Celsior® crystalloid cardioplegia versus standard hyperkalemic normothermic blood cardioplegia: Analysis of myocardial protection in elective mitral valve repair.

INTRODUCTION: With the increase and refinement of video assisted mitral valve surgery, cristalloïd cardioplegia started regaining popularity. The aim of our study was to evaluate the effectiveness of Celsior®, a crystalloid cardioplegic solution, on myocardial protection in elective surgical mitral valve repair in comparison to blood based hyperkalemic cardioplegia. METHODS: In this observational retrospective study, all consecutive elective isolated surgical mitral valve repair where Celsior® or normothermic hyperkalemic blood cardioplegia were used were included. Primary endpoint was any sign of myocardial protection failure (troponin levels, need for inotropic or mechanical support, rhythm disturbances, mortality). Secondary endpoint was Celsior® safety (allergic reactions, bleeding, organ toxicities). RESULTS: From January 2009 to August 2016, 382 patients underwent elective isolated mitral valve repair in whom normothermic hyperkalemic blood cardioplegia (n = 181) or Celsior® (n = 201) were used. There were no statistically significant differences in baseline characteristics including Euroscore 2. Peak troponin (pg/ml) release and 30-days mortality were not statistically different. Need for cardioversion was significantly more frequent in the Celsior® group (47% vs 13%, p < 0.001). There was no statistical difference in post-operative atrial fibrillation, permanent pacemaker implantation, reoperation for bleeding, transfusion, acute kidney injury, haemoglobin at discharge or length of stay. No allergic reaction to Celsior® occurred. CONCLUSION: Effective myocardial protection was achieved with the Celsior® cardioplegic solution with no unexpected toxicity. Celsior® may be an efficacious and safe cardioprotective strategy in mitral valve repair.

Synthesis and Antiviral Evaluation of 3'-Fluoro-5'-norcarbocyclic Nucleoside Phosphonates Bearing Uracil and Cytosine as Potential Antiviral Agents.

Carbocyclic nucleoside analogues are an essential class of antiviral agents and are commonly used in the treatment of viral diseases (hepatitis B, AIDS). Recently, we reported the racemic synthesis and the anti-human immunodeficiency virus activities (HIV) of 3'-fluoro-5'-norcarbocyclic nucleoside phosphonates bearing purines as heterocyclic base. Based on these results, the corresponding racemic norcarbocyclic nucleoside phosphonates bearing pyrimidine bases were synthesized. The prepared compounds were evaluated against HIV, but none of them showed marked antiviral activity compared to their purine counterparts.

Contribution of the precursors and interplay of the pathways in the phospholipid metabolism of the malaria parasite.

The malaria parasite, Plasmodium falciparum, develops and multiplies in the human erythrocyte. It needs to synthesize considerable amounts of phospholipids (PLs), principally phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Several metabolic pathways coexist for their de novo biosynthesis, involving a dozen enzymes. Given the importance of these PLs for the survival of the parasite, we sought to determine their sources and to understand the connections and dependencies between the multiple pathways. We used three deuterated precursors (choline-d9, ethanolamine-d4, and serine-d3) to follow and quantify simultaneously their incorporations in the intermediate metabolites and the final PLs by LC/MS/MS. We show that PC is mainly derived from choline, itself provided by lysophosphatidylcholine contained in the serum. In the absence of choline, the parasite is able to use both other precursors, ethanolamine and serine. PE is almost equally synthesized from ethanolamine and serine, with both precursors being able to compensate for each other. Serine incorporated in PS is mainly derived from the degradation of host cell hemoglobin by the parasite. P. falciparum thus shows an unexpected adaptability of its PL synthesis pathways in response to different disturbances. These data provide new information by mapping the importance of the PL metabolic pathways of the malaria parasite and could be used to design future therapeutic approaches.

Recent Trends in Nucleotide Synthesis.

Focusing on the recent literature (since 2000), this review outlines the main synthetic approaches for the preparation of 5'-mono-, 5'-di-, and 5'-triphosphorylated nucleosides, also known as nucleotides, as well as several derivatives, namely, cyclic nucleotides and dinucleotides, dinucleoside 5',5'-polyphosphates, sugar nucleotides, and nucleolipids. Endogenous nucleotides and their analogues can be obtained enzymatically, which is often restricted to natural substrates, or chemically. In chemical synthesis, protected or unprotected nucleosides can be used as the starting material, depending on the nature of the reagents selected from P(III) or P(V) species. Both solution-phase and solid-support syntheses have been developed and are reported here. Although a considerable amount of research has been conducted in this field, further work is required because chemists are still faced with the challenge of developing a universal methodology that is compatible with a large variety of nucleoside analogues.

Revaluation of biomass-derived furfuryl alcohol derivatives for the synthesis of carbocyclic nucleoside phosphonate analogues.

The racemic synthesis of new carbocyclic nucleoside methylphosphonate analogues bearing purine bases (adenine and guanine) was accomplished using bio-sourced furfuryl alcohol derivatives. All compounds were prepared using a Mitsunobu coupling between the heterocyclic base and an appropriate carbocyclic precursor. After deprotection, the compounds were evaluated for their activity against a large number of viruses. However, none of them showed significant antiviral activity or cytotoxicity.

Beta-hydroxyphosphonate ribonucleoside analogues derived from 4-substituted-1,2,3-triazoles as IMP/GMP mimics: synthesis and biological evaluation.

A series of seventeen ß-hydroxyphosphonate ribonucleoside analogues containing 4-substituted-1,2,3-triazoles was synthesized and fully characterized. Such compounds were designed as potential inhibitors of the cytosolic 5'-nucleotidase II (cN-II), an enzyme involved in the regulation of purine nucleotide pools. NMR and molecular modelling studies showed that a few derivatives adopted similar structural features to IMP or GMP. Five derivatives were identified as modest inhibitors with 53 to 64% of cN-II inhibition at 1 mM.

Early structural valve deterioration of Mitroflow aortic bioprosthesis: mode, incidence, and impact on outcome in a large cohort of patients.

BACKGROUND: Structural valve deterioration (SVD) is a major flaw of bioprostheses. Early SVD has been suspected in the last models of Mitroflow bioprosthesis. We sought to assess the incidence, mode, and impact of SVD on outcome in a large series of Mitroflow aortic valve replacement. METHODS AND RESULTS: Six hundred seventeen consecutive patients (aged 76.1±6.3 years) underwent aortic valve replacement with a Mitroflow prosthesis (models 12A/LX) between 2002 and 2007. By echocardiography, 39 patients developed early SVD (1.66% per patient-year), with stenosis as the main mode (n=36). Mean delay to SVD was only 3.8±1.4 years, and 5-year SVD-free survival was 91.6% (95% confidence interval [CI], 88.7-94.7) for the whole cohort and 79.8% (95% CI, 71.2-89.4) and 94.0% (95% CI, 90.3-97.8) for 19- and 21-mm sizes, respectively. Among the 39 patients with SVD, 13 patients (33%) had an accelerated SVD once the mean gradient exceeded 30 mm Hg. Valve-related death was 46.2% in this SVD subgroup. Five-year overall survival was 69.6% (95% CI, 65.7-73.9). In multivariable analysis, SVD was the strongest correlate of overall mortality (hazard ratio=7.7; 95% CI, 4.4-13.6). CONCLUSIONS: Early SVD is frequent in Mitroflow bioprosthesis (models 12A/LX), especially for small sizes (19 and 21 mm), and reduces overall survival. An unpredictable accelerated pattern of SVD constitutes a life-threatening condition. In view of the large number of Mitroflow valves implanted worldwide, one can expect an epidemic of SVD and valve-related deaths, which represents a major public health issue, especially in the elderly. Hence, a close follow-up with yearly echocardiography after Mitroflow implantation is advisable. An urgent reoperation should be discussed in patients with severe SVD even though they are still asymptomatic.

Determination of the enzymatic activity of cytosolic 5'-nucleotidase cN-II in cancer cells: development of a simple analytical method and related cell line models.

The cytosolic 5'-nucleotidase (cN-II) has been shown to be involved in the response of cancer cells to cytotoxic agents, and the quantification of its activity in biological samples is of great interest. In this context, we developed and validated an analytical method for determination of cN-II activity in cultured cancer cells. This non-radioactive method, using a Hypercarb column as stationary phase, was validated with a lower limit of quantification of 0.1 µM inosine. We used it to characterize cell line models with modified cN-II expression obtained with stable transfections. We show that the short hairpin RNA (shRNA)-mediated inhibition of cN-II expression in various malignant blood cells is associated with decreased protein expression and enzymatic activity (1.7-6.2-fold) as well as an increased sensitivity to cytotoxic agents (up to 14-fold). On the other hand, expression of green fluorescent protein (GFP)-fused wild type or hyperactive mutant (R367Q) cN-II increased the activity and also decreased the sensitivity to nucleoside analogues. Our results confirm the biological relevance of modulating cN-II in cancer cells, and we present a straightforward validated method for the determination of cN-II activity in cellular samples.

Synthesis of {[5-(adenin-9-yl)-2-furyl]methoxy}methyl phosphonic acid and evaluations against human adenylate kinases.

AMP mimics constitute an important class of therapeutic derivatives to treat diseases where the pool of ATP is involved. A new phosphonate derivative of 9-(5-hydroxymethylfuran-2-yl)adenine was synthesized in a multi-step sequence from commercially available adenosine. Its ability to behave as a substrate of human adenylate kinases 1 and 2 was assessed. The phosphonate was shown to be a moderate but selective substrate of the mitochondrial human AK2, better than well-known antiviral acyclic phosphonates 9-(2-phosphonomethoxyethyl)adenine (PMEA, Adefovir) and (R)-9-(2-phosphonomethoxypropyl)adenine (PMPA, Tenofovir). Putative binding mode within adenylate kinase NMP site revealed by molecular docking in comparison to AMP native substrate allowed to illustrate this selective behavior.

Identification of novel markers for the diagnosis of malignant pleural mesothelioma.

The diagnosis of malignant pleural mesothelioma is difficult, with the most common differential diagnoses being benign pleural diseases and metastatic adenocarcinomas (ADCA). To identify novel markers that would be able to improve diagnostic accuracy, we performed a genome-wide gene expression analysis on tumor cell lines established from pleural effusions (malignant pleural mesothelioma and lung ADCA). This analysis led to the identification of genes encoding novel and pertinent cellular and soluble markers, for which the expression was validated by real-time RT-PCR. Immunohistochemical staining of tumor biopsy specimens with anti-type III collagen antibodies showed positive labeling for mesothelioma cells but not for ADCA cells. Using enzyme-linked immunosorbent assay, we showed that the C-C motif chemokine 2 (CCL2) concentration was significantly higher in pleural effusions from patients with mesothelioma (n = 61) than in subjects with ADCA (n = 25) or with benign pleural effusions (n = 15): median (interquartile range) = 2.99 ng/ml (1.76 to 6.01) vs 0.99 ng/ml (0.51 to 1.83) and 1.47 ng/ml (0.80 to 1.56), respectively, P < 0.0001. Conversely, the galectin-3 concentration was lower in mesothelioma: 11.50 ng/ml (6.73 to 23.53) vs 24.74 ng/ml (20.42 to 70.35) and 17.64 ng/ml (14.81 to 24.68), respectively, P < 0.0001. The areas under the curve for CCL2 were 0.8030 and 0.7716 for the differentiation of mesothelioma from ADCA or benign pleural effusions, respectively. Similarly, the areas under the curve obtained for galectin-3 were 0.7980 and 0.6923, respectively. In conclusion, type III collagen, CCL2, and galectin-3 are promising new diagnostic markers for mesothelioma.

Structural insights into the inhibition of cytosolic 5'-nucleotidase II (cN-II) by ribonucleoside 5'-monophosphate analogues.

Cytosolic 5'-nucleotidase II (cN-II) regulates the intracellular nucleotide pools within the cell by catalyzing the dephosphorylation of 6-hydroxypurine nucleoside 5'-monophosphates. Beside this physiological function, high level of cN-II expression is correlated with abnormal patient outcome when treated with cytotoxic nucleoside analogues. To identify its specific role in the resistance phenomenon observed during cancer therapy, we screened a particular class of chemical compounds, namely ribonucleoside phosphonates to predict them as potential cN-II inhibitors. These compounds incorporate a chemically and enzymatically stable phosphorus-carbon linkage instead of a regular phosphoester bond. Amongst them, six compounds were predicted as better ligands than the natural substrate of cN-II, inosine 5'-monophosphate (IMP). The study of purine and pyrimidine containing analogues and the introduction of chemical modifications within the phosphonate chain has allowed us to define general rules governing the theoretical affinity of such ligands. The binding strength of these compounds was scrutinized in silico and explained by an impressive number of van der Waals contacts, highlighting the decisive role of three cN-II residues that are Phe 157, His 209 and Tyr 210. Docking predictions were confirmed by experimental measurements of the nucleotidase activity in the presence of the three best available phosphonate analogues. These compounds were shown to induce a total inhibition of the cN-II activity at 2 mM. Altogether, this study emphasizes the importance of the non-hydrolysable phosphonate bond in the design of new competitive cN-II inhibitors and the crucial hydrophobic stacking promoted by three protein residues.

F1-adenosine triphosphatase displays properties characteristic of an antigen presentation molecule for Vgamma9Vdelta2 T cells.

Human Vgamma9Vdelta2 T lymphocytes are activated by phosphoantigens provided exogenously or produced by tumors and infected cells. Activation requires a contact between Vgamma9Vdelta2 cells and neighboring cells. We previously reported a role for cell surface F1-adenosine triphosphatase (ATPase) in T cell activation by tumors and specific interactions between Vgamma9Vdelta2 TCRs and purified F1-ATPase. 721.221 cells do not express surface F1-ATPase and do not support phosphoantigen responses unless they are rendered apoptotic by high doses of zoledronate, a treatment that promotes F1-expression as well as endogenous phosphoantigen production. By monitoring calcium flux in single cells, we show in this study that contact of T cells with F1-ATPase on polystyrene beads can partially replace the cell-cell contact stimulus during phosphoantigen responses. Triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester, an adenylated derivative of isopentenyl pyrophosphate, can stably bind to F1-ATPase-coated beads and promotes TCR aggregation, lymphokine secretion, and activation of the cytolytic process provided that nucleotide pyrophosphatase activity is present. It also acts as an allosteric activator of F1-ATPase. In the absence of Vgamma9Vdelta2 cells, triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester immobilized on F1-ATPase is protected from nucleotide pyrophosphatase activity, as is the antigenic activity of stimulatory target cells. Our experiments support the notion that Vgamma9Vdelta2 T cells are dedicated to the recognition of phosphoantigens on cell membranes in the form of nucleotide derivatives that can bind to F1-ATPase acting as a presentation molecule.

Mononucleoside phosphorodithiolates as mononucleotide prodrugs.

The synthesis and in vitro anti-HIV activity of a novel series of pronucleotides are reported. These prodrugs were characterized by a phosphorodithiolate structure, incorporating two O-pivaloyl-2-oxyethyl substituents as biolabile phosphate protections. The compounds were obtained following an original one-pot three-step procedure, involving the formation of a phosphorodithioite intermediate which is in situ oxidized. In vitro, comparative anti-HIV evaluations demonstrate that such original prodrugs are able to allow the efficient intracellular release of the corresponding 5'-mononucleotide. The pronucleotide of 2',3'-dideoxyadenosine (ddA) 3 exhibited a very potent antiretroviral effect with 50% effective concentration (EC50) values in nanomolar concentration range in various cell lines. In primary monocytes/macrophages, this derivative was 500 times more potent in inhibiting HIV replication (EC50 0.23 pM) than ddA and the selectivity index of the prodrug is fifty times higher than the one of the parent nucleoside.

Discovery of an L-like Configuration for 3'-Fluoro-5'-norcarbonucleoside Phosphonates as Potent Anti-HIV Agents.

Recently, we reported the racemic synthesis of 3'-fluoro-5'-norcarbocyclic nucleoside phosphonates bearing adenine as the heterocyclic base. For this study, to evaluate the antiviral activity of each enantiomer, we synthesized both enantiomers, as well as their corresponding bis(POM) prodrugs. Anti-HIV-1 evaluation against the LAI strain and clinically NRTI-resistant HIV-1 strains are presented. The activities against these different strains show that the activities of bis(POM) prodrug (-)-9 are equivalent or even superior to those of (R)-PMPA.

Incidence and risk factors of anastomotic complications after lung transplantation.

BACKGROUND: Anastomotic complications are common after lung transplantation (1.4-33% of cases) and still associated with a high morbi-mortality. METHODS: The current study is a monocenter retrospective analysis of symptomatic anastomotic complications (SAC) occurring after lung transplantation between 2010 and 2016, using the macroscopic, diameter, and suture (M-D-S) classification from consensus of French experts in bronchoscopy. The objectives were to determine incidence from surgery, risk factors, and impact of survival of SAC. We defined SAC as M-D-S abnormalities (stenosis ⩾ 50% or dehiscence) requiring bronchoscopic or surgical interventions. RESULTS: A total of 121 patients were included. SAC occurred in 26.5% of patients (n = 32), divided in symptomatic stenosis for 23.7% (n = 29), and symptomatic dehiscence in 2.5% (n = 3). In multivariate analysis, donor bacterial lung infection [HR 2.08 (1.04-4.17), p = 0.04] and age above 50 years [HR 3.26 (1.04-10.26), p = 0.04] were associated with SAC occurrence. Cystic fibrosis etiology was associated with better survival on Kaplan-Meier curve (p < 0.001). SAC [HR 2.15 (1.07-4.32), p = 0.03] was independently associated with worst survival. The 29 symptomatic patients because of stenosis required endoscopic procedure, of whom 16 patients needed bronchial stent placement. Four patients underwent surgery: three patients because of dehiscence and one because of severe bilateral stenosis (re-transplantation). DISCUSSION: SAC occurred in 26.5% of patients. Donor lung infection was the only alterable identified factors. The increase rate of SAC in older patients above 50 years of age encourages in regular endoscopic monitoring.

Synthesis of 2',3'-dideoxynucleoside phosphoesters using H-phosphonate chemistry on soluble polymer support.

Phosphorylation of ddC and 3TC was efficiently performed on soluble poly(ethylene glycol) support. The corresponding 5'-monophosphate derivatives were obtained by oxidation of the support bound 5'-H-phosphonate intermediates. Then, di- and triphosphorylations were carried out using a carbonyldiimidazole activation step followed by nucleophilic substitution with suitable phosphate salts. Trivalent phosphorus chemistry appeared as a good alternative for monophosphate synthesis of acid-sensitive 2',3'-dideoxynucleosides.

Specific requirements for Vgamma9Vdelta2 T cell stimulation by a natural adenylated phosphoantigen.

Human Vgamma9Vdelta2 T lymphocytes recognize phosphorylated alkyl Ags. Isopentenyl pyrophosphate (IPP) was previously proposed as the main Ag responsible for Vgamma9Vdelta2 T cell activation by cancer cells. However, triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (ApppI), a metabolite in which the isopentenyl moiety is linked to ATP, was reported in cells activated with aminobisphosphonates. The contribution of this compound to tumor-stimulatory activity was thus examined. ApppI induces selective expansion of Vgamma9Vdelta2 T cells from PBMCs. In the absence of APCs, however, ApppI has little stimulatory activity on Vgamma9Vdelta2 T cells, and optimal activation with ApppI requires addition of a nucleotide pyrophosphatase releasing IPP plus AMP. Thus, ApppI has no intrinsic stimulatory activity. Nevertheless, stimulation by ApppI is strengthened by the presence of APCs. Moreover, in contrast to IPP, ApppI can be efficiently pulsed on dendritic cells as well as on nonprofessional APCs. Pulsed APCs display stable and phosphatase-resistant stimulatory activity, indicative of Ag modification. HPLC analysis of tumor cell extracts indicates that latent phosphoantigenic activity is stored intracellularly in the Vgamma9Vdelta2 cell-sensitive tumor Daudi and can be activated by a nucleotide pyrophosphatase activity. The presence of ApppI in Daudi cell extracts was demonstrated by mass spectrometry. Nucleotidic Ags such as ApppI are thus a storage form of phosphoantigen which may represent a major source of phosphoantigenic activity in tumor cells. The unique properties of ApppI may be important for the design of Ags used in anticancer immunotherapeutic protocols using Vgamma9Vdelta2 cells.

An original pronucleotide strategy for the simultaneous delivery of two bioactive drugs.

The synthesis and in vitro anti-HIV activity of a novel series of phosphoramidate pronucleotides including a S-pivaloyl-2-thioethyl (tBuSATE) group as biolabile phosphate protecting group are reported. Such constructs, obtained through different phosphorus chemistries, are characterized by the association of two different anti-HIV nucleoside analogues linked to the phosphorus atom respectively by the sugar residue and the exocyclic amino function of the nucleobase. In vitro, comparative anti-HIV evaluation demonstrates that such original prodrugs are able to allow the efficient intracellular combination release of a 5'-mononucleotide as well as another nucleoside analogue. In human T4-lymphoblastoid cells, the pronucleotide 1 shows remarkable antiviral activity with an EC50 in the nanomolar range (0.6 ηM) and without additional cytotoxicity. In addition, these two pronucleotide models exhibit higher selectivity index than the equimolar mixture of their constitutive nucleoside analogues opening the way to further studies with regard to the current use of drug combinations.

Multiplexed detection of small analytes by structure-switching aptamer-based capillary electrophoresis.

Affinity probe capillary electrophoresis (APCE) assays, combining the separation power of CE with the specificity of interactions occurring between a target and a molecular recognition element (MRE), have become important analytical tools in many application fields. In this report, a rationalized strategy, derived from the structure-switching aptamer concept, is described for the design of a novel APCE mode dedicated to small molecule detection. Two assay configurations were reported. The first one, developed for the single-analyte determination, was based on the use of a cholesteryl-tagged aptamer (Chol-Apt) as the MRE and its fluorescein-labeled complementary strand (CS*) as the tracer (laser-induced fluorescence detection). Under micellar electrokinetic chromatography (MEKC) conditions, free CS* and the hybrid formed with Chol-Apt (duplex*) were efficiently separated (and then quantified) through the specific shift of the electrophoretic mobility of the cholesteryl-tagged species in the presence of a neutral micellar phase. When the target was introduced into the preincubated sample, the hybridized form was destabilized, resulting in a decrease in the duplex* peak area and a concomitant increase in the free CS* peak area. The second format, especially designed for multianalyte sensing, employed dually cholesteryl- and fluorescein-labeled complementary strands (Chol-CS*) of different lengths and unmodified aptamers (Apt). The size-dependent electrophoretic separation of different Chol-CS* forms from each other and from their corresponding duplexes* was also accomplished under MEKC conditions. The simultaneous detection of multiple analytes in a single capillary was performed by monitoring accurately each target-induced duplex-to-complex change. This method could expand significantly the potential of small solute APCE analysis in terms of simplicity, adaptability, generalizability, and high-throughput analysis capability.

Diastereoselective synthesis of (+/-)-1',4'-dimethyluridine.

The de novo synthesis of racemic 1',4'-dimethyluridine was accomplished in 12 steps starting from 2,5-dimethylfuran and vinylene carbonate. Key steps of the sequence include the stereoconvergent preparation of a meso diacid, and a stereoselective glycosylation without neighboring group participation. Such 1',4'-disubstituted ribonucleoside analogues are undisclosed compounds, which may present interesting biological activities.