Design of an artificial phage-display library based on a new scaffold improved for average stability of the randomized proteins.

Scaffold-based protein libraries are designed to be both diverse and rich in functional/folded proteins. However, introducing an extended diversity while preserving stability of the initial scaffold remains a challenge. Here we developed an original approach to select the ensemble of folded proteins from an initial library. The thermostable CheY protein from Thermotoga maritima was chosen as scaffold. Four loops of CheY were diversified to create a new binding surface. The subset of the library giving rise to folded proteins was first selected using a natural protein partner of the template scaffold. Then, a gene shuffling approach based on a single restriction enzyme was used to recombine DNA sequences encoding these filtrated variants. Taken together, the filtration strategy and the shuffling of the filtrated sequences were shown to enrich the library in folded and stable sequences while maintaining a large diversity in the final library (Lib-Cheytins 2.1). Binders of the Oplophorus luciferase Kaz domain were then selected by phage display from the final library, showing affinities in the µM range. One of the best variants induced a loss of 92% of luminescent activity, suggesting that this Cheytin preferentially binds to the Kaz active site.

Trifluridine/tipiracil in combination with oxaliplatin and either bevacizumab or nivolumab in metastatic colorectal cancer: a dose-expansion, phase I study.

BACKGROUND: In preclinical studies trifluridine/tipiracil (FTD/TPI) plus oxaliplatin (Industriestrasse, Holzkirchen, Germany) sensitised microsatellite stable (MSS) metastatic colorectal cancer (mCRC) to anti-programmed cell death protein-1; the addition of oxaliplatin or bevacizumab (F Hoffmann- la ROCHE AG, Kaiseraugst, Switzerland) enhanced the antitumour effects of FTD/TPI. This study aimed to investigate the safety and efficacy of FTD/TPI plus oxaliplatin and either bevacizumab or nivolumab (Uxbridge business Park, Uxbridge, United Kingdom) in patients with mCRC who had progressed after at least one prior line of treatment. PATIENTS AND METHODS: In 14-day cycles, patients received FTD/TPI 35 mg/m2 (twice daily, days 1-5) plus oxaliplatin 85 mg/m2 (day 1), and, on day 1, either bevacizumab 5 mg/kg (cohort A) or nivolumab 3 mg/kg (cohort B). Patients in Cohort B had confirmed MSS status. RESULTS: In total, 54 patients were enrolled: 37 in cohort A and 17 in cohort B. Recruitment in cohort B was stopped early due to the low response rate (RR) observed at interim analyses of efficacy. The most common adverse events (AEs) in cohort A were neutropenia/decreased neutrophils (75.7%), nausea (59.5%), vomiting (40.5%), diarrhoea (37.8%), peripheral sensory neuropathy (37.8%), fatigue (35.1%) and decreased appetite (35.1%). In cohort B, the most common AEs were neutropenia/decreased neutrophils (70.6%), diarrhoea (58.8%), nausea (47.1%), vomiting (47.1%), fatigue (47.1%), asthenia (41.2%), paraesthesia (41.2%), thrombocytopenia/decreased platelets (35.3%) and decreased appetite (35.3%). Confirmed objective RR was 17.1% in cohort A and 7.1% in cohort B; the corresponding values for median progression-free survival in the two cohorts were 6.3 and 6.0 months. CONCLUSION: FTD/TPI plus oxaliplatin and bevacizumab or nivolumab had an acceptable safety profile and demonstrated antitumour activity in previously treated patients with mCRC.

Pseudo-population bootstrap methods for imputed survey data.

The most common way to treat item nonresponse in surveys is to replace a missing value by a plausible value constructed on the basis of fully observed variables. Treating the imputed values as if they were observed may lead to invalid inferences. Bootstrap variance estimators for various finite population parameters are obtained using two pseudo-population bootstrap schemes. We establish the asymptotic properties of the resulting bootstrap variance estimators for population totals and population quantiles. A simulation study suggests that the methods perform well in terms of relative bias and coverage probability.

Metabolic Reprogramming in Amyotrophic Lateral Sclerosis.

Mitochondrial dysfunction in the spinal cord is a hallmark of amyotrophic lateral sclerosis (ALS), but the neurometabolic alterations during early stages of the disease remain unknown. Here, we investigated the bioenergetic and proteomic changes in ALS mouse motor neurons and patients' skin fibroblasts. We first observed that SODG93A mice presymptomatic motor neurons display alterations in the coupling efficiency of oxidative phosphorylation, along with fragmentation of the mitochondrial network. The proteome of presymptomatic ALS mice motor neurons also revealed a peculiar metabolic signature with upregulation of most energy-transducing enzymes, including the fatty acid oxidation (FAO) and the ketogenic components HADHA and ACAT2, respectively. Accordingly, FAO inhibition altered cell viability specifically in ALS mice motor neurons, while uncoupling protein 2 (UCP2) inhibition recovered cellular ATP levels and mitochondrial network morphology. These findings suggest a novel hypothesis of ALS bioenergetics linking FAO and UCP2. Lastly, we provide a unique set of data comparing the molecular alterations found in human ALS patients' skin fibroblasts and SODG93A mouse motor neurons, revealing conserved changes in protein translation, folding and assembly, tRNA aminoacylation and cell adhesion processes.

Protein Electrochemistry: Questions and Answers.

This chapter presents the fundamentals of electrochemistry in the context of protein electrochemistry. We discuss redox proteins and enzymes that are not photoactive. Of course, the principles described herein also apply to photobioelectrochemistry, as discussed in later chapters of this book. Depending on which experiment is considered, electron transfer between proteins and electrodes can be either direct or mediated, and achieved in a variety of configurations: with the protein and/or the mediator free to diffuse in solution, immobilized in a thick, hydrated film, or adsorbed as a sub-monolayer on the electrode. The experiments can be performed with the goal to study the protein or to use it. Here emphasis is on mechanistic studies, which are easier in the configuration where the protein is adsorbed and electron transfer is direct, but we also explain the interpretation of signals obtained when diffusion processes affect the response.This chapter is organized as a series of responses to questions. Questions 1-5 are related to the basics of electrochemistry: what does "potential" or "current" mean, what does an electrochemical set-up look like? Questions 6-9 are related to the distinction between adsorbed and diffusive redox species. The answers to questions 10-13 explain the interpretation of slow and fast scan voltammetry with redox proteins. Questions 14-19 deal with catalytic electrochemistry, when the protein studied is actually an enzyme. Questions 20, 21 and 22 are general.

Validation of the omega-3 fatty acid intake measured by a web-based food frequency questionnaire against omega-3 fatty acids in red blood cells in men with prostate cancer.

BACKGROUND/OBJECTIVES: The objective of this study was to evaluate the ability of a web-based self-administered food frequency questionnaire (web-FFQ) to assess the omega-3 (ω-3) fatty acids (FAs) intake of men affected with prostate cancer (PCa) against a biomarker. SUBJECTS/METHODS: The study presented herein is a sub-study from a phase II clinical trial. Enrolled patients afflicted with PCa were included in the sub-study analysis if the FA profiles from the red blood cell (RBC) membranes and FA intakes at baseline were both determined at the time of the data analysis (n=60). Spearman's correlation coefficients were calculated to estimate the correlations between FA intakes and their proportions in the RBC membranes. RESULTS: Intakes of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were highly correlated with their respective proportions in the RBC membranes (both rs=0.593, P<0.0001). Correlation between alpha-linolenic acid (ALA) intake and its proportion in RBC was not significant (rs=0.130, P=0.332). Correlations were observed between fatty fish intake and total ω-3 FAs (rs=0.304, P=0.02), total long-chain ω-3 FAs (rs=0.290, P=0.03) and DHA (rs=0.328, P=0.01) in RBC membranes. CONCLUSIONS: This study has shown that the web-FFQ is an accurate tool to assess total long-chain ω-3 FAs, EPA and DHA but not ALA intake in clinical trials and epidemiological studies carried out in men with PCa.

HIV-Associated Burkitt Lymphoma: Good Efficacy and Tolerance of Intensive Chemotherapy Including CODOX-M/IVAC with or without Rituximab in the HAART Era.

Background. The outcome of HIV-associated non-Hodgkin lymphoma (NHL) has improved substantially in the highly active antiretroviral therapy (HAART) era. However, HIV-Burkitt lymphoma (BL), which accounts for up to 20% of HIV-NHL, has poor outcome with standard chemotherapy. Patients and Methods. We retrospectively reviewed HIV-BL treated in the HAART era with the Magrath regimen (CODOX-M/IVAC±R) at four Canadian centres. Results. Fourteen patients with HIV-BL received at least one CODOX-M/IVAC±R treatment. Median age at BL diagnosis was 45.5 years, CD4 count 375 cells/mL and HIV viral load (VL) <50 copies/mL. Patients received PCP prophylaxis and G-CSF, 13 received HAART with chemotherapy and 10 rituximab. There were 63 episodes of toxicity, none fatal, including: bacterial infection, n = 20; grade 3-4 hematologic toxicity, n = 14; febrile neutropenia, n = 7; oral thrush; and ifosfamide neurological toxicity, n = 1 each. At a median followup of 11.7 months, 12 (86%) patients are alive and in remission. All 10 patients who received HAART, chemotherapy, and rituximab are alive. CD4 counts and HIV VL 6 months following BL therapy completion (n = 5 patients) were >250 cells/mL and undetectable, respectively, in 4. Conclusion. Intensive chemotherapy with CODOX-M/IVAC±R yielded acceptable toxicity and good survival rates in patients with HIV-associated Burkitt lymphoma receiving HAART.

Improved survival in HIV-associated diffuse large B-cell lymphoma with the addition of rituximab to chemotherapy in patients receiving highly active antiretroviral therapy.

PURPOSE: Recent trials suggest serious toxicity in HIV-associated non-Hodgkin's lymphoma (NHL) with rituximab (R) and chemotherapy (CT), offsetting the benefit of rituximab. METHOD: We retrospectively reviewed experience with CHOP-R vs. CT in 40 patients with HIV-associated diffuse large B-cell lymphoma (DLBCL) diagnosed between December 1992 and February 2006, all of whom were treated with curative intent. RESULTS: In a univariate analysis, International Prognostic Index (IPI) score, prior AIDS, HAART, and rituximab were significant for overall survival (OS). In a multivariate analysis, IPI 0-1 (p < .02), no prior AIDS (p < .0002), and receiving CHOP-R (p < .01) were significant for improved OS, and HAART use (p < .09) retained a trend for improved OS. The hazard ratio (HR) for patients with high IPI receiving CHOP-R was 0.3 (95% CI 0.1-0.8). Patients without prior AIDS receiving CHOP-R had an HR of 0.5 (95% CI 0.1-1.7). The OS at 30 months in patients not receiving HAART was 0%. With HAART, OS was 33% for CT and 86% for CHOP-R; HR for CHOP-R was 0.4 (95% CI 0.1-1.2). Toxic deaths were 3 (33%) for CHOP-R and 6 (25%) for CT (p = ns); all toxic deaths with CHOP-R were in patients not receiving HAART. Rituximab-treated patients had a lower death rate from lymphoma (CHOP-R, 2 [16%] vs. CT, 15 [63%]; p < .04), and overall mortality (CHOP-R, 5 [42%] vs. CT, 21 [88%]; p < .01). CONCLUSION: These retrospective data suggest that fatal toxicity of rituximab in HIV-NHL is not increased provided HAART is used, that the addition of rituximab to CT improved outcome, and that further prospective trials investigating this issue are warranted.

[Use of antiretroviral drugs invoices for the follow-up of a treatment programme. Example in Abidjan, Côte d'Ivoire]. / Utilisation des factures des médicaments antirétroviraux pour le suivi d'un programme de traitement. Exemple a Abidjan, Côte-d'Ivoire.

A retrospective analysis of the prescriptions of the ARV treatment programme in Abidjan, Côte-d'Ivoire enabled the authors to assess the collected anonymous information in order to evaluate the efficacy of the programme. This collection of information helps to supervise each center at a lower cost, to alert on some working problems and to follow the quality of the observance. This supervision is possible regardless of the health care places, it can be carried out systematically from the administrative or financial services. In this study this follow-up reveals some problems.

A thromboxane effect of a hydroxytyrosol-rich olive oil wastewater extract in patients with uncomplicated type I diabetes.

OBJECTIVE: To assess the antioxidant/non-antioxidant effects of a hydroxytyrosol (HT)-rich phenolic extract from olive mill wastewaters administered with a breakfast. DESIGN, SETTING AND SUBJECTS: Five type I diabetic patients received 25 mg of HT the first day and 12.5 mg/day the following 3 days. Blood sampling was carried out at T(0) (baseline) and T(4d) just before the breakfast + HT administration and at time points 1, 2, 3 and 4 h after T(0). Urines (24-h) were collected from T(0) to T(4d). Baseline HbA1c was generally inferior to 10%, glycemia was within the range 6-24 mmol/l, whereas total cholesterol, HDL-chol and triglycerides were normal. RESULTS: The major finding was the 46% decrease in the serum TXB(2) production after blood clotting at T(4d). Plasma vitamin A, E, beta-carotene were not changed. Vitamin C tended to increase (P = 0.075). Plasma antioxidant capacity was enhanced at T(0)+1 h only, whereas its main determinants (albumin, bilirubin, uric acid) were not modified. Urinary 8-isoPGF(2alpha) levels were highly variable and were not affected significantly by HT administration. CONCLUSION: The major effect of HT accounts for an antiaggregating platelet action, leading to a possible prevention of thrombotic and microthrombotic processes.

Validation of a volumic reconstruction in 4-d echocardiography and gated SPECT using a dynamic cardiac phantom.

A dynamic cardiac phantom was used as a reference to compare the volumes reconstructed with 4-D echocardiography and gated single-photon emission computed tomography (SPECT). 4-D echocardiography used a new prototype of rotating scan head to acquire ultrasound (US) images during a cardiac cycle, associated with a new protocol (left ventricular 4-D or LV 4-D) to reconstruct the volume deformations of the heart as a function of time. Gated SPECT data were acquired with a standard single-head gamma camera, and the reconstructions were carried out using the Mirage software released by Segami. The influences of different LV 4-D parameters were tested and analyzed. End-diastolic volume, end-systolic volume, and ejection fraction were measured using both LV 4-D and gated SPECT. Results obtained showed a straight correlation between the two examinations. The agreement confirmed the relevance of the comparisons. This study is an initial step before conducting clinical trials to exhaustively compare the two modalities.

Heterogeneity of endothelial junctions is reflected by differential expression and specific subcellular localization of the three JAM family members.

Endothelial cells are linked to each other through intercellular junctional complexes that regulate the barrier and fence function of the vascular wall. The nature of these intercellular contacts varies with the need for permeability: For example, in brain the impervious blood-brain barrier is maintained by "tight" contacts between endothelial cells. By contrast, in high endothelial venules (HEVs), where lymphocytes continuously exit the bloodstream, the contacts are generally leaky. The precise molecular components that define the type of junction remain to be characterized. An immunoglobulin superfamily molecule named JAM-2, specifically expressed in lymphatic endothelial cells and HEVs, was recently identified. JAM-3 was cloned and characterized in the current study, and JAM-1, -2, and -3 were shown to form a novel protein family belonging to the larger cortical thymocyte Xenopus (CTX) molecular family. Using antibodies specific for each of the 3 family members, their specific participation in different types of cell-cell contact in vivo and their specific and differential localization in lateral contacts or tight junctions were demonstrated. Furthermore, it was shown that JAM-1 and JAM-2 differentially regulate paracellular permeability, suggesting that the presence of JAM-1, -2, or -3 in vascular junctions may play a role in regulating vascular function in vivo.

Wine phenolic antioxidants inhibit AP-1 transcriptional activity.

Some of the beneficial effects of moderate wine consumption may be related to the antioxidant properties of polyphenolic compounds containing tannins, flavonoids, and phenolic acids. Cellular actions have recently been reported and may involve the modulation of transcriptional factors such as AP-1 (activator protein-1), which controls the expression of various genes implicated in inflammation processes, cell differentiation, and proliferation. The aim of this study was to evaluate the modulation of AP-1 activity by the phenolic acids (gallic, caffeic, protocatechic, paracoumaric, sinapic, and ferulic acids) that are present in wine and to compare their modulating pathways to those of lipophilic or hydrophilic "chain-breaking" antioxidants (such as DL-alpha-tocopherol or trolox) vitamin C, nitric oxide, and reduced glutathione. AP-1 response was studied on a cell line (MTLN) derived from MCF-7 cells transfected with luciferase gene under TRE sequence control. After stimulation by phorbol 12-myristate 13-acetate (PMA; 100 nM, 6 h, 10(-7) M), luciferase activity was determined by a luminescence method in the presence of luciferine/coenzyme A solution using a luminometer (LKB 1251, Finland). Antioxidants to be tested were incubated with cells in the presence or absence of PMA. Stimulation with PMA resulted in an AP-1-mediated increase in luciferase gene expression corresponding to an 8-fold increase in luciferase activity. After stimulation by PMA, a dose-dependent inhibition of AP-1 was observed with the six phenolic acids in the 20 nM-20 microM concentration range: gallic acid > caffeic > protocatechic, paracoumaric, sinapic acids > ferulic acid. Inhibition was more pronounced with phenolic acids than with DL-alpha-tocopherol (IC(50) = 5 +/- 4.5 microM for gallic acid vs 85 +/- 11 microM for vitamin E). None of the hydrophilic antioxidants inhibited PMA-induced AP-1 activation. None of the antioxidants tested in the absence of PMA stimulation induced any activation or inhibition of AP-1. Our results suggest that phenolic acids may act directly on cell signaling via inhibition of AP-1 transcriptional activity. In addition to preventing LDL oxidation in the arterial wall, our observations indicate that phenolic acids have a cell-mediated capacity to prevent some of the processes involved in atherosclerosis in a plasma concentration range compatible with nutritional intakes.

Enzyme electrokinetics: energetics of succinate oxidation by fumarate reductase and succinate dehydrogenase.

Protein film voltammetry is used to probe the energetics of electron transfer and substrate binding at the active site of a respiratory flavoenzyme--the membrane-extrinsic catalytic domain of Escherichia coli fumarate reductase (FrdAB). The activity as a function of the electrochemical driving force is revealed in catalytic voltammograms, the shapes of which are interpreted using a Michaelis-Menten model that incorporates the potential dimension. Voltammetric experiments carried out at room temperature under turnover conditions reveal the reduction potentials of the FAD, the stability of the semiquinone, relevant protonation states, and pH-dependent succinate--enzyme binding constants for all three redox states of the FAD. Fast-scan experiments in the presence of substrate confirm the value of the two-electron reduction potential of the FAD and show that product release is not rate limiting. The sequence of binding and protonation events over the whole catalytic cycle is deduced. Importantly, comparisons are made with the electrocatalytic properties of SDH, the membrane-extrinsic catalytic domain of mitochondrial complex II.

A distal histidine mutant (H52Q) of yeast cytochrome c peroxidase catalyzes the oxidation of H(2)O(2) instead of its reduction.

A H52Q variant of yeast cytochrome c peroxidase (CcP), in which the distal histidine is replaced by glutamine, catalyzes oxidation of H(2)O(2) instead of reduction. This redirection of catalytic action is detected by protein film voltammetry. In the presence of H(2)O(2), wild-type CcP, adsorbed on a graphite electrode, shows a strong catalytic reduction wave commencing at about 0.8V (pH 5.4); by contrast, H52Q does not exhibit this activity but instead shows a catalytic oxidation current at potentials in the region of 0.9 V. The oxidation current is partly suppressed in the presence of tetranitromethane (a superoxide scavenger) and is not observed for other mutants studied, including H52A. The only significant structural change in the H52Q variant is that the Q-52 side chain occupies the space vacated by the H-52 imidazole; specifically, the N-epsilon atom that is believed to transfer a proton and induce O--O cleavage is replaced, to within 0.75 A, by the carbamide-O. Thus, while the weakly basic amide functionality is unable to serve in the reorganization of bound H(2)O(2), it is able to facilitate its oxidation, most obviously by serving as a H-bond acceptor to assist formation of a labile superoxide intermediate.

Specific antioxidant activity of caffeoyl derivatives and other natural phenolic compounds: LDL protection against oxidation and decrease in the proinflammatory lysophosphatidylcholine production.

Specific antioxidant activity (SAA) (i.e., activity related to the molar or gallic acid equivalent amount of antioxidant) of natural polyphenolic mixtures or pure phenolic compounds was studied using their capacity to delay the conjugated diene production brought about by in vitro LDL copper-mediated or AAPH-mediated oxidation. The cinnamic acid series (caffeic, sinapic, ferulic acids) displayed a constant SAA over a large range of concentrations, whereas the benzoic acid series (gallic and protocatechuic acids) showed much higher SAA at low concentrations. The natural phenolic mixtures had a constant SAA. The highest SAA was obtained with caffeoyl esters (caffeoylquinic, rosmarinic, and caffeoyltartaric acids) and catechin for the copper-oxidation and the AAPH-oxidation system, respectively. Phenolic mixtures and acids delayed vitamin E depletion and decreased proinflammatory lysophosphatidylcholine production. As with polyphenols, probucol delayed lysophosphatidylcholine and conjugated dienes production, at higher concentrations, but was not effective at preventing vitamin E depletion. Polyphenols prevent the oxidation of LDL and its constituents (vitamin E, phosphatidylcholine), which is compatible with an antiinflammatory and antiatherosclerotic role in pathophysiological conditions.