Analysis and prediction of helix shift errors in homology modeling

High sequence identity between two proteins (e.g. > 60%) is a strong evidence for high structural similarity. However, internal shifts in one of the two proteins can sometimes give rise to unexpectedly high structural differences. This, in turn, causes unreliable structure predictions when two such proteins are used in homology modeling. Here, we perform a computational analysis of helix shifts and we show that their occurrence can be predicted with statistical learning methods. Our results indicate that helix shifts increase the RMS error by factor 2.6 compared to those protein pairs without a helix shift. Although helix shifts are rare (1.6% of helices and a commensurately higher number of proteins are affected), they therefore pose a significant problem for reliable structure prediction systems. In this paper, we prototype a new approach for model quality assessment and demonstrate that it can successfully warn against helix shifts. A support vector machine trained on a wide range of sequence and structure properties predicts the occurrence of helix shifts with a sensitivity of 74.2% and a specificity of 83.6%. On an equalized test dataset, this corresponds to an accuracy of 78.9%. Projected to the full dataset, it translates to an accuracy of 83.4%. Our analysis shows that helix shift detection is a valuable building block for highly reliable structure prediction systems. Furthermore, the statistical learning based approach to helix shift detection that we employ here is orthogonal to well-established model quality assessment methods (which use geometric constraint checking or mean force potentials). Therefore, a further increase of prediction accuracy is expected from the combination of these methods.     

Transcutaneous measurement of renal function in conscious mice

Determination of glomerular filtration rate (GFR) in conscious mice is cumbersome for the experimenter and stressful for the animals. Here we report on a simple new technique allowing the transcutaneous measurement of GFR in conscious mice. This approach extends our previously developed technique for rats to mice. The technique relies on a miniaturized device equipped with an internal memory that permits the transcutaneous measurement of the elimination kinetics of the fluorescent renal marker FITC-sinistrin. This device is described and validated compared with FITC-sinistrin plasma clearance in healthy, unilaterally nephrectomized and pcy mice. In summary, we describe a technique allowing the measurement of renal function in freely moving mice independent of blood or urine sampling as well as of laboratory assays.     

Hyperbaric bradycardia and hypoventilation in exercising men: effects of ambient pressure and breathing gas.

We sought to determine whether hydrostatic pressure contributed to bradycardia and hypoventilation in hyperbaria. Eight men were studied during exercise at 50, 150, and 250 W while breathing 1) air at 1 bar, 2) helium-oxygen (He-O(2)) at 5.5 bar, 3) sulfur hexafluoride-oxygen (SF(6)-O(2)) at 1.3 bar, and 4) nitrogen-oxygen (N(2)-O(2)) at 5.5 bar. Gas densities were pairwise identical in 1) and 2), and 3) and 4), respectively. Increased hydrostatic pressure to 5.5 bar resulted in a modest but significant relative bradycardia on the order of 6 beats/min, in both the absence [1) vs. 2), P = 0. 0015] and presence [3) vs. 4), P = 0.029] of gases that are both denser than normal and mildly narcotic. In contrast, ventilatory responses appeared not to be influenced by hydrostatic pressure. Also, the combined exposure to increased gas density and mild-to-moderate inert gas narcosis at a given hydrostatic pressure [1) vs. 3), 2) vs. 4)] caused bradycardia (P = 0.032 and 0.061, respectively) of similar magnitude as 5.5-bar hydrostatic pressure. At the same time there was relative hypoventilation at the two higher workloads. We conclude that heart rate control, but not ventilatory control, is sensitive to relatively small increases in hydrostatic pressure.     

A Delphi Approach to Understanding Varying Expert Viewpoints in Sustainability Communication: The Case of Water Footprints of Bio‐Based Fiber Resources

In recent years, the concepts of accounting for water use and assessing its impact, also known as the water footprint (WF), have evolved. The cultivation of wood and cotton are two important bio‐based fiber resources that can use, consume, and pollute huge amounts of water. The purpose of this study is to identify the methodological options on an inventory level asociated with a WF assessment for bio‐based fiber resources. Using a three‐step Argument Delphi approach with international experts, important, but controversial, aspects of water footprinting are elaborated. During the different rounds of the Delphi procedure, the interlacement of the crucial topics became apparent, including the net green water or the total volume of green water, trade‐offs between water use and land‐use impacts, allocation of the green WF on ecosystem services, and nomination of a reference situation (e.g., potential natural vegetation). Further, this study evaluates whether the experts allowed generalizations about these methodological options. Finally, the agreement of experts on some generalized statements showed that such statements can be used legitimately as long as knowledge of the inventory methods and knowledge of production characteristics are carefully combined.     

Human skeletal muscle function and metabolism during intense exercise at high O2 and N2 pressures

The maximal contractile force (peak torque) of the quadriceps femoris was studied during 60 repeated unilateral dynamic knee extensions in nine subjects under three different conditions, viz., during air breathing at normal (1 ATA) and raised (6 ATA) ambient pressures and during O2 breathing at 1.3 ATA. In six subjects the electromyographic (EMG) activity of the working muscle was recorded. Muscle biopsies were obtained from the vastus lateralis before, immediately after, and 1 min after exercise. Tissue specimens were subsequently assayed for various muscle metabolites. Peak torque, as an average of the 60 knee extensions, was higher (P less than 0.05) at 1.3 ATA than at 6 or 1 ATA. Peak torque of the exercising muscle declined more rapidly at 1 ATA than at 1.3 ATA, differing in the final 24 contractions by 14%. At 6 ATA peak torque of the initial 12 contractions was 6% lower (P less than 0.05) than at 1 ATA but equaled 1-ATA values in the latter third of the exercise bout. Although the EMG activity at 1 ATA increased relative to that at 6 ATA as exercise proceeded, the rate of force decline was greater at 1 ATA. Despite greater total work produced at 1.3 ATA than at 1 ATA, the metabolic response to exercise was not substantially altered at increased O2 pressure. However, the restitution rate of energy-rich phosphagens and the elimination of lactate during recovery were greater (P less than 0.05) at 1.3 ATA. These results suggest that hyperoxia may enhance the rate of energy release, whereas high N2 pressure and/or high hydrostatic pressure seem to interfere with neuromuscular activity.     

Amisyn,a novel syntaxin-binding protein that may regulate SNARE complex assembly

The regulation of SNARE complex assembly likely plays an important role in governing the specificity as well as the timing of membrane fusion. Here we identify a novel brain-enriched protein, amisyn, with a tomosyn- and VAMP-like coiled-coil-forming domain that binds specifically to syntaxin 1a and syntaxin 4 both in vitro and in vivo, as assessed by co-immunoprecipitation from rat brain. Amisyn is mostly cytosolic, but a fraction co-sediments with membranes. The amisyn coil domain can form SNARE complexes of greater thermostability than can VAMP2 with syntaxin 1a and SNAP-25 in vitro, but it lacks a transmembrane anchor and so cannot act as a v-SNARE in this complex. The amisyn coil domain prevents the SNAP-25 C-terminally mediated rescue of botulinum neurotoxin E inhibition of norepinephrine exocytosis in permeabilized PC12 cells to a greater extent than it prevents the regular exocytosis of these vesicles. We propose that amisyn forms nonfusogenic complexes with syntaxin 1a and SNAP-25, holding them in a conformation ready for VAMP2 to replace it to mediate the membrane fusion event, thereby contributing to the regulation of SNARE complex formation.