Methodology to study polymers interaction by surface plasmon resonance imaging.

The surface plasmon resonance (SPR) technique has been primarily used in the field of biology, in particular for the study of antibody-antigen interactions. Recently, polymers were introduced to form inclusion complexes. We describe here, a methodology based on surface plasmon resonance imaging to study water-resistant and reversible inclusion complexes using systems which are compatible with a cosmetic use. The purpose of this study is to follow in real time the interaction between two polymers. To carry out this study: •A biochip based on a covalent binding of one "host polymer" on a gold-activated surface was developed.•The binding of the host polymer to a guest polymer was monitored.•The presence of interactions between the ß-cyclodextrins groups of the host polymer and the adamantyl functional groups of the guest polymer and the possibility of dissociating the complex were established. This technique allowed carrying out parallel assays for optimizing the amount of complexes formed, the host polymer being spotted at five concentrations. It was then possible to study the influence of the concentration in host system for two concentrations of the guest polymer. The concentration in the host polymer yielding the highest immobilization of the guest system was further determined.

Surface plasmon resonance imaging in arrays coupled with mass spectrometry (SUPRA-MS): proof of concept of on-chip characterization of a potential breast cancer marker in human plasma.

Protein biomarker discovery and validation are crucial for diagnosis, prognosis, and theranostics of human pathologies; "omics" approaches bring new insights in this field. In particular, the combination of immuno-sensors in array format with mass spectrometry efficiently extends the classical immunoassay format and includes molecular characterization. Here, we coupled surface plasmon resonance imaging (SPRi) with MALDI-TOF mass spectrometry in a hyphenated technique which enables multiplexed quantification of binding by SPRi and molecular characterization of interacting partners by subsequent MS analysis. This adds specificity, because MS enables differentiation of molecules that are difficult to distinguish by use of antibodies, for example truncation variants or protein isoforms. Proof of concept was established for detection, identification, and characterization of a potential breast cancer marker, the LAG3 protein, at ~1 µg mL(-1), added to human plasma. The analytical performance of this new method, dubbed "SUPRA-MS", was established, particularly its specificity (S/N > 10) and reliability (100 % LAG3 identification with high significant mascot score >87.9). The adjusted format for rapid, collective, and automated on-chip MALDI-MS analysis is robust at the femtomole level and has numerous potential applications in proteomics.

Endoscopic submucosal dissection in a European setting. A multi-institutional report of a technique in development.

BACKGROUND AND STUDY AIMS: Endoscopic submucosal dissection (ESD) is a technique for "en bloc" resection of superficial tumors of the gastrointestinal tract. In France, experience with this technique is still limited. We wanted to assess the development of ESD in France, with special attention to short term outcomes. PATIENTS AND METHODS: Members of the Société Française d'Endoscopie Digestive (SFED) who declared performing ESD reported their cases prospectively on a voluntary basis. Demographic, clinical, and technical data, and the results of immediate complications were collected. Case reports were completed prospectively by each investigator before pooled analysis. RESULTS: A total of 188 consecutive case reports were collected from 16 centers. The median case mix per center was 6 patients (range 1-43). The lesion sites treated by ESD were the stomach (n = 75), esophagus (n = 27), duodenum (n = 1), cecum (n = 2), right colon (n = 3), transverse colon (n = 5), sigmoid (n = 3), and rectum (n = 72). The median size of the lesions was 26 mm (range 2-150 mm). En bloc resection was achieved in 77.1% of cases, with complete R0 resection in 72.9%. Histopathology results showed high grade dysplasia or superficial cancer in 71.2%. The median duration of ESD was 105 minutes (range 20-450 minutes). The short term morbidity was 29.2% including 34 cases of perforation (18.1%), and 21 hemorrhages (11.2%) during the 24 hours following ESD, 89% of which were managed conservatively or endoscopically. CONCLUSION: In this early experience, the feasibility of ESD appeared to be good but R0 resection and complication rates did not match those reported by Japanese authors and must be improved by an extended practice.

Hyphenation of surface plasmon resonance imaging to matrix-assisted laser desorption ionization mass spectrometry by on-chip mass spectrometry and tandem mass spectrometry analysis.

Most of the recent developments aiming to the coupling between surface plasmon resonance (SPR) and mass spectrometry (MS) are based on the use of a biochip with a limited number of flow cells requiring elution steps for the recovery of the captured biomolecules. In this work, a direct on-chip MALDI-MS detection is presented using a SPRi-sensor biochip in a microarray format that allows a multiplex SPR-MS analysis. The biochip gold surface was functionalized by a self-assembled monolayer (SAM) of short polyoxyethylene (POE) chains carrying a N-hydroxysuccinimide (NHS) group for the immobilization of biomolecules. The SPR measurement of the interaction of grafted antibodies anti-beta-lactoglobulin and anti-ovalbumin with their corresponding antigens indicated that the POE-NHS SAM preserved the binding activity of the antibodies immobilized on the biochips surface. SPR-MS experiments were carried out through MALDI-MS detection of the retained antigens (beta-lactoglobulin and ovalbumin) directly from the biochip surface. Mass spectra were obtained from each distinct spot on the arrayed biochips. Femtomole amounts of specifically retained antigen proteins as determined by SPR were sufficient to obtain good quality mass spectra. These mass spectra showed protein ions corresponding to the specific antigen, without any trace of nonspecific binding. The underivatized portion of the chip was also devoid of nonspecifically bound proteins, indicating that the functionalization of the biochips surface by short polyoxyethylene chains greatly minimizes the unspecific binding. In addition, it allowed on-chip digestion of the specifically bound analyte and coupling with MS/MS experiments, opening numerous applications in the proteomic field.

5-(phenylthiomethyl)-2'-deoxyuridine as an efficient photoreactive precursor to generate single and multiple lesions within DNA fragments.

5-(Phenylthiomethyl)-2'-deoxyuridine was successfully incorporated into DNA oligomers by automated DNA synthesis using phosphoramidite chemistry. UV exposure of the latter thionucleoside containing oligonucleotides under anaerobic and aerobic conditions gives rise to specific base lesions. The photoproducts have been isolated and further characterized on the basis of NMR and mass spectrometric analyses.

Synthesis and UV photolysis of oligodeoxynucleotides that contain 5-(phenylthiomethyl)-2'-deoxyuridine: a specific photolabile precursor of 5-(2'-deoxyuridilyl)methyl radical.

[formula: see text] The title exocyclic radical (2) is generated via photochemical cleavage of 5-(phenylthiomethyl)-2'-deoxyuridine (8). The latter thionucleoside (8) was successfully incorporated into DNA oligomers by automated DNA synthesis using phosphoramidite chemistry. UV exposure of 8 containing oligonucleotides under (an)aerobic conditions gives rise to specific base lesions. The photoproducts have been isolated and further characterized on the basis of detailed NMR and mass spectrometric analyses.

The structure of phosphorylated p38gamma is monomeric and reveals a conserved activation-loop conformation.

BACKGROUND: Mitogen-activated protein (MAP) kinases mediate the cellular response to stimuli such as pro-inflammatory cytokines and environmental stress. P38gamma is a new member of the MAP kinase family, and is expressed at its highest levels in skeletal muscle. P38gamma is 63% identical in sequence to P38alpha. The structure of P38alpha MAP kinase has been determined in the apo, unphosphorylated, inactive form. The structures of apo unphosphorylated ERK2, a related MAP kinase, and apo phosphorylated ERK2 have also been determined. RESULTS: We have determined the structure of doubly phosphorylated P38gamma in complex with an ATP analog by X-ray crystallography. This is the first report of a structure of an activated kinase in the P38 subfamily, and the first bound to a nucleotide. P38gamma residue phosphoryl-Thr183 forms hydrogen bonds with five basic amino acids, and these interactions induce an interdomain rotation. The conformation of the activation loop of P38gamma is almost identical to that observed in the structure of activated ERK2. However, unlike ERK2, the crystal structure and solution studies indicate that activated P38gamma exists as a monomer. CONCLUSIONS: Interactions mediated by phosphoryl-Thr183 induce structural changes that direct the domains and active-site residues of P38gamma into a conformation consistent with catalytic activity. The conformation of the phosphorylation loop is likely to be similar in all activated MAP kinases, but not all activated MAP kinases form dimers.

First report of association of chronic pancreatitis, primary biliary cirrhosis, and systemic sclerosis.

We report an original observation of chronic pancreatitis associated with primary biliary cirrhosis and systemic sclerosis. The diagnosis of each of these conditions was unequivocally confirmed. Pancreatic involvement in this case was asymptomatic. The association of chronic pancreatitis with primary biliary cirrhosis has been previously reported and pancreatitis has been associated with other autoimmune disorders. We hypothesize about the underlying pathogenic mechanisms of chronic pancreatitis in our case.

The natural history of hereditary chronic pancreatitis: a study of 12 cases compared to chronic alcoholic pancreatitis.

The aim of this study was to document the natural history of chronic hereditary pancreatitis and to compare its evolution to that of chronic alcoholic pancreatitis. Twelve subjects with chronic hereditary pancreatitis were followed up for a mean duration of 15.8 years (range, 1-23) and compared to subjects with chronic alcoholic pancreatitis who were followed up from 1972 to 1980. The subjects with chronic hereditary pancreatitis, when compared to those with chronic alcoholic pancreatitis, were found to have an earlier onset of symptoms (10.5 vs. 46.0 years, p < 0.05); a significant delay in diagnosis (14.3 vs. 3 years); a similar prevalence of pancreatic calcification (58 vs. 57%); a similar amount of pancreatic insufficiency; both endocrine (50 vs. 70%) and exocrine 42 vs. 38%); and a higher prevalence of pseudocysts (33 vs. 10%, p < 0.05). Only one pancreatic adenocarcinoma was diagnosed in a patient with chronic alcoholic pancreatitis. Apart from the earlier onset and the delay in diagnosis, chronic hereditary pancreatitis has a natural history similar to that of chronic alcoholic pancreatitis. The disease is progressive with a high incidence of complications, but all subjects were alive after follow-up.

Crystal structure of the RAG1 dimerization domain reveals multiple zinc-binding motifs including a novel zinc binuclear cluster.

The crystal structure of the dimerization domain of the V(D)J recombination-activating protein, RAG1, was solved using zinc anomalous scattering. The structure reveals an unusual combination of multi-class zinc-binding motifs, including a zinc RING finger and a C2H2 zinc finger, that together from a single structural domain. The domain also contains a unique zinc binuclear cluster in place of a normally mononuclear zinc site in the RING finger. Together, four zinc ions help organize the entire domain, including the two helices that form the dimer interface.

Short-term effects of transcatheter arterial chemoembolisation on metabolic activity of the liver of cirrhotic patients with hepatocellular carcinoma.

BACKGROUND: Transcatheter arterial chemoembolisation, a procedure for the treatment of hepatocellular carcinoma, provokes a pronounced but transient increase in hepatic cytolysis parameters. A definite evaluation of the impairment of liver function after this treatment, performed by adequate techniques, is still lacking. AIMS: To assess and quantify the impairment of liver metabolic activity after arterial chemoembolisation in patients with cirrhosis. The variations of hepatic vein pressure gradient provoked by this procedure were evaluated. PATIENTS: 15 patients with cirrhosis (Child's class A and B) and hepatocellular carcinoma. METHODS: 17 transcatheter arterial chemoembolisations with epirubicin, iodised oil, and gelfoam were performed; liver function was assessed before, the following day, and after seven days measuring galactose elimination capacity; aminopyrine breath test was also performed in six patients before the procedure and seven days after. In 10 patients intrinsic hepatic clearance of indocyanine green and hepatic vein pressure gradient were measured by hepatic vein catheterisation before and 30 minutes after chemoembolisation. RESULTS: Intrinsic hepatic clearance of indocyanine green decreased significantly from (mean (SEM)) 355 (140) ml/min to 277 (98) ml/min after the procedure (p = 0.0007). Galactose elimination capacity did not show significant changes, being 4.00 (0.90) mg/min/kg body weight at baseline, 4.20 (0.90) mg/min/kg body weight after one day, and 3.95 (0.87) mg/min/kg body weight seven days after chemoembolisation. Aminopyrine breath test was 2.31 (1.09)% and remained unchanged after treatment, being 2.39 (2.04)% at day 7. Baseline hepatic vein pressure gradient was 17.0 (5.5) mm Hg, and 14.4 (3.7) mm Hg 30 minutes after chemoembolisation (p = 0.09). CONCLUSIONS: A single transcatheter chemoembolisation in cirrhotic patients was detected by galactose elimination capacity and aminopyrine breath test one and seven days after the procedure. Therefore it can be considered a safe therapeutic tool for hepatocellular carcinoma in Child's class A and B cirrhotic patients.

Duplex Doppler ultrasonography allows a multiorgan noninvasive approach to splanchnic pharmacodynamics in patients with cirrhosis.

UNLABELLED: Duplex Doppler ultrasonography (DDU) is a suitable method to evaluate acute splanchnic hemodynamic effects of vasoactive drugs. It allows the contemporary evaluation of arterial and venous splanchnic parameters. Forty-six cirrhotic patients with esophageal varices were investigated by DDU. Portal blood flow mean velocity (PBV) (cm/s), portal blood flow volume (PBF) (ml/min), pulsatility index (PI) [(maximum-minimum)/mean velocity] in the superior mesenteric artery, in intrahepatic arteries (main branches), in an instrasplenic artery, and in interlobar arteries of the kidneys were measured before and 120-180 min after the administration of nadolol (80 mg p.o.) in 24 patients, and before and 120 min after placebo administration in 9 patients. In 13 patients who were chronically treated with nadolol, DDU parameters were evaluated before and 90 min after the administration of isosorbide-5-mononitrate (20 mg p.o.). RESULTS: placebo caused no hemodynamic change. After nadolol, heart rate decreased (-22 +/- 8%), and so did PBV and PBF (8.8 +/- 3.4 vs. 10.9 +/- 3.2, -20 +/- 17%, p < 0.0001; 660 +/- 347 vs. 852 +/- 371, -24 +/- 18%, p < 0.0001). Mesenteric PI increased (2.72 +/- 0.67 vs. 2.28 +/- 0.56, +21 +/- 25%, p = 0.005). Hepatic, splenic, and renal PIs showed slight, not significant changes (1.42 +/- 0.41 vs. 1.38 +/- 0.32, p = NS; 1.05 +/- 0.23 vs. 0.99 +/- 0.21, p = NS; 1.24 +/- 0.26 vs. 1.19 +/- 0.20, p = NS, respectively). After the administration of isosorbide-5-mononitrate, PBV decreased (8.2 +/- 2.0 vs. 9.4 +/- 2.3, -12 +/- 13%, p = 0.006), while PBF did not modify (648 +/- 189 vs. 711 +/- 209, p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Development and validation of a prognostic index predicting death after upper gastrointestinal bleeding in patients with liver cirrhosis: a multicenter study.

Prognostic factors of the outcome of upper gastrointestinal bleeding in patients with cirrhosis are insufficiently defined. Pertinent clinical, biochemical, and endoscopic data of 332 upper gastrointestinal bleedings in 268 patients with cirrhosis observed in the participating centers during 31 months were recorded. Clinical data were analyzed until 40 days after bleeding. A further set of 82 bleedings was used as a validation group. Ninety-two of the 268 patients died within the time of the study, and 28 of the 82 patients of the validation group died. According to a stepwise logistic regression analysis, s-creatinine, ascites on admission, previous diagnosis of hepatocellular carcinoma, s-bilirubin, prothrombin index, varices as definite or probable source of bleeding, gender, and presentation with hemathemesis were the best set of covariates for predicting outcome. From them a prognostic index was developed and validated in the 82 further bleedings. Sensitivity and specificity in the cumulated training and test sets were 75 and 80%, respectively. In the present material, the prognostic index was significantly more efficient than Child-Pugh score or the prognostic index proposed by Garden et al. These data show that it is possible to predict the outcome of upper gastrointestinal bleeding in cirrhosis on the basis of few easily available data. The prognostic index we proposed and validated may become useful to predict the outcome of a bleeding and to select or stratify patients in clinical trials.

Long-term follow-up study of adult patients with non-cirrhotic obstruction of the portal system: comparison with cirrhotic patients.

Thirty-two patients with non-cirrhotic portal system obstruction and oesophageal varices of non-malignant etiology were recruited over 13 years. Diagnosis was based on the presence of oesophageal varices at endoscopy, minor alterations in liver function tests and liver histology, a low hepatic venous pressure gradient, and pertinent angiographic patterns. Twenty-three had portal vein thrombosis, nine had splenic vein thrombosis. Twenty-one had idiopathic portal vein obstruction, 11 had secondary obstruction. The outcome was compared with a group of 32 patients with cirrhosis and portal hypertension, matched for age, Child-Pugh class, previous history of gastrointestinal bleeding, and size of oesophageal varices. Patients with non-cirrhotic obstruction of the portal system were followed for up to 171 months (mean 94 months). During follow-up ten patients had gastrointestinal bleeding, and eight died (five of gastrointestinal bleeding). After 6 years of follow-up, the cumulative risk of gastrointestinal bleeding was 24%, the cumulative risk of death was 17%, and the cumulative risk of death from gastrointestinal bleeding was 14%. Cumulative probability of death by any cause and the probability of gastrointestinal bleeding were significantly lower in patients with non-cirrhotic obstruction of the portal system than in patients with cirrhosis comparable for liver function and portal hypertension (p = 0.04 for both). The cumulative probability of death by gastrointestinal bleeding was not significantly different. In conclusion, the prognosis for non-cirrhotic obstruction of the portal system is significantly better than for patients with cirrhosis with comparable levels of liver function impairment and severity of portal hypertension.

Aminopyrine breath test in the prognostic evaluation of patients with cirrhosis.

This prospective study assessed the role of aminopyrine breath test in the prognosis of patients with cirrhosis, and evaluated whether the test provided useful information not included in the Pugh score. During a period of 36 months, 125 patients with biopsy proven liver cirrhosis were included, and followed for up to 48 months (median 17 months). During follow up 43 patients died (20 of liver failure). Survival was univariately related to aminopyrine breath test (p less than 0.02), Pugh score (p less than 0.01), presence of ascites (p less than 0.01), and sex (p less than 0.05). Using Cox's regression analysis, Pugh score, aminopyrine breath test, and sex, were independent significant predictors of survival. From the Cox's model a prognostic index was computed. According to a receiver operating characteristic curve analysis, the prognostic index predicting death showed an improvement in area under the curve when compared with a prognostic index calculated excluding aminopyrine breath test, but the improvement did not reach statistical significance (p = 0.12). A similar prognostic index was calculated to predict death from liver failure. Cox's regression analysis selected aminopyrine breath test, Pugh score, and aetiology as the best set of predictor covariates. According to a receiver operating characteristic curve analysis, a prognostic index cut off value of 2.6 had a 94% sensitivity and a 88% specificity. The prognostic index significantly improved prognostic accuracy when compared with a prognostic index calculated from Pugh score and aetiology, but excluding aminopyrine breath test (p = 0.05). These data disclose that the aminopyrine breath test offers additional prognostic information to the Pugh score, and the prognosis of patients with cirrhosis.

Prognostic usefulness of hepatic vein catheterization in patients with cirrhosis and esophageal varices.

Clinical and anamnestic data, Pugh score, and size of esophageal varices were obtained in 129 cirrhotics. Hepatic vein catheterization was performed to measure hepatic venous pressure gradient (HVPG), indocyanine green (ICG) intrinsic hepatic clearance, and hepatic plasma flow. During a follow-up period of up to 60 months, 44 patients experienced gastrointestinal bleeding and 54 died. Applying Cox regression analysis, ICG intrinsic hepatic clearance, Pugh score, previous variceal bleeding, and HVPG were the only significant prognostic determinants of survival. In addition, Cox's regression analysis showed that HVPG, Pugh score, size of varices, and previous variceal bleeding all contained significant prognostic information regarding risk of gastrointestinal bleeding. The models were validated using a split-sample technique, and prognostic indexes for death and gastrointestinal bleeding were calculated. The prognostic index predicting death had significantly improved prognostic accuracy over a prognostic index calculated excluding the data obtained from hepatic vein catheterization (P less than 0.05). In conclusion, prognostic accuracy in cirrhosis with portal hypertension is significantly improved by information obtained from hepatic vein catheterization.