Immunoreactive parathyroid hormone in early and advanced renal failure

Immunoreactive parathyroid hormone (iPTH) was measured in the serum of 20 patients with early renal failure (ERF) using three assays with different specificity. Half of these patients had elevated iPTH in one or more assays, up to twice the upper limit of normal. In contrast, 36 patients with a creatinine clearance below less than 20 ml/min had an 80% elevated iPTH, up to 5 times the upper limit of normal. The patients with ERF and elevated iPTH had a lower serum calcium but no higher serum phosphate than those with normal iPTH. The differences in iPTH in early and end-stage renal failure can be explained by known differences in metabolism of different PTH forms in uremia.     

Accurate determination of succinimide degradation products using high fidelity trypsin digestion peptide map analysis

We report an efficient, high fidelity trypsin digestion method for peptide map analysis. This method minimizes artifacts caused by the sample preparation process, and we show its utility for the accurate determination of succinimide formation in a degraded monoclonal antibody product. A basic charge variant was detected by imaged capillary isoelectric focusing and was shown with reduced antigen binding and biological activity. Samples were reduced under denaturing conditions at pH 5.0, and digestion of the reduced protein with porcine trypsin was performed at pH 7.0 for 1 h. Following reversed phase high-performance liquid chromatography and online mass spectrometric analysis, succinimide formation was identified at Asp30 in the light chain. This result contrasts with the observation of only iso-Asp and Asp residues under conventional sample preparation conditions, which are therefore concluded to be artificially generated. The Asp30 residue is seen in the cocrystal structure model to participate in favorable charge interaction with an antigen molecule. Formation of succinimide and the resulting loss of negative charge are therefore hypothesized to be the degradation mechanism. After treatment of the degraded antibody sample to mildly alkaline pH conditions, we observed only Asp residue as the succinimide hydrolysis product and concurrent recovery of biological activity.     

Toxicity and reductions in intracellular calcium levels following uptake of a tetracycline antibiotic in Arabidopsis

Plant responses to natural stresses have been the focus of numerous studies; however less is known about plant responses to artificial (i.e., man-made) stress. Chlortetracycline (CTC) is widely used in agriculture and becomes an environmental contaminant when introduced into soil from manure used as fertilizer. We show here that in the model plant Arabidopsis (Arabidopsis thaliana), root uptake of CTC leads to toxicity, with growth reductions and other effects. Analysis of protein accumulation and in vivo synthesis revealed numerous changes in soluble and membrane-associated proteins in leaves and roots. Many representative proteins associated with different cellular processes and compartments showed little or no change in response to CTC. However, differences in accumulation and synthesis of NAD-malic enzyme in leaves versus roots suggest potential CTC-associated effects on metabolic respiration may vary in different tissues. Fluorescence resonance energy transfer (FRET) analysis indicated reduced levels of intracellular calcium are associated with CTC uptake and toxicity. These findings support a model in which CTC uptake through roots leads to reductions in levels of intracellular calcium due to chelation. In turn, changes in overall patterns and levels of protein synthesis and accumulation due to reduced calcium ultimately lead to growth reductions and other toxicity effects.     

Universal nanopatternable interfacial bonding

A nanopatternable polydimethylsiloxane (PDMS) oligomer layer is demonstrated as an interfacial adhesive for its intrinsic transferability and universal adhesiveness. Utilizing the well-established surface modification and bonding techniques of PDMS surfaces, irreversible bonding is formed (up to 400 kPa) between a wide range of substrate pairs, representing ones within and across different materials categories, including metals, ceramics, thermoset, and thermoplastic polymers.     

Aspects of transition cow metabolomics—Part I: Effects of a metaphylactic butaphosphan and cyanocobalamin treatment on the metabolome in liver,blood, and urine in cows with different liver metabotypes

Dairy cows in modern production systems are at risk to develop metabolic disorders during the transition period. Reasons for individual differences in susceptibility, as well as the underlying pathomechanisms, are still only partially understood. The development of metaphylactic treatment protocols is needed. In this context, an on-farm prospective 3-fold blinded randomized study involving 80 German Holstein cows was performed throughout 1 yr. The trial involved a thorough recording of the production and clinical traits, clinical chemistry, and liver biopsies and blood and urine sampling at d 14 (mean: 12 d, range: 1–26 d) antepartum (AP), and d 7 (7, 4–13) and 28 (28, 23–34) postpartum (PP) for metabolomics analyses. Two groups received a treatment with butaphosphan and cyanocobalamin (BCC) at either the dosage recommended by the manufacturer or the double dosage (5 or 10 mL/100 kg of body weight 10% butaphosphan and 0.005% cyanocobalamin (Catosal, Bayer Animal Health), n = 20 in each group, parity: 4.2 ± 2.0 and 3.4 ± 1.3, respectively (mean ± SD)] and one group a placebo treatment (NaCl 0.9%, n = 40, parity: 4.0 ± 1.9). The animals were treated at 6 time points (7, 6, and 5 d AP, and 1, 2, and 3 d PP) via intravenous injection. Mass spectroscopy-based targeted metabolomics analysis of blood plasma and liver samples were performed using the AbsoluteIDQ p180 kit (Biocrates Life Sciences), whereas the urine samples were analyzed by nuclear magnetic resonance spectroscopy. Statistical analysis was performed using multivariate [partial least squares discriminant analysis (PLS-DA)] and univariate methods (linear mixed model). Multivariate data analysis (PLS-DA plots) of the liver metabolome revealed 3 different metabotypes (A = medium, B = minor, C = large alterations in liver metabolome profile between AP and PP status). Metabotype B animals were characterized by higher PP lipomobilization (stronger PP body condition decrease and higher blood bilirubin, fatty acids, gamma-glutamyltransferase, and triglyceride levels) and a higher occurrence of transition cow diseases, compared with the animals in metabotype C. Analysis of the feeding data showed that the period of metabotype B animals (calving in a distinct time frame) was characterized by a decreased grass silage quality. The PP liver metabolome of the metabotype C animals was characterized by higher concentrations of AA, acylcarnitines, lysoPC and sphingomyelins compared with metabotype B. For the metaphylactic treatment with BCC a dose-dependent effect was confirmed, differing between the metabotypes. In all matrices and metabotypes at various time points significant treatment effects were observed, with different profiles in clinical chemistry and as well in metabolomics data. The most clear-cut treatment effect was observed in metabotype B in the liver at 7 d PP, characterized by an increase in several acylcarnitines and phosphatidylcholines, indicating a more efficient influx and oxidation of fatty acids in mitochondria and thereby an increase in energy supply and more efficient triglyceride export in the liver. The results from the liver metabolomics analysis support the application of an indication-based metaphylactic treatment with BCC.     

Differential Effects of Halofuginone Enantiomers on Muscle Fibrosis and Histopathology in Duchenne Muscular Dystrophy

Progressive loss of muscle and muscle function is associated with significant fibrosis in Duchenne muscular dystrophy (DMD) patients. Halofuginone, an analog of febrifugine, prevents fibrosis in various animal models, including those of muscular dystrophies. Effects of (+)/(−)-halofuginone enantiomers on motor coordination and diaphragm histopathology in mdx mice, the mouse model for DMD, were examined. Four-week-old male mice were treated with racemic halofuginone, or its separate enantiomers, for 10 weeks. Controls were treated with saline. Racemic halofuginone-treated mice demonstrated better motor coordination and balance than controls. However, (+)-halofuginone surpassed the racemic form’s effect. No effect was observed for (−)-halofuginone, which behaved like the control. A significant reduction in collagen content and degenerative areas, and an increase in utrophin levels were observed in diaphragms of mice treated with racemic halofuginone. Again, (+)-halofuginone was more effective than the racemic form, whereas (−)-halofuginone had no effect. Both racemic and (+)-halofuginone increased diaphragm myofiber diameters, with no effect for (−)-halofuginone. No effects were observed for any of the compounds tested in an in-vitro cell viability assay. These results, demonstrating a differential effect of the halofuginone enantiomers and superiority of (+)-halofuginone, are of great importance for future use of (+)-halofuginone as a DMD antifibrotic therapy.     

Predicting acidification recovery at the Hubbard Brook Experimental Forest, New Hampshire: evaluation of four models

The performance and prediction uncertainty (owing to parameter and structural uncertainties) of four dynamic watershed acidification models (MAGIC, PnET-BGC, SAFE, and VSD) were assessed by systematically applying them to data from the Hubbard Brook Experimental Forest (HBEF), New Hampshire, where long-term records of precipitation and stream chemistry were available. In order to facilitate systematic evaluation, Monte Carlo simulation was used to randomly generate common model input data sets (n = 10,000) from parameter distributions; input data were subsequently translated among models to retain consistency. The model simulations were objectively calibrated against observed data (streamwater: 1963-2004, soil: 1983). The ensemble of calibrated models was used to assess future response of soil and stream chemistry to reduced sulfur deposition at the HBEF. Although both hindcast (1850-1962) and forecast (2005-2100) predictions were qualitatively similar across the four models, the temporal pattern of key indicators of acidification recovery (stream acid neutralizing capacity and soil base saturation) differed substantially. The range in predictions resulted from differences in model structure and their associated posterior parameter distributions. These differences can be accommodated by employing multiple models (ensemble analysis) but have implications for individual model applications.