Bringing biocatalytic deuteration into the toolbox of asymmetric isotopic labelling techniques.

Enzymes dependent on nicotinamide cofactors are important components of the expanding range of asymmetric synthetic techniques. New challenges in asymmetric catalysis are arising in the field of deuterium labelling, where compounds bearing deuterium (2H) atoms at chiral centres are becoming increasingly desirable targets for pharmaceutical and analytical chemists. However, utilisation of NADH-dependent enzymes for 2H-labelling is not straightforward, owing to difficulties in supplying a suitably isotopically-labelled cofactor ([4-2H]-NADH). Here we report on a strategy that combines a clean reductant (H2) with a cheap source of 2H-atoms (2H2O) to generate and recycle [4-2H]-NADH. By coupling [4-2H]-NADH-recycling to an array of C=O, C=N, and C=C bond reductases, we demonstrate asymmetric deuteration across a range of organic molecules under ambient conditions with near-perfect chemo-, stereo- and isotopic selectivity. We demonstrate the synthetic utility of the system by applying it in the isolation of the heavy drug (1S,3'R)-[2',2',3'-2H3]-solifenacin fumarate on a preparative scale.

Discriminating changes in intracellular NADH/NAD+ levels due to anoxicity and H2 supply in R. eutropha cells using the Frex fluorescence sensor.

The hydrogen-oxidizing "Knallgas" bacterium Ralstonia eutropha can thrive in aerobic and anaerobic environments and readily switches between heterotrophic and autotrophic metabolism, making it an attractive host for biotechnological applications including the sustainable H2-driven production of hydrocarbons. The soluble hydrogenase (SH), one out of four different [NiFe]-hydrogenases in R. eutropha, mediates H2 oxidation even in the presence of O2, thus providing an ideal model system for biological hydrogen production and utilization. The SH reversibly couples H2 oxidation with the reduction of NAD+ to NADH, thereby enabling the sustainable regeneration of this biotechnologically important nicotinamide cofactor. Thus, understanding the interaction of the SH with the cellular NADH/NAD+ pool is of high interest. Here, we applied the fluorescent biosensor Frex to measure changes in cytoplasmic [NADH] in R. eutropha cells under different gas supply conditions. The results show that Frex is well-suited to distinguish SH-mediated changes in the cytoplasmic redox status from effects of general anaerobiosis of the respiratory chain. Upon H2 supply, the Frex reporter reveals a robust fluorescence response and allows for monitoring rapid changes in cellular [NADH]. Compared to the Peredox fluorescence reporter, Frex displays a diminished NADH affinity, which prevents the saturation of the sensor under typical bacterial [NADH] levels. Thus, Frex is a valuable reporter for on-line monitoring of the [NADH]/[NAD+] redox state in living cells of R. eutropha and other proteobacteria. Based on these results, strategies for a rational optimization of fluorescent NADH sensors are discussed.

Virology analyses of HCV genotype 4 isolates from patients treated with simeprevir and peginterferon/ribavirin in the Phase III RESTORE study.

Simeprevir is a hepatitis C virus NS3/4A protease inhibitor. Hepatitis C virus baseline NS3/4A polymorphisms and emerging mutations were characterized in treatment-naїve and treatment-experienced genotype 4-infected patients treated with simeprevir+peginterferon/ribavirin in the RESTORE study. Population sequencing of the NS3/4A region was performed and in vitro simeprevir activity against site-directed mutants or chimeric replicons with patient-derived NS3 protease sequences was assessed in a transient replicon assay. Simeprevir remained active against most (83/91 [91%]) baseline isolates tested in the chimeric replicon assay. Eight baseline isolates reduced simeprevir activity; these carried I132L or D168E substitutions reducing simeprevir median activity by 4.6- and 39-fold, respectively. Six of these eight isolates were from patients achieving sustained virologic response. Baseline NS3 Q80K polymorphism was not observed in the genotype 4-infected patients. Of the 107 simeprevir-treated patients, 37 did not achieve sustained virologic response for any reason. Of the 32 patients who failed treatment and had sequencing information, 28 (88%) had emerging mutations at NS3 positions 80, 122, 155, 156 and/or 168 at time of failure, similar to those in genotype 1. Emerging mutations were mainly D168V and D168E alone or combined with mutations at position 80. In general, isolates obtained at time of failure displayed high-level in vitro resistance to simeprevir (fold change ≥50) in a chimeric replicon assay with a median simeprevir fold change value of 440, consistent with observed mutations. In conclusion, emerging mutations in genotype 4 patients failing simeprevir+peginterferon/ribavirin treatment were similar to those in genotype 1 and conferred high-level resistance to simeprevir.

Identification and characterization of a new member of the genus Luteovirus from cherry.

The complete genomic sequence of a new virus from cherry trees was determined. Its genome is 5857 nt long and resembles that of members of the genus Luteovirus in its genomic organization and nucleotide sequence. Based on the species demarcation criteria for luteoviruses, the virus represents a new luteovirus species. Furthermore, a 47-nt-long inverted repeat was found at the 3' end of its genome. The virus has been provisionally named cherry-associated luteovirus (ChALV) and is the fourth member of the family Luteoviridae reported to naturally infect woody plants.

[Hippocampal stroke]. / Der hippokampale Schlaganfall.

Unilateral cerebral ischemia of the hippocampus is very rare. This paper reviews the literature and presents the case of a 59-year-old woman with an amnestic syndrome due to a left hippocampal stroke. The patient suffered from retrograde amnesia which was most severe over the 2 days prior to presenting and a slight anterograde amnesia. In addition, a verbal memory disorder was confirmed 1 week after admission by neurological tests. As risk factors, arterial hypertension and a relative hyper-beta lipoproteinemia were found. This case shows that unilateral amnestic stroke, e.g. in the hippocampus region, may be the cause of an amnestic syndrome and should be included in the differential diagnostics.

Simeprevir with peginterferon/ribavirin for treatment of chronic hepatitis C virus genotype 1 infection: pooled safety analysis from Phase IIb and III studies.

This pooled analysis of five Phase IIb and III studies evaluated the safety and tolerability of simeprevir, a once daily, oral hepatitis C virus (HCV) NS3/4A protease inhibitor. Data were summarised for patients who received simeprevir 150 mg once daily (n = 924) or placebo (n = 540) plus pegylated interferon-α/ribavirin for 12 weeks. During the first 12 weeks of treatment, few patients discontinued simeprevir or placebo due to adverse events (AEs) (both 2.2%). Pruritus (23.8% vs 17.4%), rash (any; 22.9% vs 16.7%) and photosensitivity (3.2% vs 0.6%) [Correction added on 16 January 2015, after first online publication: In the above sentence, the values in 'Photosensitivity' were previously incorrect and have now been changed to 3.2% vs 0.6%.] were more prevalent in the simeprevir vs the placebo groups. Most AEs were grade 1/2 (72.4% for simeprevir vs 71.3% for placebo). All grade 3/4 AEs occurred in <5.0% of patients, except neutropenia (9.8% vs 7.6%). Overall incidence of neutropenia was similar (17.3% vs 15.7%). Incidence of anaemia was 13.2% for simeprevir vs 10.9% for placebo, and incidence of increased bilirubin was 8.4% vs 2.8%. Bilirubin increases were mild-to-moderate and transient without concurrent transaminase increases or association with hepatic injury. Safety and tolerability did not vary with METAVIR score, although increased bilirubin and anaemia were more frequent in simeprevir-treated patients with METAVIR F4 (increased bilirubin, 13.0% vs 3.3%; anaemia, 19.0% vs 14.8%). Serious AEs were infrequent (2.1% for simeprevir vs 3.0% for placebo). No deaths were reported during the first 12 weeks of treatment. Patient-reported fatigue and other outcomes were comparable for both groups, but were of shorter duration for simeprevir due to the use of response-guided therapy. Simeprevir is well tolerated in HCV genotype 1-infected patients.

Time-of-flight angiography: a viable alternative to contrast-enhanced MR angiography and fat-suppressed T1w images for the diagnosis of cervical artery dissection?

OBJECTIVES: To compare the use of an unenhanced high-resolution time-of-flight MR angiography sequence (Hr-TOF MRA) with fat-suppressed axial/coronal T1-weighted images and contrast-enhanced angiography (standard MRI) for the diagnosis of cervical artery dissection (cDISS). METHODS: Twenty consecutive patients (9 women, 11 men, aged 24-66 years) with proven cDISS on standard MRI underwent Hr-TOF MRA at 3.0 T using dedicated surface coils. Sensitivity (SE), specificity (SP), positive and negative predictive values (PPV, NPV), Cohen's kappa (к) and accuracy of Hr-TOF MRA were calculated using the standard protocol as the gold standard. Image quality and diagnostic confidence were assessed on a four-point scale. RESULTS: Image quality was rated better for standard MRI (P = 0.02), whereas diagnostic confidence did not differ significantly (P = 0.27). There was good agreement between Hr-TOF images and the standard protocol for the presence/absence of cDISS, with к = 0.95 for reader 1 and к = 0.89 for reader 2 (P < 0.001). This resulted in SE, SP, PPV, NPV and accuracy of 97 %, 98 %, 97 %, 98 % and 97 % for reader 1 and 93 %, 96 %, 93 %, 96 % and 95 % for reader 2. CONCLUSIONS: Hr-TOF MRA can be used to diagnose cDISS with excellent agreement compared with the standard protocol. This might be useful in patients with renal insufficiency or if contrast-enhanced MR angiography is of insufficient image quality. KEY POINTS: • New magnetic resonance angiography sequences are increasingly used for vertebral artery assessment. • A high-resolution time-of-flight sequence allows the diagnosis of cervical artery dissection. • This technique allows the diagnosis without intravenous contrast medium. • It could help in renal insufficiency or when contrast-enhanced MRA fails.

NAD(H)-coupled hydrogen cycling - structure-function relationships of bidirectional [NiFe] hydrogenases.

Hydrogenases catalyze the activation or production of molecular hydrogen. Due to their potential importance for future biotechnological applications, these enzymes have been in the focus of intense research for the past decades. Bidirectional [NiFe] hydrogenases are of particular interest as they couple the reversible cleavage of hydrogen to the redox conversion of NAD(H). In this account, we review the current state of knowledge about mechanistic aspects and structural determinants of these complex multi-cofactor enzymes. Special emphasis is laid on the oxygen-tolerant NAD(H)-linked bidirectional [NiFe] hydrogenase from Ralstonia eutropha.

A microarray for screening the variability of 16S-23S rRNA internal transcribed spacer in Pseudomonas syringae.

The 16S-23S ribosomal internal transcribed spacer (ITS1) is often used as a subspecies or strain-specific molecular marker for various kinds of bacteria. However, the presence of different copies of ITS1 within a single genome has been reported. Such mosaicism may influence correct typing of many bacteria and therefore knowledge about exact configuration of this region in a particular genome is essential. In order to screen the variability of ITS1 among and within Pseudomonas syringae genomes, an oligonucleotide microarray targeting different configurations of ITS1 was developed. The microarray revealed seven distinct variants in 13 pathovars tested and detected mosaicism within the genomes of P. syringae pv. coronafaciens, pisi, syringae and tabaci. In addition, the findings presented here challenge the using of rRNA analysis for pathovar and strain determination.

Review: Lupus nephritis: pathologic features, epidemiology and a guide to therapeutic decisions.

Systemic lupus erythematosus may present with renal manifestations that frequently are difficult to categorize and lupus nephritis is an important predictor of poor outcome. The type and spectrum of renal injury may remain undiagnosed until full-blown nephritic and/or nephrotic syndrome appear with increased risk of end-stage renal disease. These abnormalities occur within the first few years after the diagnosis of lupus is made on clinical grounds and with the support of laboratory tests in high risk patients. An early renal biopsy is helpful in patients with an abnormal urinalysis and/or reduced glomerular filtration rate and the results form the basis for therapeutic decisions. The biopsy also provides vital prognostic information based on histological categorization of different types of lupus nephritis, the degree of activity, chronicity and the immunopathogenesis. In the current armamentarium, the use of cyclophosphamide and azathioprine and recently mycophenolate mofetil, reduce morbidity and maintenance therapies reduce the risk of end-stage renal disease. Clinical trials underway promise new, effective and safe immunosuppressive regimens for the treatment of proliferative lupus nephritis.

[Nephrogenic fibrosing dermopathy or nephrogenic systemic fibrosis? What do we know and what do we have to learn?]. / Dermopatía fibrosante nefrogénica o fibrosis sistémica nefrogénica? Qué es lo que sabemos y qué debemos aprender?

Nephrogenic systemic fibrosis (NSF) was first recognized as a unique entity in 1997 and subsequently defined in the literature in 2000 as a novel fibrosing disorder occurring in the setting of renal disease. Prevention, early recognition and treatment are essential to limiting its impact. The most important risk factors for developing NSF are chronic or significant acute kidney disease (especially dialysis dependent patients) and the administration of gadolinium (GD3) containing contrast agents, agents that cause NSF by releasing free gadolinium (GD3) into tissues based on their pharmacokinetics. International commissions in drug control and medicinal products recommend to avoid gadolinium based contrast agents in patients with GFR < 30 ml/minute/1.73 m(2). Unfortunately there is lack of universally effective therapy at this time and the literature is based on case reports and small case series. Recommendations to guide the use of gadolinium based contrast agents in patients with underlying kidney disease should be individualized and considered in consultation with the ordering physician, radiologist and nephrologist.

¿Dermopatía fibrosante nefrogénica o fibrosis sistémica nefrogénica? ¿Qué es lo que sabemos y qué debemos aprender? / No disponible

La Fibrosis Sistémica Nefrogénica (FSN) fue descrita por primera vez como entidad singular en el año 1997, y después se confirmó y documentó en la literatura médica durante el año 2000como una enfermedad escleromixedematosa fibrosante que se presentaba en pacientes con afecciones renales. La prevención, el diagnóstico precoz y el tratamiento son esenciales para limitar su impacto, siendo el fracaso renal agudo y la enfermedad renal crónica, junto con la administración de agentes de contraste que contienen Gadolinio (GD) los factores de riesgo más importantes que pueden hacer desarrollar la enfermedad. Estos agentes causan FSN mediante la eliminación y el depósito de radicales libres de GD3 (gadolinio). Las comisiones internacionales de agencias del fármaco recomiendan la no utilización de estos agentes en pacientes con un filtrado glomerular menor de30 ml/min/1,73 m2. Desafortunadamente, carecemos de terapias contrastadas y efectivas, y la evidencia disponible se basa en series pequeñas y grupos de casos aislados. Las recomendaciones para el uso de agentes de contraste que contengan gadolinio, en pacientes con insuficiencia renal avanzada, deberán individualizarse y tomarse en cuenta multidisciplinariamente por equipos formados por internistas, radiólogos y nefrólogos (AU)

Nephrogeni c systemic fibrosis was first recognized as a unique entity in 1997and subsequently defined in the literature in 2000 as a novel fibrosing disorder occurring in the setting of renal disease. Prevent ion, early recognition and treatment are essential to limiting its impact. The most important risk factors for developing NSF are chronic or significant acute kidney disease (espec ially dialysis dependent patient s ) and the adminitration of gadolinium containing contrast agents , agent s that cause NFS by releasing free GD3 into tissues based on their pharmacokinetics. International commissions in drug control and medicinal products recommend to avoid gadolinium based contrast agents in patients with GFR <30 ml /minute/1,73 m2.Unfortunately there i s lack of universally effective therapy at this time and the literature is based on case reports and small case series . Recommendations to guide the use of gadolinium based contrast agents in patients with underlying kidney disease should be individualized and considered in consul tat ion with the ordering physician, radiologist and nephrologist (AU)

P3M algorithm for dipolar interactions.

An extension to the P(3)M algorithm for electrostatic interactions is presented that allows to efficiently compute dipolar interactions in periodic boundary conditions. Theoretical estimates for the root-mean-square error of the forces, torques, and the energy are derived. The applicability of the estimates is tested and confirmed in several numerical examples. A comparison of the computational performance of the new algorithm to a standard dipolar-Ewald summation methods shows a performance crossover from the Ewald method to the dipolar P(3)M method for as few as 300 dipolar particles. In larger systems, the new algorithm represents a substantial improvement in performance with respect to the dipolar standard Ewald method. Finally, a test comparing point-dipole-based and charged-pair based models shows that point-dipole-based models exhibit a better performance than charged-pair based models.

Failure to phosphorylate AKT in podocytes from mice with early diabetic nephropathy promotes cell death.

Loss of podocytes by apoptosis characterizes the early stages of diabetic nephropathy. To examine its mechanism we studied glomeruli and podocytes isolated from db/db mice with early diabetic nephropathy and albuminuria. Phosphorylation of AKT (protein kinase B, a key survival protein) was found to be lower in the glomeruli of 12 week old db/db compared to db/+ mice. In vitro, insulin phosphorylated AKT solely in podocytes from db/+ mice. Serum deprivation and exposure to tumor necrosis factor-alpha significantly compromised cell viability in podocytes from db/db but not from db/+ mice, and this was associated with a significant decrease in AKT phosphorylation. Inhibition of AKT was necessary to achieve the same degree of cell death in db/+ podocytes. Our study shows that podocyte inability to respond to insulin and susceptibility to cell death may partially account for the decreased podocyte number seen in early diabetic nephropathy.

Simulation of charge reversal in salty environments: giant overcharging?

We have performed MD simulations of a highly charged colloid in a solution of 3:1 and additional 1:1 salt. The dependency of the colloid's inverted charge on the concentration of the additional 1:1 salt has been studied. Most theories predict, that the inverted charge increases when the concentration of monovalent salt grows, up to what is called giant overcharging, while experiments and simulational studies observe the opposite. Our simulations agree with the experimental findings and shed light onto the weaknesses of the theories.

The optimal P3M algorithm for computing electrostatic energies in periodic systems.

We optimize Hockney and Eastwood's particle-particle particle-mesh algorithm to achieve maximal accuracy in the electrostatic energies (instead of forces) in three-dimensional periodic charged systems. To this end we construct an optimal influence function that minimizes the root-mean-square (rms) errors of the energies. As a by-product we derive a new real-space cutoff correction term, give a transparent derivation of the systematic errors in terms of Madelung energies, and provide an accurate analytical estimate for the rms error of the energies. This error estimate is a useful indicator of the accuracy of the computed energies and allows an easy and precise determination of the optimal values of the various parameters in the algorithm (Ewald splitting parameter, mesh size, and charge assignment order).

Investigating the sensitivity of a fluorescence-based microarray for the detection of fruit-tree viruses.

An oligonucleotide microarray for the detection of some fruit-tree viruses was designed and its theoretical detection limit was assessed using Cy3-labelled oligonucleotides. The real sensitivity of the microarray was compared for different kinds of fluorescently labelled targets: (a) cDNA and PCR amplified targets, (b) PCR amplified targets labelled using three different labelling methods. In the first case (a), the number of viral cDNA molecules was below the assessed detection limit of the microarray and only PCR amplified targets were detected. A second comparison (b), done on 3 selected viruses, included indirect labelling, the direct incorporation of labelled-dUTPs, and the use of Cy3-labelled primer. The targets labelled most intensively were produced by the Cy3-primer labelling (2 of 3 viruses) or by the indirect labelling method (1 of 3 viruses), the weakest signal showed targets labelled directly (all 3 viruses). The use of Cy3-primer labelling involved the simplest preparation and the lowest cost, however occasional weak cross-hybridization appeared. The indirect labelling method was of the highest specificity. The probes hybridizing near the 3-end of the targets showed the lowest intensities of fluorescent signal.

New directions in the management of severe lupus nephritis.

Systemic lupus erythematosus, which predominantly affects young women, frequently is complicated by renal involvement. The presence of acute glomerulonephritis significantly adds to morbidity and mortality. Based on currently published clinical trials, induction therapy with cyclophosphamide combined with pulse corticosteroids is an efficacious treatment option to preserve renal function, and long-term data are available to support this choice. Mycophenolate mofetil is a promising new agent, and recent data suggest that it is at least as efficacious as cyclophosphamide in the induction and maintenance phase, but with fewer side effects. Cell-depleting agents may be added in patients who fail the traditional regimens, preferentially in the setting of one of the ongoing clinical trials. The number of treatment regimens that the clinician can choose from when confronted with a patient with severe lupus nephritis has increased significantly, and more options are on the horizon. This promises more efficacious and better tolerable therapies, but it also puts an additional obligation on the physician to consider risk-to-benefit ratio, patient preference and adherence, feasibility, and cost; and to engage the patient in an active discussion about the different alternatives.

Outcomes in African Americans and Hispanics with lupus nephritis.

Poor outcomes have been reported in African Americans and Hispanics compared to Caucasians with lupus nephritis. The purpose of this retrospective analysis was to identify independent predictors of outcomes in African Americans and Hispanics with lupus nephritis. In total, 93 African Americans, 100 Hispanics, and 20 Caucasians with a mean age of 28 +/- 13 years and an annual household income of 32.9 +/- 17.3 (in 1000 US dollars) were studied. World Health Organization (WHO) lupus nephritis classes II, III, IV, and V were seen in 9, 13, 52, and 26%, respectively. Important baseline differences were higher mean arterial pressure (MAP) in African Americans compared to Hispanics and Caucasians (107 +/- 19, 102 +/- 15, and 99 +/- 13 mmHg, P < 0.05), and higher serum creatinine (1.66 +/- 1.3, 1.25 +/- 1.0, and 1.31 +/- 1.0 mg/dl, P < 0.025). African Americans had lower hematocrit compared to Hispanics and Caucasians (29 +/- 5, and 31 +/- 6, and 32 +/- 7%, P < 0.05), and lower annual household income (30.8 +/- 14.9, 33.1 +/- 15.9, and 42.2 +/- 29.3 in 1000 US dollars; P < 0.05). Lower prevalence of WHO class IV was seen in Caucasians (30%) compared to Hispanics (57%, P = 0.03) and African Americans (51%, P = 0.09). Development of doubling creatinine or end-stage renal disease was higher in African Americans and Hispanics than in Caucasians (31, 18, and 10%; P < 0.05), as was the development of renal events or death (34, 20, and 10%; P < 0.025). Our results suggest that both biological factors indicating an aggressive disease and low household income are common in African Americans and Hispanics with lupus nephritis, and outcomes in these groups are worse than in Caucasians.

Percutaneous management of perianastomotic stenosis in arteriovenous fistulae: results of a prospective study.

Surgical creation of new anastomosis has been proposed as the preferred treatment for perianastomotic stenoses of fistulae. However, disadvantages of surgical approach have included (1) frequent conversion of fistula to a graft by using synthetic graft material to create a new anastomosis, (2) shortening the length of the cannulation segment by proximal autologous arteriovenous neoanastomosis, and (3) abandoning the fistula altogether in favor of a synthetic graft. We report the results of a prospective study using percutaneous balloon angioplasty (PTA) to treat fistulae with perianastomotic lesions. Seventy-three consecutive patients undergoing 112 PTA procedures for the treatment of perianastomotic lesions were studied. Primary and secondary patency rates were calculated. Procedure success, procedure-related complications, and conversion of fistulae to grafts were recorded. The initial success rate was 97%. The degree of stenosis before and after PTA was 81 +/- 9 and 11+/-11%, respectively. Primary patency rates at 6, 12, and 18 months were 75, 51, and 41%, respectively. Secondary patency rates at 6, 12, and 18 months were 94, 90, and 90%, respectively. Grade I hematoma occurred in three and vein rupture in two cases. No grafts were inserted. These outcomes are superior to those that have been reported for surgery. The outpatient PTA is safe and effective for the management of perianastomotic stenosis. Because of its advantage of fistula preservation, the percutaneous approach should be considered as the preferred first-line therapy for the management of perianastomotic fistula lesions.