Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms.

Xylanases are of great value in various industries, including paper, food, and biorefinery. Due to their biotechnological production, these enzymes can contain a variety of post-translational modifications, which may have a profound effect on protein function. Understanding the structure-function relationship can guide the development of products with optimal performance. We have developed a workflow for the structural and functional characterization of an endo-1,4-ß-xylanase (ENDO-I) produced by Aspergillus niger with and without applying thermal stress. This workflow relies on orthogonal native separation techniques to resolve proteoforms. Mass spectrometry and activity assays of separated proteoforms permitted the establishment of structure-function relationships. The separation conditions were focus on balancing efficient separation and protein functionality. We employed size exclusion chromatography (SEC) to separate ENDO-I from other co-expressed proteins. Charge variants were investigated with ion exchange chromatography (IEX) and revealed the presence of low abundant glycated variants in the temperature-stressed material. To obtain better insights into the effect on glycation on function, we enriched for these species using boronate affinity chromatography (BAC). The activity measurements showed lower activity of glycated species compared to the non-modified enzyme. Altogether, this workflow allowed in-depth structural and functional characterization of ENDO-I proteoforms.

Native Structural and Functional Proteoform Characterization of the Prolyl-Alanyl-Specific Endoprotease EndoPro from Aspergillus niger.

The prolyl-alanyl-specific endoprotease (EndoPro) is an industrial enzyme produced in Aspergillus niger. EndoPro is mainly used for food applications but also as a protease in proteomics. In-depth characterization of this enzyme is essential to understand its structural features and functionality. However, there is a lack of analytical methods capable of maintaining both the structural and functional integrity of separated proteoforms. In this study, we developed an anion exchange (AEX) method coupled to native mass spectrometry (MS) for profiling EndoPro proteoforms. Moreover, we investigated purified EndoPro proteoforms with complementary MS-based approaches, including released N-glycan and glycopeptide analysis, to obtain a comprehensive overview of the structural heterogeneity. We showed that EndoPro has at least three sequence variants and seven N-glycosylation sites occupied by high-mannose glycans that can be phosphorylated. Each glycosylation site showed high microheterogeneity with ∼20 glycans per site. The functional characterization of fractionated proteoforms revealed that EndoPro proteoforms remained active after AEX-separation and the specificity of these proteoforms did not depend on N-glycan phosphorylation. Nevertheless, our data confirmed a strong pH dependence of EndoPro cleavage activity. Altogether, our study demonstrates that AEX-MS is an excellent tool to characterize complex industrial enzymes under native conditions.

Endovascular Treatment of Asymptomatic Abdominal Aortic Aneurysms in Octogenarians: Factors Influencing Long-term Survival.

BACKGROUND: Beyond the age of 80 years, the preventive treatment of an asymptomatic abdominal aortic aneurysm (AAA) has to be decided in light of the life expectancy which it is difficult to evaluate, but it is important to determine who in this population will benefit from it. The objective of our study was to determine the factors influencing short-term mortality and long-term survival in patients aged 80 years and older after the endovascular treatment of AAAs (EVAR). MATERIAL AND METHODS: We present a retrospective analysis of the prospective databases of 4 French academic departments of vascular surgery, bringing together the data of all the patients presenting an AAA who were treated by EVAR between 1998 and 2011. Logistic regression and multivariate analysis with a Cox survival model were used to determine the factors influencing perioperative and long-term mortality. The cumulative rate of events for the measurement of survival was calculated with the technique of Kaplan-Meier. RESULTS: We treated 345 octogenarians and 339 younger patients. The average follow-up was 40 months. Average survival was 75% at 36 months and 49% at 60 months. There was no evidence of any risk factor influencing mortality at 30 days in the octogenarians. However, chronic kidney disease (odds ratio [OR] = 3.95, P <0.001) and chronic respiratory failure (OR = 2.62, P <0.001) proved to be independent factors of a poor long-term prognosis. CONCLUSIONS: The treatment by stent graft in octogenarians is effective in the long term. The presence of an impaired renal function or respiratory failure in this population could put into question the operative indication.

Obesity is Not an Independent Factor for Adverse Outcome after Abdominal Aortic Aneurysm Repair.

BACKGROUND: The prevalence of obesity is increasing, and its impact on the outcome of open and endovascular abdominal aortic aneurysm (AAA) repair remains unclear, particularly in the European population. We herein assessed the impact of obesity on the postoperative course for both techniques. METHODS: From a database that consecutively collects all patients undergoing AAA repair; we selected all patients undergoing elective surgery for open or endovascular AAA repair, between January 2003 and December 2011. We considered obese patients (body mass index >30 kg/m(2)), overweight (25.1-30 kg/m(2)), and normal-weight patients (18.7-25 kg/m(2)), and compared mortality and/or severe complications at 30 days between obese and nonobese patients (overweight and normal weight) separately for each type of surgery by logistic regression analysis. We analyzed wound complications in the 2 groups. RESULTS: We included 748 patients, 174 obese, and 574 nonobese patients. Obese patients were younger (P < 0.001) and were less likely to have renal failure (P < 0.001) in both techniques. Obese patients in the open repair (OR) group showed a trend toward lower mortality and/or complication rates than in nonobese patients (4.8% vs. 7.5%, P = 0.34). In contrast, in the endovascular aortic aneurysm repair (EVAR) group, obese patients showed a trend toward higher mortality and/or complication rates than nonobese patients (7.1% vs. 3.2%, P = 0.17). In multivariate analysis, obesity was not an independent predictor of outcomes in OR (P = 0.18) or in EVAR (P = 0.20). Wound complications were not higher in obese patients in OR and in EVAR. CONCLUSIONS: Obesity should not be considered an independent risk factor of death and severe complications at 30 days in either open or endovascular AAA repair. Therefore, obesity should not systematically lead to the decision to use EVAR.

Delay between symptoms and surgery for carotid artery stenosis: modification of our practice.

BACKGROUND: Recent data from the literature concerning symptomatic carotid stenosis show that the long-term benefits of surgery are greater when the surgery is performed soon after the neurologic event, ideally within 2 weeks. Since 2009, following recommendations, we decided to perform surgery as quick as possible. The aim of the study was to determine whether this approach increased postoperative morbimortality and the way it could change our practice. METHODS: Using a prospective database containing a consecutive and continuous series of 1,500 carotid endarterectomies performed between 2003 and 2012, we extracted the records concerning the 417 symptomatic carotid stenoses (27.8%). We compared the 30-day and long-term outcome in 3 groups of patients: those operated within 2 weeks of the neurologic event (early surgery group [ESG], n = 158, 37.9%), those operated between 16 days and 6 weeks after the event (deferred surgery group [DSG], n = 79, 18.9%), and those operated more than 6 weeks after the event (late surgery group [LSG], n = 180, 43.2%). In the second part, to assess the new management beginning 2009, patients were divided in 2 periods 2003-2008 (period A) and 2009-2012 (period B) and we compared the 2 period's descriptive data and outcome. The primary outcome was the 30-day combined stroke and death rate. Secondary end points were follow-up freedoms from mortality and stroke. RESULTS: The mean time between symptom onset and surgery was 7.7 ± 3.8 days for the ESG, 28.3 ± 8.6 days for the DSG, and 89.4 ± 36.7 days for the LSG. In the 3 groups, the types of symptoms leading to the indication for carotid surgery were comparable, with a stroke in 221 cases (53.0%), a transient ischemic attack in 146 cases (35.40%), and amaurosis fugax in 50 cases (12.0%). The groups were comparable in terms of comorbidities. The overall 30-day stroke rate was 1.4% (6 cases), the 30-day death rate was 1.7% (7 cases), and the combined stroke and death rate was 3.4% (3.2% in the ESG, 5.1% in the DSG, and 2.8% in the LSG [P = 0.808]). Survival rates at 24, 48, and 60 months were, respectively, 95%, 78%, and 78% in ESG, 86%, 81%, and 81% in DSG, and 91%, 83%, and 74% in LSG (P = 0.78). Freedom from stroke at 60 months showed to be, respectively, 97% in ESG, 96% in DSG, and 91% in LSG (P = 0.32). During the period A (2003-2008), we had taken care of 217 symptomatic carotid artery stenosis patients (22.3% of stenosis) and during the period B (2009-2012), 200 symptomatic stenosis (37.9% of stenosis). During the period A, an early surgery (<15 days) had place in 31 cases (14.3%), and during the period B, in 127 cases (63.5%). The 30-day stroke and death rate was of 3.7% during the period A and of 3.0% during the period B (P = 0.455). The 24-month survival and stroke-free survival rates were comparable between the 2 periods. CONCLUSIONS: In our experience, surgery for symptomatic carotid stenosis can be performed early without increasing the rate of postoperative and long-term outcome. We have modified our practice, performing more and more early surgery for symptomatic stenosis without any impact on the outcome.

Development of abdominal aortic aneurysm is decreased in mice with plasma phospholipid transfer protein deficiency.

Plasma phospholipid transfer protein (PLTP) increases the circulating levels of proatherogenic lipoproteins, accelerates blood coagulation, and modulates inflammation. The role of PLTP in the development of abdominal aortic aneurysm (AAA) was investigated by using either a combination of mechanical and elastase injury at one site of mouse aorta (elastase model) or continuous infusion of angiotensin II in hyperlipidemic ApoE-knockout mice (Ang II model). With the elastase model, complete PLTP deficiency was associated with a significantly lower incidence and a lesser degree of AAA expansion. With the Ang II model, findings were consistent with those in the elastase model, with a lower severity grade in PLTP-deficient mice, an intermediate phenotype in PLTP-deficient heterozygotes, and a blunted effect of the PLTP-deficient trait when restricted to bone marrow-derived immune cells. The protective effect of whole-body PLTP deficiency in AAA was illustrated further by a lesser degree of adventitia expansion, reduced elastin degradation, fewer recruited macrophages, and less smooth muscle cell depletion in PLTP-deficient than in wild-type mice, as evident from comparative microscopic analysis of aorta sections. Finally, cumulative evidence supports the association of PLTP deficiency with reduced expression and activity levels of matrix metalloproteinases, known to degrade elastin and collagen. We conclude that PLTP can play a significant role in the pathophysiology of AAA.

Post-production modification of industrial enzymes.

Industry has an increasing interest in the use of enzymes as environmentally friendly, highly efficient, and specific bio-catalysts. Enzymes have primarily evolved to function in aqueous environments at ambient temperature and pressure. These conditions however do not always correspond with industrial processes or applications, and only a small portion of all known enzymes are therefore suitable for industrial use. Protein engineering can sometimes be applied to convey more desirable properties to enzymes, such as increased stability, but is limited to the 20 naturally occurring amino acids or homologs thereof. Using post-production modification, which has the potential to combine desirable properties from the enzyme and the conjugated compounds, enzymes can be modified with both natural and synthetic molecules. This offers access to a myriad of possibilities for tuning the properties of enzymes. At this moment, however, the effects of post-production modification cannot yet be reliably predicted. The increasing number of applications will improve this so that the potential of this technology can be fully exploited. This review will focus on post-production modification of enzymes and its use and opportunities in industry.

Risk index for predicting shunt in carotid endarterectomy.

BACKGROUND: To prevent ischemia during carotid endarterectomy, a routine or selective shunt can be set up in cases of insufficient cerebral perfusion during the carotid clamping. The aim of this study was to analyze predictive factors for shunting under locoregional anesthesia and to validate a risk index to predict shunt. METHODS: Using a prospective database, we studied carotid endarterectomy performed under locoregional anesthesia between January 1, 2003, and December 31, 2010 (n=1,223). A shunt was used because of clinical intolerance of clamping in 88 cases (group S, 7.2%). Clinical, comorbidities, demographics, and duplex scan data were used to compare group S to a control group (group C, n=1,135, 92.8%). A multivariable logistic regression was performed to identify predictors of shunt. Coefficients were assigned to each predictor to propose a predictive score. RESULTS: Patients in group S were significantly older than those in group C (75.6±7.8 years vs. 72.6±9.4 years, P<0.001). Other factors associated with a carotid shunt were female sex (odds ratio [OR]=2.41, 95% confidence interval [CI]: 1.54-3.78, P<0.001), systemic arterial hypertension (OR=2.478, 95% CI: 1.16-4.46, P=0.016), occlusion of the contralateral carotid artery (OR=6.03, 95% CI: 2.91-12.48, P<0.001), and 1 factor against the likelihood of a carotid shunt, a history of contralateral carotid surgery (OR=0.34, 95% CI: 0.12-0.93, P=0.037). The mean flow in the contralateral common carotid artery was 696.5±298.0 mL/sec in group S and 814.7±285.5 mL/sec in group C (P<0.001). Using those 6 items, we propose a prognostic score validated in our series and allowing to divided risk of intolerance of clamping into low-risk (≤6%), intermediate-risk (6.1%-15%), and high-risk (>15%) groups. CONCLUSIONS: We have established the first version of a score that predicts the need for a shunt by studying factors associated with intolerance to clamping. The relevance of this score, validated in our series, must be confirmed and adjusted by studies based on a larger sample size.

Evaluating the effect of glycation on lipase activity using boronate affinity chromatography and mass spectrometry.

Protein glycation may occur naturally when reducing sugars and proteins coexist, which is often the case for industrial enzymes. The impact of post-translational modifications on enzyme performance (e.g., stability or function) is often not predictable, highlighting the importance of having appropriate analytical methodologies to monitor the influence of glycation on performance. Here, a boronate affinity chromatography method was developed to enrich glycated species followed by mass spectrometry for structural characterization and activity assays for functional assessment. This approach was applied to a (temperature-stressed) lipase used for food applications revealing that storage at -20 °C and 4 °C resulted in minor glycation (below 9%), whereas storage at 25 °C led to a higher glycation level with up to four sugars per lipase molecule. Remarkably, activity measurements revealed that glycation did not reduce lipase activity or stability. Altogether, this novel strategy is a helpful extension to the current analytical toolbox supporting development of enzyme products.

Influence of the contralateral carotid artery on carotid surgery outcome.

BACKGROUND: Any obstacle in the contralateral artery has long been considered a high risk for carotid surgery. Here, we report the results of a monocentric, continuous, consecutive, prospective series and present a review of the literature. METHODS: All carotid endarterectomies performed under locoregional anesthesia in our department between 2003 and 2010 were recorded in a prospective database (n = 1,212). Different statuses of the contralateral carotid artery--occlusion (group O, n = 81) and stenosis of >60% (group S, n = 173)--were compared with a control group (group C, n = 958). A shunt was used in cases of clinical intolerance to clamping. The assessment criteria concerned the need for a shunt and the combined 30-day nonfatal stroke and death rate. A stepwise logistic regression was performed. RESULTS: Overall, a shunt was necessary in 7.3% of cases. The proportion was greater in group O (25.9%, P < 0.001). Severe renal insufficiency (odds ratio [OR] = 1.94) and contralateral carotid occlusion (OR = 5.53) were the sole factors predicting the need for shunting. The overall 30-day nonfatal stroke and death rate was 2.5%, with no difference between groups (P = 0.738), and severe renal insufficiency was the single predictor of a poor outcome (OR = 3.11; 95% confidence interval: 1.21-7.97; P = 0.18). CONCLUSION: In this series, and in a large review of literature, occlusion of the contralateral internal carotid artery increased the incidence of intolerance to clamping and thus the use of shunts but did not worsen postoperative morbidity and mortality. The presence of a stenosed contralateral carotid was not predictive of postoperative events. In our experience, the status of the contralateral carotid artery cannot be considered a high risk for carotid surgery.

Long-term results of stenting of the aortic bifurcation.

BACKGROUND: To evaluate the long-term results in a multicentric continuous series of narrowing lesions of the aortic bifurcation treated with a kissing stent. METHODS: From January, 1st 1999 to the December, 31st 2001, all of the patients (n = 80) presenting with stenosis of the aortic bifurcation (n = 15) and/or the 2 common iliac arteries (n = 65), treated with a kissing stent, in 8 teaching hospitals were collected retrospectively. The risk factors were smoking (91%), dyslipidemia (60%), arterial hypertension (42%) and diabetes (27%). In 84% of cases, the indication for treatment was claudication. The lesions were stenotic < 70% (n = 76) and/or thrombotic (n = 18). The associated lesions were external iliac stenoses (n = 21), common femoral stenoses (n = 19), femoro-popliteal stenoses (n = 42), arteriopathy in the leg (n = 35). Follow-up was clinical examination and Doppler US scan. RESULTS: The success rate of the technique was 89%. There were 4 cases (5.3%) of residual stenosis and 4 cases (5.3%) of dissection. The length of the lesions treated in the aorta and the iliac arteries was respectively 17.1 ± 7 and 17.3 ± 9 mm. The stents were all placed as kissing stents, and had a mean diameter and a mean length of 13.75 mm and 56 mm in the aorta and 9 mm and 48 mm in the iliac arteries, respectively. At 5 years, 19 patients had required repeat angioplasty in the treated area, and 13 had undergone open surgery. Primary and assisted primary patency at 5 years were 64.5% and 81.8%, respectively. CONCLUSION: Long-term follow-up of endovascular treatment with kissing stents for stenosis of the aortic bifurcation shows that this technique gives good results, though it does not justify doing away with classical revascularisation surgery, in a population with major cardiovascular risk factors.

The impact of renal insufficiency on the outcome of carotid surgery is influenced by the definition used.

BACKGROUND: Renal insufficiency (RI) seems to be a source of complications after carotid endarterectomy (CEA). However, published studies do not use a common definition of RI. Our objective was to analyze the effects of RI on carotid surgery using three classifications of renal function. METHODS: Using a prospective database, we studied renal function and postoperative complications in patients operated on between January 1, 2003 and December 31, 2008. Renal function was studied using the level of plasma creatinine, creatinine clearance calculated according to the Cockcroft formula, and to the Modification of Diet in Renal Disease (MDRD) equation. For each method, the patients were divided into three groups: normal renal function, moderate RI, and severe RI. The principal judgment criterion was the 30-day non-fatal stroke and death rate. RESULTS: The analysis concerned 961 CEAs carried out in 901 patients. The 30-day non-fatal stroke and death rate was 2%. In the analysis of renal function using the level of creatinine, there was no statistical difference between the groups in the 30-day stroke and death rate (normal renal function: 1.8%, moderate: 2.7%, severe: 8.3%, P = .21). The analysis of renal function according to creatinine clearance calculated using the Cockcroft formula showed that in the severe RI group, the stroke and death rate was higher than in the other two groups (normal renal function: 1.7%, moderate RI: 1.4%, severe RI: 7.5%, P = .004). Analysis using the MDRD formula showed similar differences between the severe RI group and the other two with a higher rate of 30-day stroke and death (normal renal function: 1.4%, moderate RI: 1.7%, severe RI: 12.5%, P < .001). Subgroup analysis showed that among patients with severe RI according to the creatinine clearance, those with symptomatic carotid stenosis had the highest incidences of non-fatal stroke and death (Cockcroft, n = 19: 21.1%, MDRD, n = 10: 40%). CONCLUSION: Severe RI is a risk factor for complications after carotid surgery. Creatinine clearance calculated according to the MDRD formula correlates most closely with these complications. Symptomatic patients with severe RI, according to the creatinine clearance, are at high risk with a very high level of postoperative complications.

High incidence of venous thrombosis after surgery for abdominal aortic aneurysm.

OBJECTIVE: The incidence of venous thromboembolism (VT) after aortic abdominal aneurysm (AAA) surgery is imprecisely reported. On one hand, thromboprophylaxis has improved, on the other hand, AAA patients have become older and/or present worse comorbidities. Herein, we prospectively analyzed the incidence of VT in a continuous series of patients operated on for AAA repair and looked for predictive factors. MATERIALS AND METHODS: Between January 1, 2005, and December, 31, 2006, 193 consecutive patients (177 men and 16 women), mean age 73 (range, 47-93) underwent elective AAA repair, 137 open (71%) and 56 endovascular (29%), in our institution. Thromboprophylaxis consisted of thigh-length compression bandages or stockings, early mobilization, and a daily subcutaneous injection of low-molecular-weight heparin (enoxaparin 40 mg per day). Patients with renal insufficiency or aged over 80 were given unfractionated heparin (5000 IU twice a day). Heparin was started between day 1 and day 5 (median = day 1) after surgery, according to the prescription of the surgeon. A bilateral lower limb duplex venous compression ultrasonography scan using 3 to 7.5 MHz transducers was systematically done before and after surgery in each patient. Two groups were considered: group 1 with postoperative VT (n = 17) and group 2 without (n = 176). The 17 patients with VT were compared with 51 patients randomly chosen among the 176 patients without VT. Different characteristics such as venous risk factors, preoperative antithrombotic treatment, anatomical features of the AAA, and perioperative data were studied. RESULTS: Seventeen patients (8.1%) were diagnosed with postoperative VT (15 asymptomatic deep vein thrombosis [DVT] and 2 symptomatic pulmonary embolisms [PE]). VTE tended to be more frequent in open (10.2%) than endovascular (5.3%) repair (P = .28). Among perioperative data, delay to thromboprophylaxis was related to bleeding complication (P = .05) and blood transfusion (P = .02), and tended to be longer in VT patients (1.7 +/- 1.4 vs 0.9 +/- 0.9 day; P = .09). CONCLUSION: Despite systematic prevention with heparin, surgery for AAA repair induces a high incidence of postoperative VT. This series mandates for vigilance about VT, with particular attention to the patients who received transfusion with fresh frozen plasma. Even though this series is one of the largest ever published on this topic, the rarity of the events calls for confirmation with a larger prospective study.

Study protocol: a cluster randomized controlled trial to assess the effectiveness of a therapeutic educational program in oral health for persons with schizophrenia.

BACKGROUND: Schizophrenia is a severe mental disorder that affects 1 % of the world's population, including 600,000 people in France. Persons with schizophrenia (PWS) have excess mortality (their life expectancy is reduced by 20 %) and excess morbidity. In addition, such persons may have a large number of missing or decayed teeth. Dental caries and periodontal measurement indexes are often twice as high as the level found in the general population. Poor oral health can also affect quality of life and oral health is inseparable from general health. The management of oral health problems needs a multidisciplinary approach. According to the World Health Organization, the aim of therapeutic education (TE) is to help patients take care of themselves and to improve empowerment and recovery. In this educational approach, it is important to take into account the patient's personal experience. Though rarely investigated, the personal experience of PWS in oral health quality of life (OHRQoL) must be used to build a therapeutic educational programme in oral health (TEPOH) in a multidisciplinary approach, and the effectiveness of this program must be evaluated. METHODS/DESIGN: We report the protocol of a randomized controlled cluster study. This study will be conducted in twelve hospitals in France. We hypothesized that a decrease of 20 % in the proportion of patients with CPI ≥ 3 would establish the effectiveness of TEPOH. Therefore, 12 hospitals will be randomly allocated to either TEPOH or no TEPOH. Altogether, they will have to recruit 230 PWS, who will be randomly allocated with a ratio of 1:1 to one of two conditions: control without intervention versus the group benefitting from TEPOH. DISCUSSION: If successful, the study will generate methodologically sound results that provide knowledge on the effectiveness of a TEP in oral health for PWS. The results can be used to promote OHRQoL in a global health approach and develop appropriate strategies to encourage and facilitate financial support for healthcare, the multidisciplinary treatment of dental disorders, and the development of training in oral and mental health for caregivers. Trial registration Clinical Trials Gov NCT02512367. Date registered 19 July, 2015.

Efficacy of transcranial direct current stimulation (tDCS) in reducing consumption in patients with alcohol use disorders: study protocol for a randomized controlled trial.

BACKGROUND: Approximately 15 million persons in the European Union and 10 million persons in the USA are alcohol-dependent. The global burden of disease and injury attributable to alcohol is considerable: worldwide, approximately one in 25 deaths in 2004 was caused by alcohol. At the same time, alcohol use disorders remain seriously undertreated. In this context, alternative or adjunctive therapies such as brain stimulation may play a prominent role. The early results of studies using transcranial direct current stimulation found that stimulations delivered to the dorsolateral prefrontal cortex result in a significant reduction of craving and an improvement of the decision-making processes in various additive disorders. We, therefore, hypothesize that transcranial direct current stimulation can lead to a decrease in alcohol consumption in patients suffering from alcohol use disorders. METHODS/DESIGN: We report the protocol of a randomized, double-blind, placebo-controlled, parallel-group trial, to evaluate the efficacy of transcranial direct current stimulation on alcohol reduction in patients with an alcohol use disorder. The study will be conducted in 14 centers in France and Monaco. Altogether, 340 subjects over 18 years of age and diagnosed with an alcohol use disorder will be randomized to receive five consecutive twice-daily sessions of either active or placebo transcranial direct current stimulation. One session consists in delivering a current flow continuously (anode F4; cathode F3) twice for 13 minutes, with treatments separated by a rest interval of 20 min. Efficacy will be evaluated using the change from baseline (alcohol consumption during the 4 weeks before randomization) to 24 weeks in the total alcohol consumption and number of heavy drinking days. Secondary outcome measures will include alcohol craving, clinical and biological improvements, and the effects on mood and quality of life, as well as cognitive and safety assessments, and, for smokers, an assessment of the effects of transcranial direct current stimulation on tobacco consumption. DISCUSSION: Several studies have reported a beneficial effect of transcranial direct current stimulation on substance use disorders by reducing craving, impulsivity, and risk-taking behavior, and suggest that transcranial direct current stimulation may be a promising treatment in addiction. However, to date, no studies have included sufficiently large samples and sufficient follow-up to confirm the hypothesis. Results from this large randomized controlled trial will give a better overview of the therapeutic potential of transcranial direct current stimulation in alcohol use disorders. TRIAL REGISTRATION: Clinical Trials Gov, NCT02505126 (registration date: July 15 2015).

Specific enrichment of 2-arachidonoyl-lysophosphatidylcholine in carotid atheroma plaque from type 2 diabetic patients.

BACKGROUND AND AIMS: Diabetic patients are at high risk of stroke and coronary artery disease. Recent data suggest that arachidonic acid metabolism is altered in diabetic conditions and that these alterations contribute to accelerated atherosclerosis. Little is known about how these alterations affect the metabolism and the proportions of different lipid species within the atherosclerotic plaque. The aim of our study was to perform a targeted lipidomic analysis of human atherosclerotic lesions, with a specific focus on PUFA-containing lipid species, to reveal differences in the fatty-acid composition of plaque in diabetic patients compared with non-diabetic controls. METHODS: Carotid atheroma plaque samples were obtained from 31 diabetic and 48 non-diabetic patients undergoing carotid endarterectomy. Targeted lipidomic analysis was then performed to determine the fatty acid composition of major glycerophospholipids and cholesteryl ester species by liquid chromatography-tandem mass spectrometry. RESULTS: Atheroma plaques from diabetic patients were significantly enriched with 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) (2.3 ± 0.8% Vs. 1.8 ± 0.6% p = 0.0002). Multivariable logistic regression showed that an increased 2-AA-LPC level was independently associated with diabetes. Finally, a positive relationship was found between 2-AA-LPC and HbA1c levels. Interestingly, endothelial lipase and calcium independent PLA2 gamma which could account for the production of 2-AA-LPC were detected in carotid plaques by immunohistochemistry. CONCLUSIONS: 2-AA-LPC stands at the crossroads of major metabolic pathways that lead to the synthesis of bioactive molecules such as AA-derived eicosanoids, 2-AA-lysophosphatidic acid and 2-AA-glycerol. 2-AA-LPC therefore appears to be a promising molecule to investigate in the context of diabetes.

Enrichment and detection of tyrosine-nitrated proteins.

Nitrotyrosine is a post-translationally modified amino acid with distinctly different properties than tyrosine or any other of the genetically encoded amino acids. Detecting proteins containing nitrotyrosine is the first step towards a better understanding of the role of nitrotyrosine in health and disease. Moreover, quantifying the extent of nitrotyrosine and determining its location in a protein forms the basis for a better understanding of the effect of tyrosine nitration on biological function. Described in this unit is a method to detect tyrosine-nitrated proteins in tissue sections and on western blots after creating a fluorescent complex between aminotyrosine, salicylaldehyde, and Al(3+). In addition, an approach is detailed for labeling aminotyrosine with biotin to enrich peptides from complex samples. Both methods require reduction of nitrotyrosine to aminotyrosine, which can be achieved with sodium dithionite or hemin plus dithiothreitol.

Chemical labeling and enrichment of nitrotyrosine-containing peptides.

Protein tyrosine nitration (PTN) is a post-translational modification of proteins associated with a number of inflammatory diseases. While PTN is rather selective (not all proteins are modified and within a protein, only certain tyrosines are subject to nitration), no consensus sequence has been identified. Since PTN is a low-abundance post-translational modification, it is necessary to enrich modified proteins and/or to detect them with high selectivity and sensitivity. Until now this has been mostly accomplished with anti-nitrotyrosine antibodies in combination with two-dimensional gel electrophoresis and mass spectrometry. We propose a chemical labeling approach designed to allow enrichment of tyrosine-nitrated peptides independent of the sequence context, which is a potential shortcoming of antibody-based approaches. In this procedure, all amines are blocked by acetylation followed by conversion of nitrotyrosine to aminotyrosine and biotinylation of aminotyrosine. The entire reaction sequence is performed in a single buffer with no need for sample cleanup or pH changes thereby reducing sample loss. Free biotin is subsequently removed with a strong cation exchanger, the labeled peptides are enriched on an immobilized avidin column and the enriched peptides analyzed by LC-MS/MS. As a proof of concept, this method was successfully applied to the enrichment of tyrosine-nitrated angiotensin II in a tryptic digest of bovine serum albumin (BSA). The approach presented here is well adapted to peptide analysis, for instance in shotgun proteomics.

Protein tyrosine nitration: selectivity, physicochemical and biological consequences, denitration, and proteomics methods for the identification of tyrosine-nitrated proteins.

Protein tyrosine nitration (PTN) is a post-translational modification occurring under the action of a nitrating agent. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group (NO2). In the present article, we review the main nitration reactions and elucidate why nitration is not a random chemical process. The particular physical and chemical properties of 3-nitrotyrosine (e.g., pKa, spectrophotometric properties, reduction to aminotyrosine) will be discussed, and the biological consequences of PTN (e.g., modification of enzymatic activity, sensitivity to proteolytic degradation, impact on protein phosphorylation, immunogenicity and implication in disease) will be reviewed. Recent data indicate the possibility of an in vivo denitration process, which will be discussed with respect to the different reaction mechanisms that have been proposed. The second part of this review article focuses on analytical methods to determine this post-translational modification in complex proteomes, which remains a major challenge.