Synthesis and biological evaluation of a water-soluble derivative of the potent V-ATPase inhibitor archazolid

The water-solubility of the highly potent V-ATPase inhibitors archazolid A and the glucosylated derivative archazolid C was studied in the presence of a wide range of cosolvents, revealing very low solubilites. The first water-soluble analogue was then designed, synthesized, and evaluated for V-ATPase inhibitory activity in vitro.     

Candidate gene sequencing of SLC11A2 and TMPRSS6 in a family with severe anaemia: common SNPs, rare haplotypes, no causative mutation

Background

Iron-refractory iron deficiency anaemia (IRIDA) is a rare disorder which was linked to mutations in two genes (SLC11A2 and TMPRSS6). Common polymorphisms within these genes were associated with serum iron levels. We identified a family of Serbian origin with asymptomatic non-consanguineous parents with three of four children presenting with IRIDA not responding to oral but to intravenous iron supplementation. After excluding all known causes responsible for iron deficiency anaemia we searched for mutations in SLC11A2 and TMPRSS6 that could explain the severe anaemia in these children.

Methodology/Results

We sequenced the exons and exon–intron boundaries of SLC11A2 and TMPRSS6 in all six family members. Thereby, we found seven known and fairly common SNPs, but no new mutation. We then genotyped these seven SNPs in the population-based SAPHIR study (n = 1,726) and performed genetic association analysis on iron and ferritin levels. Only two SNPs, which were top-hits from recent GWAS on iron and ferritin, exhibited an effect on iron and ferritin levels in SAPHIR. Six SAPHIR participants carrying the same TMPRSS6 genotypes and haplotype-pairs as one anaemic son showed lower ferritin and iron levels than the average. One individual exhibiting the joint SLC11A2/TMPRSS6 profile of the anaemic son had iron and ferritin levels lying below the 5^th percentile of the population''s iron and ferritin level distribution. We then checked the genotype constellations in the Nijmegen Biomedical Study (n = 1,832), but the profile of the anaemic son did not occur in this population.

Conclusions

We cannot exclude a gene-gene interaction between SLC11A2 and TMPRSS6, but we can also not confirm it. As in this case candidate gene sequencing did not reveal causative rare mutations, the samples will be subjected to whole exome sequencing.     

Solid-state NMR [13C,15N] resonance assignments of the nucleotide-binding domain of a bacterial cyclic nucleotide-gated channel

Channels regulated by cyclic nucleotides are key signalling proteins in several biological pathways. The regulatory aspect is conferred by a C-terminal cyclic nucleotide-binding domain (CNBD). We report resonance assignments of the CNBD of a bacterial mlCNG channel obtained using 2D and 3D solid-state NMR under Magic-angle Spinning conditions. A secondary chemical shift analysis of the 141 residue protein suggests a three-dimensional fold seen in earlier X-ray and solution-state NMR work and points to spectroscopic polymorphism for a selected set of resonances.     

Can electromagnetic fields influence the structure and enzymatic digest of proteins? A critical evaluation of microwave-assisted proteomics protocols

This study reevaluates the putative advantages of microwave-assisted tryptic digests compared to conventionally heated protocols performed at the same temperature. An initial investigation of enzyme stability in a temperature range of 37-80°C demonstrated that trypsin activity declines sharply at temperatures above 60°C, regardless if microwave dielectric heating or conventional heating is employed. Tryptic digests of three proteins of different size (bovine serum albumin, cytochrome c and β-casein) were thus performed at 37°C and 50°C using both microwave and conventional heating applying accurate internal fiber-optic probe reaction temperature measurements. The impact of the heating method on protein degradation and peptide fragment generation was analyzed by SDS-PAGE and MALDI-TOF-MS. Time-dependent tryptic digestion of the three proteins and subsequent analysis of the corresponding cleavage products by MALDI-TOF provided virtually identical results for both microwave and conventional heating. In addition, the impact of electromagnetic field strength on the tertiary structure of trypsin and BSA was evaluated by molecular mechanics calculations. These simulations revealed that the applied field in a typical laboratory microwave reactor is 3-4 orders of magnitude too low to induce conformational changes in proteins or enzymes.     

Indicators of acute and persistent renal damage in adult thrombotic microangiopathy

Background

Thrombotic microangiopathies (TMA) in adults such as thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are life-threatening disorders if untreated. Clinical presentation is highly variable and prognostic factors for clinical course and outcome are not well established.

Methods

We performed a retrospective observational study of 62 patients with TMA, 22 males and 40 females aged 16 to 76 years, treated with plasma exchange at one center to identify clinical risk factors for the development of renal insufficiency.

Results

On admission, 39 of 62 patients (63%) had acute renal failure (ARF) with 32 patients (52%) requiring dialysis treatment. High systolic arterial pressure (SAP, p = 0.009) or mean arterial pressure (MAP, p = 0.027) on admission was associated with acute renal failure. Patients with SAP>140 mmHg on admission had a sevenfold increased risk of severe kidney disease (OR 7.464, CI 2.097–26.565). MAP>100 mmHg indicated a fourfold increased risk for acute renal failure (OR 4.261, CI 1.400–12.972). High SAP, diastolic arterial pressure (DAP), and MAP on admission were also independent risk factors for persistent renal insufficiency with the strongest correlation for high MAP. Moreover, a high C-reactive protein (CRP) level on admission correlated with renal failure in the course of the disease (p = 0.003). At discharge, renal function in 11 of 39 patients (28%) had fully recovered, 14 patients (23%) remained on dialysis, and 14 patients (23%) had non-dialysis-dependent chronic kidney disease. Seven patients (11%) died. We identified an older age as risk factor for death.

Conclusions

High blood pressure as well as high CRP serum levels on admission are associated with renal insufficiency in TMA. High blood pressure on admission is also a strong predictor of sustained renal insufficiency. Thus, adult TMA patients with high blood pressure may require special attention to prevent persistent renal failure.     

Changes in neuronal DNA content variation in the human brain during aging

The human brain has been proposed to represent a genetic mosaic, containing a small but constant number of neurons with an amount of DNA exceeding the diploid level that appear to be generated through various chromosome segregation defects initially. While a portion of these cells apparently die during development, neurons with abnormal chromosomal copy number have been identified in the mature brain. This genomic alteration might to lead to chromosomal instability affecting neuronal viability and could thus contribute to age-related mental disorders. Changes in the frequency of neurons with such structural genomic variation in the adult and aging brain, however, are unknown. Here, we quantified the frequency of neurons with a more than diploid DNA content in the cerebral cortex of normal human brain and analyzed its changes between the fourth and ninth decades of life. We applied a protocol of slide-based cytometry optimized for DNA quantification of single identified neurons, which allowed to analyze the DNA content of about 500 000 neurons for each brain. On average, 11.5% of cortical neurons showed DNA content above the diploid level. The frequency of neurons with this genomic alteration was highest at younger age and declined with age. Our results indicate that the genomic variation associated with DNA content exceeding the diploid level might compromise viability of these neurons in the aging brain and might thus contribute to susceptibilities for age-related CNS disorders. Alternatively, a potential selection bias of "healthy aging brains" needs to be considered, assuming that DNA content variation above a certain threshold associates with Alzheimer's disease.     

Prothrombotic effects of tumor necrosis factor alpha in vivo are amplified by the absence of TNF-alpha receptor subtype 1 and require TNF-alpha receptor subtype 2

Introduction

Elevated serum levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) correlate with an increased risk for atherothrombotic events and TNFα is known to induce prothrombotic molecules in endothelial cells. Based on the preexisting evidence for the impact of TNFα in the pathogenesis of autoimmune disorders and their known association with an acquired hypercoagulability, we investigated the effects of TNFα and the role of the TNF receptor subtypes TNFR1 and TNFR2 for arteriolar thrombosis in vivo.

Methods

Arteriolar thrombosis and platelet-rolling in vivo were investigated in wildtype, TNFR1-/-, TNFR2-/- and TNFR1-/R2-/- C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. In vitro, expression of prothrombotic molecules was assessed in human endothelial cells by real-time PCR and flow cytometry.

Results

In wildtype mice, stimulation with TNFα significantly accelerated thrombotic vessel occlusion in vivo upon ferric chloride injury. Arteriolar thrombosis was much more pronounced in TNFR1-/- animals, where TNFα additionally led to increased platelet-endothelium-interaction. TNFα dependent prothrombotic effects were not observed in TNFR2-/- and TNFR1-/R2- mice. In vitro, stimulation of human platelet rich plasma with TNFα did not influence aggregation properties. In human endothelial cells, TNFα induced superoxide production, p-selectin, tissue factor and PAI-1, and suppressed thrombomodulin, resulting in an accelerated endothelial dependent blood clotting in vitro. Additionally, TNFα caused the release of soluble mediators by endothelial cells which induced prothrombotic and suppressed anticoagulant genes comparable to direct TNFα effects.

Conclusions

TNFα accelerates thrombus formation in an in vivo model of arteriolar thrombosis. Its prothrombotic effects in vivo require TNFR2 and are partly compensated by TNFR1. In vitro studies indicate endothelial mechanisms to be responsible for prothrombotic TNFα effects. Our results support a more selective therapeutic approach in anticytokine therapy favouring TNFR2 specific antagonists.     

Reorientation of cellulose nanowhiskers in agarose hydrogels under tensile loading

Agarose hydrogels filled with cellulose nanowhiskers were strained in uniaxial stretching under different humidity conditions. The orientation of the cellulose whiskers was examined before and after testing with an X-ray laboratory source and monitored in situ during loading by synchrotron X-ray diffraction. The aim of this approach was to determine the process parameters for reorienting the cellulose nanowhiskers toward a preferential direction. Results show that a controlled drying of the hydrogel is essential to establish interactions between the matrix and the cellulose nanowhiskers which allow for a stress transfer during stretching and thereby promote their alignment. Rewetting of the sample after reorientation of the cellulose nanowhiskers circumvents a critical increase of stress. This improves the extensibility of the hydrogel and is accompanied by a further moderate alignment of the cellulose nanowhiskers. Following this protocol, cellulose nanowhiskers with an initial random distribution can be reoriented toward a preferential direction, creating anisotropic nanocomposites.     

Targeted Proteomic Quantification on Quadrupole-Orbitrap Mass Spectrometer

There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and address the pressing demand of systems biology or biomarker evaluation studies.Shotgun proteomics has emerged over the past decade as the most effective method for the qualitative study of complex proteomes (i.e., the identification of the protein content), as illustrated by a wealth of publications (1, 2). In this approach, after enzymatic digestion of the proteins, the generated peptides are analyzed by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)¹ in a data dependent mode. However, the complexity of the digested proteomes under investigation and the wide range of protein abundances limit the reproducibility and the sensitivity of this stochastic approach (3), which is critical if one aims at the systematic quantification of the proteins. Thus, alternative MS approaches have emerged for the systematic quantitative study of complex proteomes, the MS-based targeted proteomics (4). In this hypothesis-driven approach, only specific subsets of analytes (a few targeted peptides used as surrogates for the proteins of interest) are selectively measured in predefined m/z ranges and retention time windows, which overcomes the bias toward most abundant compounds commonly observed with shotgun proteomics. When applied to complex biological samples—for example, bodily fluids such as urine or plasma—targeted proteomics requires high performance instruments allowing measurements of a wide dynamic range (many orders of magnitude), with high sensitivity in order to detect peptides in the low amol range and sufficient selectivity to cope with massive biochemical background (5). Selected reaction monitoring (SRM) on triple quadrupole (6) or triple quadrupole-linear ion trap mass spectrometers (7) has emerged as a means to conduct such analyses (8). Initially applied in the MS analysis of small molecules (9, 10), SRM has gradually emerged as the reference quantitative technique for analyzing proteins (or peptides) in biological samples. When coupled with the isotope dilution strategy (11, 12), this very effective technique allows the precise quantification of proteins (13–18). However, despite the increased selectivity provided by the two-stage mass filtering of SRM (at the precursor and fragment ion levels), the low resolution of mass selection does not allow the systematic removal of interferences (19, 20). Moreover, in proteomics, the biochemical background has a composition similar to that of the analytes of interest, which remains a major hurdle limiting the sensitivity of assays, especially in a bodily fluid matrix. High resolution/accurate mass (HR/AM) analysis represents a promising alternative approach that might more efficiently distinguish the compounds of interest from interferences in targeted proteomics. Such analyses can be conducted on orbitrap-based mass spectrometers because of their high sensitivity and high mass accuracy capabilities (21). The introduction of the benchtop standalone orbitrap mass spectrometer (Exactive) (22) further strengthened the attractiveness of the approach, especially in the field of small molecule analysis (23, 24). However, as quantification using trapping devices intrinsically suffers from a limited dynamic range because of the overall ion capacity, the complexity of biological samples remains very challenging even with the HR/AM approach (25). Targeted protein analysis with triple quadrupole mass spectrometers keeps on showing significant superiority for such samples.² The recently developed quadrupole-orbitrap mass spectrometer (Q-Exactive) can potentially address this issue.³ It is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection (26, 27). This configuration combines advantages of triple quadrupole instruments for mass filtering and orbitrap-based mass spectrometers for HR/AM measurement. The ability of the instrument to select a restricted m/z range or (sequentially) a small number of precursor ions offers new opportunities for quantification in complex samples by selectively enriching low abundant components. The resulting data, acquired in the so-called single ion monitoring (SIM) mode, fully benefit from the trapping capability while keeping a high acquisition rate as a result of the fast switching time between targeted precursor ions of the quadrupole. Although this mode of data acquisition is possible with a configuration combining a linear ion trap with the orbitrap (as in the LTQ-Orbitrap mass spectrometer), its effectiveness is far more limited in this case. The quadrupole-orbitrap configuration presents significant benefits by selectively isolating a narrow population of precursor ions. Other features of the instrument include its multiplexed trapping capability (26) using either the C-trap or the higher energy collisional dissociation (HCD) cell (28, 29), which opens new avenues in the design of innovative acquisition methods for quantification studies. For the first time, a panel of acquisition methods is designed and applied to targeted quantification at the MS and MS/MS levels. In the latter case, the simultaneous monitoring of multiple MS/MS fragmentation channels, also called parallel reaction monitoring⁴ (PRM), is particularly promising for quantifying large sets of peptides with increased selectivity.