The disulfide oxidoreductase SdbA is active in Streptococcus gordonii using a single C‐terminal cysteine of the CXXC motif

Recently, we identified a novel disulfide oxidoreductase, SdbA, in the oral bacterium Streptococcus gordonii. Disulfide oxidoreductases form disulfide bonds in nascent proteins using a CXXC catalytic motif. Typically, the N‐terminal cysteine interacts with substrates, whereas the C‐terminal cysteine is buried and only reacts with the first cysteine of the motif. In this study, we investigated the SdbA C⁸⁶P⁸⁷D⁸⁸C⁸⁹ catalytic motif. In vitro, SdbA single cysteine variants at the N or C‐terminal position (SdbA_C86P and SdbA_C89A) were active but displayed different susceptibility to oxidation, and N‐terminal cysteine was prone to sulfenylation. In S. gordonii, mutants with a single N‐terminal cysteine were inactive and formed unstable disulfide adducts with other proteins. Activity was partially restored by inactivation of pyruvate oxidase, a hydrogen peroxide generator. Presence of the C‐terminal cysteine alone (in the SdbA_C86P variant) could complement the ΔsdbA mutant and restore disulfide bond formation in recombinant and natural protein substrates. These results provide evidence that certain disulfide oxidoreductases can catalyze disulfide bond formation using a single cysteine of the CXXC motif, including the buried C‐terminal cysteine.     

Taking geometry to its edge: fast unbound rigid (and hinge-bent) docking

We present a very efficient rigid "unbound" soft docking methodology, which is based on detection of geometric shape complementarity, allowing liberal steric clash at the interface. The method is based on local shape feature matching, avoiding the exhaustive search of the 6D transformation space. Our experiments at CAPRI rounds 1 and 2 show that although the method does not perform an exhaustive search of the 6D transformation space, the "correct" solution is never lost. However, such a solution might rank low for large proteins, because there are alternatives with significantly larger geometrically compatible interfaces. In many cases this problem can be resolved by successful a priori focusing on the vicinity of potential binding sites as well as the extension of the technique to flexible (hinge-bent) docking. This is demonstrated in the experiments performed as a lesson from our CAPRI experience.     

Rare Variant Association Testing Under Low-Coverage Sequencing

Deep sequencing technologies enable the study of the effects of rare variants in disease risk. While methods have been developed to increase statistical power for detection of such effects, detecting subtle associations requires studies with hundreds or thousands of individuals, which is prohibitively costly. Recently, low-coverage sequencing has been shown to effectively reduce the cost of genome-wide association studies, using current sequencing technologies. However, current methods for disease association testing on rare variants cannot be applied directly to low-coverage sequencing data, as they require individual genotype data, which may not be called correctly due to low-coverage and inherent sequencing errors. In this article, we propose two novel methods for detecting association of rare variants with disease risk, using low coverage, error-prone sequencing. We show by simulation that our methods outperform previous methods under both low- and high-coverage sequencing and under different disease architectures. We use real data and simulation studies to demonstrate that to maximize the power to detect associations for a fixed budget, it is desirable to include more samples while lowering coverage and to perform an analysis using our suggested methods.     

ATP-dependent association between subunits of Clp protease in pea chloroplasts

The chloroplast ATP-dependent Clp protease (EC 3.4.21.92) is composed of the proteolytic subunit ClpP and the regulatory ATPase, ClpC. Although both subunits are found in the stroma, the interaction between the two is dynamic. When immunoprecipitation with antibodies against ClpC was performed on stroma from dark-adapted pea (Pisum sativum L. cv. Alaska) chloroplasts, ClpC but not ClpP was precipitated. However, when stroma was supplemented with ATP, both ClpC and ClpP were precipitated. Co-immunoprecipitation was even more efficient in the presence of ATP-gamma-S, suggesting that the association between regulatory and proteolytic subunits is dependent on binding of ATP to ClpC, but not its hydrolysis. To further test this association, stroma was fractionated by column chromatography, and the presence of Clp subunits in the different fractions was monitored immunologically. When stroma depleted of ATP was fractionated on an ion-exchange column, ClpP and ClpC migrated separately, whereas in the presence of ATP-gamma-S both subunits co-migrated. Similar results were observed in size-exclusion chromatography. To further characterize the precipitated enzyme, its proteolytic activity was assayed by testing its ability to degrade beta-casein. No degradation was observed in the absence of ATP, and degradation was inhibited in the presence of phenylmethylsulfonyl fluoride, consistent with Clp being an ATP-dependent serine protease. The activity of the isolated enzyme was further tested using chimeric OE33 as a model substrate. This protein was also degraded in an ATP-dependent manner, supporting the suggested role of Clp protease as a major housekeeping protease in the stroma.     

Development and evaluation of a multimedia e-learning resource for electrolyte and acid-base disorders

This article reports on the development and evaluation of a Web-based application that provides instruction and hands-on practice in managing electrolyte and acid-base disorders. Our teaching approach, which focuses on concepts rather than details, encourages quantitative analysis and a logical problem-solving approach. Identifying any dangers to the patient is a vital first step. Concepts such as an "appropriate response" to a given perturbation and the need for electroneutrality in body fluids are used repeatedly. Our Electrolyte Workshop was developed using Flash and followed an iterative design process. Two case-based tutorials were built in this first phase, with one tutorial including an interactive treatment simulation. Users select from a menu of therapies and see the impact of their choices on the patient. Appropriate text messages are displayed, and changes in body compartment sizes, brain size, and plasma sodium concentrations are illustrated via Flash animation. Challenges encountered included a shortage of skilled Flash developers, budgetary constraints, and challenges in communication between the authors and the developers. The application was evaluated via user testing by residents and specialists in internal medicine. Satisfaction was measured with a questionnaire based on the System Usability Scale. The mean System Usability Scale score was 78.4 ± 13.8, indicating a good level of usability. Participants rated the content as being scientifically sound; they liked the teaching approach and felt that concepts were conveyed clearly. They indicated that the application held their interest, that it increased their understanding of hyponatremia, and that they would recommend this learning resource to others.     

Physical characterization of the "immunosignaturing effect"

Identifying new, effective biomarkers for diseases is proving to be a challenging problem. We have proposed that antibodies may offer a solution to this problem. The physical features and abundance of antibodies make them ideal biomarkers. Additionally, antibodies are often elicited early in the ontogeny of different chronic and infectious diseases. We previously reported that antibodies from patients with infectious disease and separately those with Alzheimer's disease display a characteristic and reproducible "immunosignature" on a microarray of 10,000 random sequence peptides. Here we investigate the physical and chemical parameters underlying how immunosignaturing works. We first show that a variety of monoclonal and polyclonal antibodies raised against different classes of antigens produce distinct profiles on this microarray and the relative affinities are determined. A proposal for how antibodies bind the random sequences is tested. Sera from vaccinated mice and people suffering from a fugal infection are individually assayed to determine the complexity of signals that can be distinguished. Based on these results, we propose that this simple, general and inexpensive system could be optimized to generate a new class of antibody biomarkers for a wide variety of diseases.     

Sodium chloride reduces growth and cytosolic calcium,but does not affect cytosolic pH,in root hairs of Arabidopsis thaliana L

The effects of salinity (NaCl) stress on growth, cytosolic Ca(2+) gradients and cytosolic pH homeostasis of root hairs of Arabidopsis thaliana are assessed here. Neither cytosolic Ca(2+) nor pH at the hair apex were significantly affected by 20 min exposure of up to 90 mM NaCl or of up to 5 mM extracellular Ca(2+). Exposure to increasing NaCl concentrations, up to 90 mM, for 2 d or 6 d reduced hair extension, and this inhibition was relieved by supplemental extracellular Ca(2+). Such extended salinity stress reduced the magnitude of the Ca(2+) gradient in the apical 12 microm of hairs at all NaCl concentrations tested (up to 90 mM), including NaCl concentrations that did not reduce hair extension. The magnitude of the tip-focused gradient was also reduced in root hairs of plants grown with low (0.5 mM) extracellular Ca(2+) when compared to those in 5 mM extracellular Ca(2+), regardless of the presence of NaCl. Up to 90 mM NaCl did not affect cytosolic pH of root hairs in any of the treatments. It is concluded that NaCl inhibition of root hair extension in the long term may operate via alterations in the tip-focused Ca(2+) gradient that regulates root hair growth. However, NaCl-induced alterations in this gradient do not always lead to detectably altered growth kinetics. Short-term signalling events in response to NaCl may operate by a means other than altering Ca(2+) at the root hair apex. Salinity stress in root hairs does not appear to be mediated by effects on cytosolic pH.     

Importance of medullary events in ammonium excretion: studies in acute respiratory and acute metabolic acidosis

The renal medulla can play an important role in acid excretion by modulating both hydrogen ion secretion in the medullary collecting duct and the medullary PNH3. The purpose of these experiments was to characterize the intrarenal events associated with ammonium excretion in acute acidosis. Cortical events were monitored in two ways: first, the rates of glutamine extraction and ammoniagenesis were assessed by measuring arteriovenous differences and the rate of renal blood flow; second, the biochemical response of the ammoniagenesis pathway was examined by measuring glutamate and 2-oxoglutarate, key renal cortical metabolites in this pathway. There were no significant differences noted in any of these cortical parameters between acute respiratory and metabolic acidosis. Despite a comparable twofold rise in ammonium excretion in both cases, the urine pH, PNH3, and the urine minus blood PCO2 difference (U-B PCO2) were lower during acute hypercapnia. In these experiments, the urine PCO2 was 34 mmHg (1 mmHg = 133.322 Pa) lower than that of the blood during acute respiratory acidosis while the U-B PCO2 was 5 +/- 3 mmHg in acute metabolic acidosis. Thus there were significant differences in medullary events during these two conditions. Although the urine pH is critical in determining ammonium excretion in certain circumstances, these results suggest that regional variations in the medullary PNH3 can modify this relationship.