοανατομικά χαρακτηριστικά των ευάλωτων καρωτιδικών πλακών και πιο συγκεκριμένα με τους δείκτες CD68 και τα aSMA. Υλικό Και Μέθοδος. Στο πρωτόκολλο της παρούσας εργασίας έλαβαν μέρος 70 ασθενείς που παρουσίαζαν ασυμπτωματική και συμπτωματική στένωση του καρωτιδικού διχασμού ≥ 70%. Σε όλους τους ασθενείς έγινε υπερηχογραφικός έλεγχος, περιοδοντολογική εξέταση - μέτρηση βάθους θυλάκου (PPD), μέτρηση απώλειας πρόσφυσης (CAL), μέτρηση της αιμορραγίας κατά την ανίχνευση (BOP), μέτρηση δείκτη έκτασης φλεγμονής γύρω από τα δόντια (PISA - periodontal inamed surface area) και προσδιορισμός των ασθενών με περιοδοντίτιδα (4 θύλακες με βάθος θυλάκου ≥4 και απώλεια πρόσφυσης ≥3 και τέλος έγινε ανοσοϊστοχημική εξέταση για τον προσδιορισμό των μικροφάγων και των λείων μυϊκών κυττάρων. Εν συνεχεία έγινε συσχέτιση του GSM της καρωτιδικής πλάκας και των δεικτών CD68 και αSMA της καρωτιδικής πλάκας με τους δείκτες περιοδοντίτιδας. Αποτέλεσμα. Η αστάθεια της πλάκας, όπως διαγνώσθηκε με βάση το GSM, το δείκτη CD-68 και το δείκτη α-SMA, σχετίζεται με μεγαλύτερες τιμές βάθος θυλάκου - PD, απώλεια πρόσφυσης - CAL, αιμορραγίας κατά την ανίχνευση - BoP, δείκτη έκτασης φλεγμονής γύρω από τα δόντια - PISA και με την παρουσία περιοδοντίτιδας (PERIO1). Συμπέρασμα. Η περιοδοντίτιδα συσχετίζεται με την ευάλωτη καρωτιδική πλάκα και θα μπορούσαμε να την δούμε ως έναν ανεξάρτητο παράγοντα για την συμπτωματική καρωτιδική νόσο

RNA-seq data analysis of stimulated hepatocellular carcinoma cells treated with epigallocatechin gallate and fisetin reveals target genes and action mechanisms

Hepatocellular carcinoma (HCC) is an essentially incurable inflammation-related cancer. We have previously shown by network analysis of proteomic data that the flavonoids epigallocatechin gallate (EGCG) and fisetin (FIS) efficiently downregulated pro-tumor cytokines released by HCC through inhibition of Akt/mTOR/RPS6 phospho-signaling. However, their mode of action at the global transcriptome level remains unclear. Herein, we endeavor to compare gene expression alterations mediated by these compounds through a comprehensive transcriptome analysis based on RNA-seq in HEP3B, a responsive HCC cell line, upon perturbation with a mixture of prototypical stimuli mimicking conditions of tumor microenvironment or under constitutive state. Analysis of RNA-seq data revealed extended changes on HEP3B transcriptome imposed by test nutraceuticals. Under stimulated conditions, EGCG and FIS significantly modified, compared to the corresponding control, the expression of 922 and 973 genes, respectively, the large majority of which (695 genes), was affected by both compounds. Hierarchical clustering based on the expression data of shared genes demonstrated an almost identical profile in nutraceutical-treated stimulated cells which was virtually opposite in cells exposed to stimuli alone. Downstream enrichment analyses of the co-modified genes uncovered significant associations with cancer-related transcription factors as well as terms of Gene Ontology/Reactome Pathways and highlighted ECM dynamics as a nodal modulation point by nutraceuticals along with angiogenesis, inflammation, cell motility and growth. RNA-seq data for selected genes were independently confirmed by RT-qPCR. Overall, the present systems approach provides novel evidence stepping up the mechanistic understanding of test nutraceuticals, thus rationalizing their clinical exploitation in new preventive/therapeutic modalities against HCC. © 2020 The Author