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1.  The Elusive Role of the Prion Protein and the Mechanism of Toxicity in Prion Disease 
PLoS Pathogens  2015;11(5):e1004745.
PMCID: PMC4423772  PMID: 25951168
2.  Transgenic Fatal Familial Insomnia Mice Indicate Prion Infectivity-Independent Mechanisms of Pathogenesis and Phenotypic Expression of Disease 
PLoS Pathogens  2015;11(4):e1004796.
Fatal familial insomnia (FFI) and a genetic form of Creutzfeldt-Jakob disease (CJD178) are clinically different prion disorders linked to the D178N prion protein (PrP) mutation. The disease phenotype is determined by the 129 M/V polymorphism on the mutant allele, which is thought to influence D178N PrP misfolding, leading to the formation of distinctive prion strains with specific neurotoxic properties. However, the mechanism by which misfolded variants of mutant PrP cause different diseases is not known. We generated transgenic (Tg) mice expressing the mouse PrP homolog of the FFI mutation. These mice synthesize a misfolded form of mutant PrP in their brains and develop a neurological illness with severe sleep disruption, highly reminiscent of FFI and different from that of analogously generated Tg(CJD) mice modeling CJD178. No prion infectivity was detectable in Tg(FFI) and Tg(CJD) brains by bioassay or protein misfolding cyclic amplification, indicating that mutant PrP has disease-encoding properties that do not depend on its ability to propagate its misfolded conformation. Tg(FFI) and Tg(CJD) neurons have different patterns of intracellular PrP accumulation associated with distinct morphological abnormalities of the endoplasmic reticulum and Golgi, suggesting that mutation-specific alterations of secretory transport may contribute to the disease phenotype.
Author Summary
Genetic prion diseases are degenerative brain disorders caused by mutations in the gene encoding the prion protein (PrP). Different PrP mutations cause different diseases, including Creutzfeldt-Jakob disease (CJD) and fatal familial insomnia (FFI). The reason for this variability is not known, but assembly of the mutant PrPs into distinct aggregates that spread in the brain by promoting PrP aggregation may contribute to the disease phenotype. We previously generated transgenic mice modeling genetic CJD, clinically identified by dementia and motor abnormalities. We have now generated transgenic mice carrying the PrP mutation associated with FFI, and found that they develop severe sleep abnormalities and other key features of the human disorder. Thus, transgenic mice recapitulate the phenotypic differences seen in humans. The mutant PrPs in FFI and CJD mice are aggregated but unable to promote PrP aggregation. They accumulate in different intracellular compartments and cause distinct morphological abnormalities of transport organelles. These results indicate that mutant PrP has disease-encoding properties that are independent of its ability to self-propagate, and suggest that the phenotypic heterogeneity may be due to different effects of aggregated PrP on intracellular transport. Our study provides new insights into the mechanisms of selective neuronal dysfunction due to protein aggregation.
PMCID: PMC4400166  PMID: 25880443
3.  Possible graft-related complications in visceral debranching for hybrid B dissection repair 
Annals of Cardiothoracic Surgery  2014;3(4):393-399.
Hybrid repair (HR) of thoracoabdominal aortic aneurysm (TAAA) and dissection (TAAD), consisting of rerouting renovisceral branches followed by endograft aortic repair, has been shown to be a feasible option. It is especially appealing in patients unfit for both open and total endovascular repair. In order to determine the role of dissecting etiology and intraoperative variables as risk factors for graft-related complications in visceral debranching, we retrospectively analyzed the clinical outcomes, patency rate and hemodynamic alterations of the renovisceral debranching grafts in our series.
We analyzed 55 consecutive patients who underwent thoracoabdominal aortic HR between 2001 and 2013 in our center. Forty-four procedures were performed for TAAA and 11 procedures for TAAD. In TAAD patients, dissection involved 9/44 (20.5%) renovisceral vessels. One hundred and fifty-nine visceral bypasses were made (156 retrograde; three anterograde).
Thirty-day mortality was 12.7% (n=7). Potential graft-related complications included four cases of pancreatitis (7.3%) and five of peri-operative renal failure (9.1%). At a mean follow-up of 36.1 months, the global rate of visceral graft occlusion was 9.4% (15/159), leading to fatal bowel infarction in two patients and kidney loss in seven patients. Actuarial primary patency in renovisceral grafts at 12, 24, and 36 months was 96.3%, 92.6%, and 90.2% respectively. At the level of the anastomosis of the graft to the superior mesenteric artery, significant flow alterations (systolic peak velocity >250 cm/s) were observed during computed flow dynamics analysis in 18.5% of cases. Overall, an additional procedure to ensure patency was required in 19 bypasses intraoperatively and three during follow-up. The presence of aortic dissection had no significant impact on debranching graft-related complications. During multivariate analysis, retropancreatic routing to CT was the only independent predictor of graft-related complications (P=0.006).
Specific visceral graft-related complications were not uncommon in our series and were often associated with clinical consequences. Hemodynamic alterations of debranching grafts were observed in particular at the level of the anastomosis with the superior mesenteric artery. Careful follow-up is mandatory in order to monitor visceral bypasses and facilitate patency when required.
PMCID: PMC4128930  PMID: 25133102
Thoracoabdominal aortic aneurysm (TAAA); hybrid repair (HR); visceral bypass; stent
4.  Role of Lipid Rafts and GM1 in the Segregation and Processing of Prion Protein 
PLoS ONE  2014;9(5):e98344.
The prion protein (PrPC) is highly expressed within the nervous system. Similar to other GPI-anchored proteins, PrPC is found in lipid rafts, membrane domains enriched in cholesterol and sphingolipids. PrPC raft association, together with raft lipid composition, appears essential for the conversion of PrPC into the scrapie isoform PrPSc, and the development of prion disease. Controversial findings were reported on the nature of PrPC-containing rafts, as well as on the distribution of PrPC between rafts and non-raft membranes. We investigated PrPC/ganglioside relationships and their influence on PrPC localization in a neuronal cellular model, cerebellar granule cells. Our findings argue that in these cells at least two PrPC conformations coexist: in lipid rafts PrPC is present in the native folding (α-helical), stabilized by chemico-physical condition, while it is mainly present in other membrane compartments in a PrPSc-like conformation. We verified, by means of antibody reactivity and circular dichroism spectroscopy, that changes in lipid raft-ganglioside content alters PrPC conformation and interaction with lipid bilayers, without modifying PrPC distribution or cleavage. Our data provide new insights into the cellular mechanism of prion conversion and suggest that GM1-prion protein interaction at the cell surface could play a significant role in the mechanism predisposing to pathology.
PMCID: PMC4032283  PMID: 24859148
5.  Protein Misfolding and Neurodegenerative Diseases 
PMCID: PMC3988718  PMID: 24799906
6.  The inhibition of functional expression of calcium channels by prion protein demonstrates competition with α2δ for GPI-anchoring pathways 
Biochemical Journal  2014;458(Pt 2):365-374.
It has been shown recently that PrP (prion protein) and the calcium channel auxiliary α2δ subunits interact in neurons and expression systems [Senatore, Colleoni, Verderio, Restelli, Morini, Condliffe, Bertani, Mantovani, Canovi, Micotti, Forloni, Dolphin, Matteoli, Gobbi and Chiesa (2012) Neuron 74, 300–313]. In the present study we examined whether there was an effect of PrP on calcium currents. We have shown that when PrP is co-expressed with calcium channels formed from CaV2.1/β and α2δ-1 or α2δ-2, there is a consistent decrease in calcium current density. This reduction was absent when a PrP construct was used lacking its GPI (glycosylphosphatidylinositol) anchor. We have reported previously that α2δ subunits are able to form GPI-anchored proteins [Davies, Kadurin, Alvarez-Laviada, Douglas, Nieto-Rostro, Bauer, Pratt and Dolphin (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 1654–1659] and show further evidence in the present paper. We have characterized recently a C-terminally truncated α2δ-1 construct, α2δ-1ΔC, and found that, despite loss of its membrane anchor, it still shows a partial ability to increase calcium currents [Kadurin, Alvarez-Laviada, Ng, Walker-Gray, D’Arco, Fadel, Pratt and Dolphin (2012) J. Biol. Chem. 1287, 33554–33566]. We now find that PrP does not inhibit CaV2.1/β currents formed with α2δ-1ΔC, rather than α2δ-1. It is possible that PrP and α2δ-1 compete for GPI-anchor intermediates or trafficking pathways, or that interaction between PrP and α2δ-1 requires association in cholesterol-rich membrane microdomains. Our additional finding that CaV2.1/β1b/α2δ-1 currents were inhibited by GPI–GFP, but not cytosolic GFP, indicates that competition for limited GPI-anchor intermediates or trafficking pathways may be involved in PrP suppression of α2δ subunit function.
PrP, but not GPI-anchorless PrP, suppresses CaV2.1/β/α2δ-1 or α2δ-2 calcium channel currents. However, PrP does not inhibit CaV2.1/β currents formed with anchorless α2δ-1, rather than full-length α2δ-1. The results of the present study suggest that α2δ subunits and PrP may compete for GPI-anchor pathways.
PMCID: PMC3924758  PMID: 24329154
α2δ; auxiliary subunit; calcium channel; GPI anchor; prion protein; CaV, voltage-gated Ca2+; DRM, detergent-resistant membrane; GPI, glycosylphosphatidylinositol; HA, haemagglutinin; KO, knockout; MBS, Mes-buffered saline; PI-PLC, phosphatidylinositol-specific phospholipase C; PNGase F, peptide N-glycosidase F; PrP, prion protein; PrPC, normal cellular PrP; PrPSc, PrP scrapie; WT, wild-type
7.  The Effect of Statins on Mortality in Septic Patients: A Meta-Analysis of Randomized Controlled Trials 
PLoS ONE  2013;8(12):e82775.
Statins are among the most prescribed drugs worldwide and their recently discovered anti-inflammatory effect seems to have an important role in inhibiting proinflammatory cytokine production, chemokines expression and counteracting the harmful effects of sepsis on the coagulation system. We decided to perform a meta-analysis of all randomized controlled trials ever published on statin therapy in septic patients to evaluate their effect on survival and length of hospital stay.
Data sources and study selection
Articles were assessed by four trained investigators, with divergences resolved by consensus. BioMedCentral, PubMed, Embase and the Cochrane Central Register of clinical trials were searched for pertinent studies. Inclusion criteria were random allocation to treatment and comparison of statins versus any comparator in septic patients.
Data extraction and synthesis
Data from 650 patients in 5 randomized controlled studies were analyzed. No difference in mortality between patients receiving statins versus control (44/322 [14%] in the statins group vs 50/328 [15%] in the control arm, RR = 0.90 [95% CI 0.65 to 1.26], p = 0.6) was observed. No differences in hospital stay (p = 0.7) were found.
Published data show that statin therapy has no effect on mortality in the overall population of adult septic patients. Scientific evidence on statins role in septic patients is still limited and larger randomized trials should be performed on this topic.
PMCID: PMC3876996  PMID: 24391721
8.  Synaptic Dysfunction in Prion Diseases: A Trafficking Problem? 
Synaptic dysfunction is an important cause of neurological symptoms in prion diseases, a class of clinically heterogeneous neurodegenerative disorders caused by misfolding of the cellular prion protein (PrPC). Experimental data suggest that accumulation of misfolded PrPC in the endoplasmic reticulum (ER) may be crucial in synaptic failure, possibly because of the activation of the translational repression pathway of the unfolded protein response. Here, we report that this pathway is not operative in mouse models of genetic prion disease, consistent with our previous observation that ER stress is not involved. Building on our recent finding that ER retention of mutant PrPC impairs the secretory trafficking of calcium channels essential for synaptic function, we propose a model of pathogenicity in which intracellular retention of misfolded PrPC results in loss of function or gain of toxicity of PrPC-interacting proteins. This neurotoxic modality may also explain the phenotypic heterogeneity of prion diseases.
PMCID: PMC3863542  PMID: 24369467
Growing evidence suggests that a physiological activity of the cellular prion protein (PrPC) plays a crucial role in several neurodegenerative disorders, including prion and Alzheimer’s diseases. However, how the functional activity of PrPC is subverted to deliver neurotoxic signals remains uncertain. Transgenic mice expressing PrP with a deletion of residues 105–125 in the central region (referred to as ΔCR PrP) provide important insights into this problem. Tg(ΔCR) mice exhibit neonatal lethality and massive degeneration of cerebellar granule neurons, a phenotype that is dose-dependently suppressed by the presence of wild-type PrP. When expressed in cultured cells, ΔCR PrP induces large, ionic currents that can be detected by patch-clamping techniques. Here, we have tested the hypothesis that abnormal ion channel activity underlies the neuronal death seen in Tg(ΔCR) mice. We find that ΔCR PrP induces abnormal ionic currents in neurons in culture and in cerebellar slices, and that this activity sensitizes the neurons to glutamate-induced, calcium-mediated death. In combination with ultrastructural and biochemical analyses, these results demonstrate a role for glutamate-induced excitotoxicity in PrP-mediated neurodegeneration. A similar mechanism may operate in other neurodegenerative disorders due to toxic, β-rich oligomers that bind to PrPC.
PMCID: PMC3711660  PMID: 23392670
10.  Video-atlas of hybrid thoracoabdominal aortic aneurysm repair 
Annals of Cardiothoracic Surgery  2012;1(3):404-405.
Although open surgical repair of thoracoabdominal aortic aneurysm (TAAA) has evolved significantly over the last few decades, technical challenges and current morbidity and mortality of the inclusion technique are still significant, particularly in patients with extensive aneurysms or prior aortic surgery and in poor surgical candidates. Hybrid TAAA repair is an appealing technique and may represent a “bridge” solution as we wait for larger series and reproducible results from the evolving experience with total endovascular TAAA repair with fenestrated and branched endografts.
PMCID: PMC3741765  PMID: 23977527
Thoracoabdominal aortic aneurysm (TAAA); extensive aneurysms; total endovascular TAAA repair
11.  Clinical outcomes of hybrid repair for thoracoabdominal aortic aneurysms 
Annals of Cardiothoracic Surgery  2012;1(3):293-303.
Thoracoabdominal aortic aneurysm (TAAA) hybrid repair consists of aortic visceral branch rerouting followed by TAAA endograft exclusion. This technique has been shown to represent a technically feasible strategy in selected patients.
We analyzed 52 high-risk patients who underwent hybrid TAAA repair between 2001 and 2012 in our centre with a variety of visceral rerouting configurations and of commercially available thoracic endografts. Thirty-seven simultaneous (71.2%) and 15 staged procedures (21.8%) were performed with a four-vessel revascularization in 18 cases (34.6%), a three-vessel revascularization in 11 cases (21.2%) and a two-vessel revascularization in 23 cases (44.2%).
No intraoperative deaths were observed. We recorded a perioperative mortality rate of 13.5% (n=7), including deaths from multiorgan failure (n=2), myocardial infarction (n=2), coagulopathy (n=1), pancreatitis (n=1) and bowel infarction (n=1). Perioperative morbidity rate was 28.8% (n=15), including 2 cases of transient paraparesis and 1 case of permanent paraplegia. Renal failure (n=5), pancreatitis (n=3), respiratory failure (n=3) and dysphagia (n=1) were also observed. At median follow-up of 23.9 months procedure-related mortality rate was 9.6%: two patients died from visceral graft occlusion and three from aortic rupture. There were three endoleaks and one endograft migration, none of which resulted in death. Five patients (9.6%) died as a consequence of unrelated events.
Typical complications of conventional TAAA open surgery have not been eliminated by hybrid repair, and significant mortality and morbidity rates have been recorded. Fate of visceral bypasses and incidence of endoleak and other endograft-related complications needs to be carefully assessed. Hybrid TAAA repair should currently be limited to high-risk surgical patients with unfit anatomy for endovascular repair.
PMCID: PMC3741770  PMID: 23977511
Thoracoabdominal; aorta; aneurysm; hybrid; endovascular
12.  Video-atlas of open thoracoabdominal aortic aneurysm repair 
Annals of Cardiothoracic Surgery  2012;1(3):398-403.
Open surgical repair of thoracoabdominal aortic aneurysms has evolved significantly over the last decades thanks to technical improvements, especially in the area of organ protection. However, despite adjunctive strategies, morbidity and mortality rates are still not negligible. Repair of the thoracoabdominal aorta represents a formidable challenge for surgeons, anesthesiologists and patients alike. While operative repair is generally carried out in specialized institutions, knowledge of the state-of-the-art diagnostic, anesthesiologic, surgical and endovascular aspects will certainly be of great value to all physicians involved in the care of these patients at any level. This “How to” video will explain all of these diagnostic, anesthesiologic and surgical aspects in our daily practice.
PMCID: PMC3741779  PMID: 23977526
Thoracoabdominal aortic aneurysm; diagnosis; open surgical rapair
13.  Cross-Reacting Antibacterial Auto-Antibodies Are Produced within Coronary Atherosclerotic Plaques of Acute Coronary Syndrome Patients 
PLoS ONE  2012;7(8):e42283.
Coronary atherosclerosis, the main condition predisposing to acute myocardial infarction, has an inflammatory component caused by stimuli that are yet unknown. We molecularly investigated the nature of the immune response within human coronary lesion in four coronary plaques obtained by endoluminal atherectomy from four patients. We constructed phage-display libraries containing the IgG1/kappa antibody fragments produced by B-lymphocytes present in each plaque. By immunoaffinity, we selected from these libraries a monoclonal antibody, arbitrarily named Fab7816, able to react both with coronary and carotid atherosclerotic tissue samples. We also demonstrated by confocal microscopy that this monoclonal antibody recognized human transgelin type 1, a cytoskeleton protein involved in atherogenesis, and that it co-localized with fibrocyte-like cells transgelin+, CD68+, CD45+ in human sections of coronary and carotid plaques. In vitro fibrocytes obtained by differentiating CD14+ cells isolated from peripheral blood mononuclear cells also interacted with Fab7816, thus supporting the hypothesis of a specific recognition of fibrocytes into the atherosclerotic lesions. Interestingly, the same antibody, cross-reacted with the outer membrane proteins of Proteus mirabilis and Klebsiella pneumoniae (and possibly with homologous proteins of other enterobacteriaceae present in the microbiota). From all the other three libraries, we were able to clone, by immunoaffinity selection, human monoclonal antibodies cross-reacting with bacterial outer membrane proteins and with transgelin. These findings demonstrated that in human atherosclerotic plaques a local cross-reactive immune response takes place.
PMCID: PMC3412836  PMID: 22879930
14.  Surgical treatment of thoracic outlet syndrome in young adults: single centre experience with minimum three-year follow-up 
International Orthopaedics  2010;35(8):1179-1186.
Thoracic outlet syndrome is an often misdiagnosed syndrome which consists of a neurovascular compression at the upper thoracic outlet. The clinical presentation can be variable, ranging from mild symptoms to venous thrombosis and muscle atrophy. Many aetiologies, both congenital and acquired, related either to bony or soft tissue anomalies, have been associated with this syndrome. As a consequence, the diagnosis is often challenging and sometimes it can be obtained only with surgical exploration. Additionally, no specific clinical test is considered diagnostic of thoracic outlet syndrome. However, the recent advances in imaging techniques together with a careful clinical evaluation give the surgeon the chance to recognize the constricting anatomy before surgery in many cases. No standard surgical procedure has been identified; however, in literature the largest series have been treated with transaxillary first rib resection. Here we report our experience in the surgical treatment of this syndrome with a minimum follow-up of three years. Our approach consists of performing a supraclavicular decompression without routine first rib resection. This allows for identifying and removing the constricting anatomy in most cases, with satisfactory results in 96.9% of patients and a low complication rate.
PMCID: PMC3167441  PMID: 21184222
15.  Mutant PrP Suppresses Glutamatergic Neurotransmission in Cerebellar Granule Neurons by Impairing Membrane Delivery of VGCC α2δ-1 Subunit 
Neuron  2012;74(2):300-313.
How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α2δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α2δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies.
► Mutant PrP dysrupts glutamatergic transmission in cerebellar granule neurons (CGNs) ► Mutant PrP impairs depolarization-evoked calcium dynamics in CGNs ► Mutant PrP binds to the α2δ-1 subunit of voltage-gated calcium channels (VGCCs) ► Mutant PrP misfolding and intracellular retention impairs membrane delivery of VGCCs
How mutant prion protein causes neurological dysfunction in genetic prion diseases is not fully known. Senatore et al. report intracellular accumulation of misfolded prion impairs voltage-gated calcium channel transport to synapses, altering glutamatergic neurotransmission and cerebellum-dependent motor function.
PMCID: PMC3339322  PMID: 22542184
16.  The Toxicity of a Mutant Prion Protein Is Cell-Autonomous, and Can Be Suppressed by Wild-Type Prion Protein on Adjacent Cells 
PLoS ONE  2012;7(3):e33472.
Insight into the normal function of PrPC, and how it can be subverted to produce neurotoxic effects, is provided by PrP molecules carrying deletions encompassing the conserved central region. The most neurotoxic of these mutants, Δ105–125 (called ΔCR), produces a spontaneous neurodegenerative illness when expressed in transgenic mice, and this phenotype can be dose-dependently suppressed by co-expression of wild-type PrP. Whether the toxic activity of ΔCR PrP and the protective activity or wild-type PrP are cell-autonomous, or can be exerted on neighboring cells, is unknown. To investigate this question, we have utilized co-cultures of differentiated neural stem cells derived from mice expressing ΔCR or wild-type PrP. Cells from the two kinds of mice, which are marked by the presence or absence of GFP, are differentiated together to yield neurons, astrocytes, and oligodendrocytes. As a surrogate read-out of ΔCR PrP toxicity, we assayed sensitivity of the cells to the cationic antibiotic, Zeocin. In a previous study, we reported that cells expressing ΔCR PrP are hypersensitive to the toxic effects of several cationic antibiotics, an effect that is suppressed by co-expression of wild type PrP, similar to the rescue of the neurodegenerative phenotype observed in transgenic mice. Using this system, we find that while ΔCR-dependent toxicity is cell-autonomous, the rescuing activity of wild-type PrP can be exerted in trans from nearby cells. These results provide important insights into how ΔCR PrP subverts a normal physiological function of PrPC, and the cellular mechanisms underlying the rescuing process.
PMCID: PMC3299791  PMID: 22428057
17.  Emergent Repair of a Complex Dissecting Aneurysm in the Thoracic Aorta 
Texas Heart Institute Journal  2012;39(5):687-691.
Endovascular treatment of complex thoracic pathologic conditions involving the aortic arch can often be appropriate and safe; however, minimally invasive procedures are not always feasible, especially in emergent cases. We report the case of a 78-year-old woman who emergently presented in hemorrhagic shock with a ruptured chronic dissecting aneurysm that involved the aortic arch. Eight years earlier, she had undergone aortic valve replacement and plication of the ascending aorta, which was complicated a day later by Stanford type B dissection, malperfusion, and ischemia that required an axillobifemoral bypass. At the current admission, we successfully treated her surgically through a left thoracotomy, using moderate hypothermic extracorporeal circulation and advanced organ-protection methods. We discuss the surgical indications and our operative strategy in relation to open surgical repair versus endovascular treatment in patients with complex conditions.
PMCID: PMC3461696  PMID: 23109769
Aneurysm, dissecting/radiography/surgery; aortic aneurysm, thoracic/radiography/surgery; aortic diseases/surgery; aortic rupture/surgery; treatment outcome; vascular surgical procedures
18.  Expression of Mutant or Cytosolic PrP in Transgenic Mice and Cells Is Not Associated with Endoplasmic Reticulum Stress or Proteasome Dysfunction 
PLoS ONE  2011;6(4):e19339.
The cellular pathways activated by mutant prion protein (PrP) in genetic prion diseases, ultimately leading to neuronal dysfunction and degeneration, are not known. Several mutant PrPs misfold in the early secretory pathway and reside longer in the endoplasmic reticulum (ER) possibly stimulating ER stress-related pathogenic mechanisms. To investigate whether mutant PrP induced maladaptive responses, we checked key elements of the unfolded protein response (UPR) in transgenic mice, primary neurons and transfected cells expressing two different mutant PrPs. Because ER stress favors the formation of untranslocated PrP that might aggregate in the cytosol and impair proteasome function, we also measured the activity of the ubiquitin proteasome system (UPS). Molecular, biochemical and immunohistochemical analyses found no increase in the expression of UPR-regulated genes, such as Grp78/Bip, CHOP/GADD153, or ER stress-dependent splicing of the mRNA encoding the X-box-binding protein 1. No alterations in UPS activity were detected in mutant mouse brains and primary neurons using the UbG76V-GFP reporter and a new fluorogenic peptide for monitoring proteasomal proteolytic activity in vivo. Finally, there was no loss of proteasome function in neurons in which endogenous PrP was forced to accumulate in the cytosol by inhibiting cotranslational translocation. These results indicate that neither ER stress, nor perturbation of proteasome activity plays a major pathogenic role in prion diseases.
PMCID: PMC3084828  PMID: 21559407
19.  Hybrid Repair of Aortic Aneurysms and Dissections 
Texas Heart Institute Journal  2011;38(6):687-690.
PMCID: PMC3233314  PMID: 22199437
Aneurysm, dissecting/surgery; aortic aneurysm, thoracic/surgery; blood vessel prosthesis implantation; endovascular procedures; stents
20.  Endovascular Repair of Aortoesophageal and Aortobronchial Fistulae 
Texas Heart Institute Journal  2011;38(6):655-657.
PMCID: PMC3233331  PMID: 22199426
Aortic diseases/therapy; blood vessel prosthesis implantation; bronchial fistula/therapy; esophageal fistula/therapy; vascular fistula/therapy
21.  Pseudoaneurysm overlying an osteochondroma: a noteworthy complication 
Pseuodaneurysms are an extremely rare complication of osteochondromas. We describe a case of traumatic pseudoaneurysm of the brachial artery presenting as a soft tissue mass in a patient who was treated for an osteochondroma 3 years earlier. This case demonstrates that radiographic follow-up of large osteochondromas is mandatory and that, in patients with soft tissue masses and a history of osteochondroma, pseudoaneurysms should be included in the differential diagnosis.
PMCID: PMC3014466  PMID: 21103904
Pseudoaneurysm; Osteochondroma; Soft tissue mass; Brachial artery
22.  Pseudoaneurysm overlying an osteochondroma: a noteworthy complication 
Pseuodaneurysms are an extremely rare complication of osteochondromas. We describe a case of traumatic pseudoaneurysm of the brachial artery presenting as a soft tissue mass in a patient who was treated for an osteochondroma 3 years earlier. This case demonstrates that radiographic follow-up of large osteochondromas is mandatory and that, in patients with soft tissue masses and a history of osteochondroma, pseudoaneurysms should be included in the differential diagnosis.
PMCID: PMC3014466  PMID: 21103904
Pseudoaneurysm; Osteochondroma; Soft tissue mass; Brachial artery
23.  Cell Type-Specific Neuroprotective Activity of Untranslocated Prion Protein 
PLoS ONE  2010;5(10):e13725.
A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP). However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions.
Principal Findings
Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells.
These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.
PMCID: PMC2965675  PMID: 21060848
24.  Bioreducible Liposomes for Gene Delivery: From the Formulation to the Mechanism of Action 
PLoS ONE  2010;5(10):e13430.
A promising strategy to create stimuli-responsive gene delivery systems is to exploit the redox gradient between the oxidizing extracellular milieu and the reducing cytoplasm in order to disassemble DNA/cationic lipid complexes (lipoplexes). On these premises, we previously described the synthesis of SS14 redox-sensitive gemini surfactant for gene delivery. Although others have attributed the beneficial effects of intracellular reducing environment to reduced glutathione (GSH), these observations cannot rule out the possible implication of the redox milieu in its whole on transfection efficiency of bioreducible transfectants leaving the determinants of DNA release largely undefined.
Methodology/Principal Findings
With the aim of addressing this issue, SS14 was here formulated into binary and ternary 100 nm-extruded liposomes and the effects of the helper lipid composition and of the SS14/helper lipids molar ratio on chemical-physical and structural parameters defining transfection effectiveness were investigated. Among all formulations tested, DOPC/DOPE/SS14 at 25∶50∶25 molar ratio was the most effective in transfection studies owing to the presence of dioleoyl chains and phosphatidylethanolamine head groups in co-lipids. The increase in SS14 content up to 50% along DOPC/DOPE/SS14 liposome series yielded enhanced transfection, up to 2.7-fold higher than that of the benchmark Lipofectamine 2000, without altering cytotoxicity of the corresponding lipoplexes at charge ratio 5. Secondly, we specifically investigated the redox-dependent mechanisms of gene delivery into cells through tailored protocols of transfection in GSH-depleted and repleted vs. increased oxidative stress conditions. Importantly, GSH specifically induced DNA release in batch and in vitro.
The presence of helper lipids carrying unsaturated dioleoyl chains and phosphatidylethanolamine head groups significantly improved transfection efficiencies of DOPC/DOPE/SS14 lipoplexes. Most importantly, this study shows that intracellular GSH levels linearly correlated with transfection efficiency while oxidative stress levels did not, highlighting for the first time the pivotal role of GSH rather than oxidative stress in its whole in transfection of bioreducible vectors.
PMCID: PMC2955545  PMID: 20976172
25.  Combined Endovascular and Surgical Treatment of Primary Aortoesophageal Fistula 
Texas Heart Institute Journal  2010;37(6):722-724.
Primary aortoesophageal fistula is a rare cause of massive upper gastrointestinal bleeding. Conservative treatment of aortoesophageal fistula results in a 60% in-hospital mortality rate with no late survival, and conventional surgical treatment has a reported in-hospital mortality rate that approaches 40%.
Thoracic endovascular aortic repair is an innovative and less invasive technique for the treatment of aortoesophageal fistula. It enables the rapid control of aortic bleeding and prevents fatal early exsanguination. However, the technique does not repair the esophagus, and there remains a substantial risk of mediastinitis and infection of the stent-graft. Herein, we report the cases of 2 patients in whom we used a combined treatment: thoracic endovascular aortic repair and delayed surgical repair of the esophagus. The esophageal repair involved direct suture of the esophageal wall and reinforcement with an intercostal muscle flap. Early follow-up evaluations suggest that our treatment of both patients was successful. We discuss the advantages and limitations of our technical choices and briefly review the pertinent medical literature.
PMCID: PMC3014136  PMID: 21224956
Aorta, thoracic/surgery; aortic diseases/diagnosis/etiology/surgery; esophageal fistula/diagnosis/etiology/therapy; stents; surgical flaps; treatment outcome

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