Ion Competition in Condensed DNA Arrays in the Attractive Regime

Physical origin of DNA condensation by multivalent cations remains unsettled. Here, we report quantitative studies of how one DNA-condensing ion (Cobalt³⁺ Hexammine, or Co³⁺Hex) and one nonDNA-condensing ion (Mg²⁺) compete within the interstitial space in spontaneously condensed DNA arrays. As the ion concentrations in the bath solution are systematically varied, the ion contents and DNA-DNA spacings of the DNA arrays are determined by atomic emission spectroscopy and x-ray diffraction, respectively. To gain quantitative insights, we first compare the experimentally determined ion contents with predictions from exact numerical calculations based on nonlinear Poisson-Boltzmann equations. Such calculations are shown to significantly underestimate the number of Co³⁺Hex ions, consistent with the deficiencies of nonlinear Poisson-Boltzmann approaches in describing multivalent cations. Upon increasing the concentration of Mg²⁺, the Co³⁺Hex-condensed DNA array expands and eventually redissolves as a result of ion competition weakening DNA-DNA attraction. Although the DNA-DNA spacing depends on both Mg²⁺ and Co³⁺Hex concentrations in the bath solution, it is observed that the spacing is largely determined by a single parameter of the DNA array, the fraction of DNA charges neutralized by Co³⁺Hex. It is also observed that only ∼20% DNA charge neutralization by Co³⁺Hex is necessary for spontaneous DNA condensation. We then show that the bath ion conditions can be reduced to one variable with a simplistic ion binding model, which is able to describe the variations of both ion contents and DNA-DNA spacings reasonably well. Finally, we discuss the implications on the nature of interstitial ions and cation-mediated DNA-DNA interactions.     

Comprehensive analysis of NMR data using advanced line shape fitting

NMR spectroscopy is uniquely suited for atomic resolution studies of biomolecules such as proteins, nucleic acids and metabolites, since detailed information on structure and dynamics are encoded in positions and line shapes of peaks in NMR spectra. Unfortunately, accurate determination of these parameters is often complicated and time consuming, in part due to the need for different software at the various analysis steps and for validating the results. Here, we present an integrated, cross-platform and open-source software that is significantly more versatile than the typical line shape fitting application. The software is a completely redesigned version of PINT (https://pint-nmr.github.io/PINT/). It features a graphical user interface and includes functionality for peak picking, editing of peak lists and line shape fitting. In addition, the obtained peak intensities can be used directly to extract, for instance, relaxation rates, heteronuclear NOE values and exchange parameters. In contrast to most available software the entire process from spectral visualization to preparation of publication-ready figures is done solely using PINT and often within minutes, thereby, increasing productivity for users of all experience levels. Unique to the software are also the outstanding tools for evaluating the quality of the fitting results and extensive, but easy-to-use, customization of the fitting protocol and graphical output. In this communication, we describe the features of the new version of PINT and benchmark its performance.     

Studies on 5alpha-androst-16-en-3-one binding to porcine serum, plasma and testicular cytosolic fraction and to human serum

The present study evaluated whether a specific androstenone-binding protein is present in porcine and human serum, and in the cytosolic fraction of porcine testis. The binding of [(3)H]-androstenone to serum and testicular cytosol was measured in the absence (total binding) and presence (non-specific binding) of unlabelled androstenone. The optimization of the assay is described. As a part of the assay validation, the binding of [(3)H]-dihydrotestosterone ([(3)H]-DHT) to porcine and human serum was also examined. As expected, specific binding of [(3)H]-DHT was detected in human serum, but not in porcine serum. No specific androstenone-binding protein was detected, either in porcine or human serum, or in the cytosolic fraction of porcine testis. The amount of non-specific binding of [(3)H]-androstenone was slightly lower in porcine serum compared to human serum. Between-animal variations in [(3)H]-androstenone binding were studied in plasma samples from 15 animals with androstenone concentrations ranging from 1.1 to 23.1 ng/mL. Mean values+/-standard deviations of binding in these samples were 15.2+/-0.9% for total binding and 15.9+/-0.8% for non-specific bindings. Low between-animal variations indicate that androstenone binding does not affect androstenone accumulation in fat.     

Medium-chain acyl-CoA dehydrogenase (MCAD) mutations identified by MS/MS-based prospective screening of newborns differ from those observed in patients with clinical symptoms: identification and characterization of a new, prevalent mutation that results in mild MCAD deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently diagnosed mitochondrial beta-oxidation defect, and it is potentially fatal. Eighty percent of patients are homozygous for a common mutation, 985A-->G, and a further 18% have this mutation in only one disease allele. In addition, a large number of rare disease-causing mutations have been identified and characterized. There is no clear genotype-phenotype correlation. High 985A-->G carrier frequencies in populations of European descent and the usual avoidance of recurrent disease episodes by patients diagnosed with MCAD deficiency who comply with a simple dietary treatment suggest that MCAD deficiency is a candidate in prospective screening of newborns. Therefore, several such screening programs employing analysis of acylcarnitines in blood spots by tandem mass spectrometry (MS/MS) are currently used worldwide. No validation of this method by mutation analysis has yet been reported. We investigated for MCAD mutations in newborns from US populations who had been identified by prospective MS/MS-based screening of 930,078 blood spots. An MCAD-deficiency frequency of 1/15,001 was observed. Our mutation analysis shows that the MS/MS-based method is excellent for detection of MCAD deficiency but that the frequency of the 985A-->G mutant allele in newborns with a positive acylcarnitine profile is much lower than that observed in clinically affected patients. Our identification of a new mutation, 199T-->C, which has never been observed in patients with clinically manifested disease but was present in a large proportion of the acylcarnitine-positive samples, may explain this skewed ratio. Overexpression experiments showed that this is a mild folding mutation that exhibits decreased levels of enzyme activity only under stringent conditions. A carrier frequency of 1/500 in the general population makes the 199T-->C mutation one of the three most prevalent mutations in the enzymes of fatty-acid oxidation.     

Identification of pathogenic microbial cells and spores by electrochemical detection on a biochip

Background  

Bacillus cereus constitutes a significant cause of acute food poisoning in humans. Despite the recent development of different detection methods, new effective control measures and better diagnostic tools are required for quick and reliable detection of pathogenic micro-organisms. Thus, the objective of this study was to determine a simple method for rapid identification of enterotoxic Bacillus strains. Here, a special attention is given to an electrochemical biosensor since it meets the requirements of minimal size, lower costs and decreased power consumption.     

Tissue oxygenation and perfusion in inferior pedicle reduction mammaplasty by near-infrared reflection spectroscopy and color-coded duplex sonography

Near-infrared reflection spectroscopy has been used in various experimental and clinical settings to investigate tissue perfusion and oxygenation noninvasively. Its application in plastic surgery has only recently been reported. The current study used near-infrared reflection spectroscopy to monitor cutaneous microcirculation in breast skin flaps after inferior pedicle reduction mammaplasty. Thirty patients underwent bilateral reduction mammaplasty by a modified Robbins technique. Near-infrared reflection spectroscopy measurements were performed preoperatively and postoperatively at several defined positions of the breast. The reflection spectroscopy system was capable of detecting absolute values of total hemoglobin in milligrams per milliliter of tissue and tissue hemoglobin oxygen saturation in percent. Color-coded duplex sonography was used to visualize nutrient vessels of the inferior dermoglandular pedicle and to measure systolic peak flow in the arteries supplying the nipple-areola complex. Reflection spectroscopy values were examined for changes during the postoperative course. Reflection spectroscopy and duplex sonography values were analyzed for differences between patients with normal and compromised skin flap perfusion and wound healing, which was assessed clinically and by ultrasound. Preoperative reflection spectroscopy values demonstrated local, regional, and interindividual variations. Postoperatively, characteristic changes of tissue hemoglobin oxygen saturation and total hemoglobin were observed in all patients during the 2-week follow-up. Reflection spectroscopy values differed significantly between breast and nipple-areola skin. Tissue hemoglobin oxygen saturation was significantly lower, and total hemoglobin significantly higher, in patients with impaired wound healing compared with patients having normal wound healing. However, systolic peak flow in arteries of the inferior dermoglandular pedicle did not reveal differences between patients with impaired or normal wound healing of the nipple-areola complex. Near-infrared reflection spectroscopy allows the detection of hemoglobin content and oxygenation in skin flaps. Changes in tissue hemoglobin oxygen saturation and total hemoglobin reflect hemodynamic changes in skin flaps during normal and pathological wound healing. Because of considerable intraindividual and interindividual variations, trend values seem to be superior to single measurements. Although in this study, near-infrared reflection spectroscopy was capable of distinguishing between normal and impaired perfusion in skin flaps in a clinical model, its future implication may be the early detection of vascular compromise in free flaps.     

Robust and convenient analysis of protein thermal and chemical stability

We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard assumptions and equations applied to two‐state and various types of three‐state denaturation models in order to determine thermodynamic parameters. It can analyze denaturation monitored by both circular dichroism and fluorescence spectroscopy and is extremely flexible in terms of input format. Furthermore, it is intuitive and easy to use because of the graphical user interface and extensive documentation. As illustrated by the examples herein, CDpal should be a valuable tool for analysis of protein stability.     

Atrial natriuretic peptide binding, cross-linking, and stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity in cultured cells

The stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity by atrial natriuretic peptide (ANP) was compared to the affinity and number of ANP receptors in eight cultured cell types. At 100 nM, ANP increased cyclic GMP by 13-fold in bovine adrenal cortical, 35-fold in human lung fibroblast, 58-fold in canine kidney epithelial, 60-fold in bovine aortic smooth muscle, 120-fold in rat mammary epithelial, 260-fold in rat Leydig, 300-fold in bovine kidney epithelial, and 475-fold in bovine aortic endothelial cells. ANP (1 microM) increased particulate guanylate cyclase activity by 1.5-, 2.5-, 3.1-, 3.2-, 5.0-, 7.0-, 7.8-, and 8.0-fold in bovine adrenal cortical, bovine aortic smooth muscle, human lung fibroblast, canine kidney epithelial, rat mammary epithelial, rat Leydig, bovine kidney epithelial, and bovine aortic endothelial cells, respectively. Specific 125I-ANP binding to intact rat Leydig (3,000 sites/cell; Kd = 0.11 nM), bovine aortic endothelial (14,000 sites/cell; Kd = 0.09 nM), bovine adrenal cortical (50,000 sites/cell; Kd = 0.12 nM), human lung fibroblast (80,000 sites/cell; Kd = 0.32 nM), and bovine aortic smooth muscle (310,000 sites/cell; Kd = 0.82 nM) cells was saturable and high affinity. No specific and saturable ANP binding was detected in bovine and canine kidney epithelial and rat mammary epithelial cells. Two ANP-binding sites of 66,000 and 130,000 daltons were specifically labeled by 125I-ANP after cross-linking with disuccinimidyl suberate. The 130,000-dalton ANP-binding sites bound to a GTP-agarose affinity column, and the specific activity of guanylate cyclase was increased by 90-fold in this fraction. Our results demonstrate that the increase in cyclic GMP accumulation and particulate guanylate cyclase activity by ANP does not correlate with the affinity and number of ANP-binding sites. These results suggest that multiple populations of ANP receptors exist in these cells and that only one receptor subtype (130,000 daltons) is associated with particulate guanylate cyclase activity.     

Sulfate-reducing anaerobic ammonium oxidation as a potential treatment method for high nitrogen-content wastewater

After sulfate-reducing ammonium oxidation (SRAO) was first assumed in 2001, several works have been published describing this process in laboratory-scale bioreactors or occurring in the nature. In this paper, the SRAO process was performed using reject water as a substrate for microorganisms and a source of NH(4) (+), with SO(4) (2-) being added as an electron acceptor. At a moderate temperature of 20°C in a moving bed biofilm reactor (MBBR) sulfate reduction along with ammonium oxidation were established. In an upflow anaerobic sludge blanket reactor (UASBR) the SRAO process took place at 36°C. Average volumetric TN removal rates of 0.03?kg-N/m3/day in the MBBR and 0.04?kg-N/m3/day in the UASBR were achieved, with long-term moderate average removal efficiencies, respectively. Uncultured bacteria clone P4 and uncultured planctomycete clone Amx-PAn30 were detected from the biofilm of the MBBR, from sludge of the UASBR uncultured Verrucomicrobiales bacterium clone De2102 and Uncultured bacterium clone ATB-KS-1929 were found also. The stoichiometrical ratio of NH(4) (+) removal was significantly higher than could be expected from the extent of SO(4) (2-) reduction. This phenomenon can primarily be attributed to complex interactions between nitrogen and sulfur compounds and organic matter present in the wastewater. The high NH(4) (+) removal ratio can be attributed to sulfur-utilizing denitrification/denitritation providing the evidence that SRAO is occurring independently and is not a result of sulfate reduction and anammox. HCO(3) (-) concentrations exceeding 1,000?mg/l were found to have an inhibiting effect on the SRAO process. Small amounts of hydrazine were naturally present in the reaction medium, indicating occurrence of the anammox process. Injections of anammox intermediates, hydrazine and hydroxylamine, had a positive effect on SRAO process performance, particularly in the case of the UASBR.