The Universal Stress Protein UspC Scaffolds the KdpD/KdpE Signaling Cascade of Escherichia coli under Salt Stress

The sensor kinase KdpD and the response regulator KdpE control induction of the kdpFABC operon encoding the high-affinity K⁺-transport system KdpFABC in response to K⁺ limitation or salt stress. Under K⁺ limiting conditions the Kdp system restores the intracellular K⁺ concentration, while in response to salt stress K⁺ is accumulated far above the normal content. The kinase activity of KdpD is inhibited at high concentrations of K⁺, so it has been puzzling how the sensor can be activated in response to salt stress. Here, we demonstrate that the universal stress protein UspC acts as a scaffolding protein of the KdpD/KdpE signaling cascade by interacting with a Usp domain in KdpD of the UspA subfamily under salt stress. Escherichia coli encodes three single domain proteins of this subfamily, UspA, UspC, and UspD, whose expression is up-regulated under various stress conditions. Among these proteins only UspC stimulated the in vitro reconstructed signaling cascade (KdpD→KdpE→DNA) resulting in phosphorylation of KdpE at a K⁺ concentration that would otherwise almost prevent phosphorylation. In agreement, in a ΔuspC mutant KdpFABC production was down-regulated significantly when cells were exposed to salt stress, but unchanged under K⁺ limitation. Biochemical studies revealed that UspC interacts specifically with the Usp domain in the stimulus perceiving N-terminal domain of KdpD. Furthermore, UspC stabilized the KdpD/KdpE∼P/DNA complex and is therefore believed to act as a scaffolding protein. This study describes the stimulation of a bacterial two-component system under distinct stress conditions by a scaffolding protein, and highlights a new role of the universal stress proteins.     

Depth Perception Not Found in Human Observers for Static or Dynamic Anti-Correlated Random Dot Stereograms

One of the greatest challenges in visual neuroscience is that of linking neural activity with perceptual experience. In the case of binocular depth perception, important insights have been achieved through comparing neural responses and the perception of depth, for carefully selected stimuli. One of the most important types of stimulus that has been used here is the anti-correlated random dot stereogram (ACRDS). In these stimuli, the contrast polarity of one half of a stereoscopic image is reversed. While neurons in cortical area V1 respond reliably to the binocular disparities in ACRDS, they do not create a sensation of depth. This discrepancy has been used to argue that depth perception must rely on neural activity elsewhere in the brain. Currently, the psychophysical results on which this argument rests are not clear-cut. While it is generally assumed that ACRDS do not support the perception of depth, some studies have reported that some people, some of the time, perceive depth in some types of these stimuli. Given the importance of these results for understanding the neural correlates of stereopsis, we studied depth perception in ACRDS using a large number of observers, in order to provide an unambiguous conclusion about the extent to which these stimuli support the perception of depth. We presented observers with random dot stereograms in which correlated dots were presented in a surrounding annulus and correlated or anti-correlated dots were presented in a central circular region. While observers could reliably report the depth of the central region for correlated stimuli, we found no evidence for depth perception in static or dynamic anti-correlated stimuli. Confidence ratings for stereoscopic perception were uniformly low for anti-correlated stimuli, but showed normal variation with disparity for correlated stimuli. These results establish that the inability of observers to perceive depth in ACRDS is a robust phenomenon.     

Necrotic core thickness and positive arterial remodeling index: emergent biomechanical factors for evaluating the risk of plaque rupture

Fibrous cap thickness is often considered as diagnostic of the degree of plaque instability. Necrotic core area (Core(area)) and the arterial remodeling index (Remod(index)), on the other hand, are difficult to use as clinical morphological indexes: literature data show a wide dispersion of Core(area) thresholds above which plaque becomes unstable. Although histopathology shows a strong correlation between Core(area) and Remod(index), it remains unclear how these interact and affect peak cap stress (Cap(stress)), a known predictor of rupture. The aim of this study was to investigate the change in plaque vulnerability as a function of necrotic core size and plaque morphology. Cap(stress) value was calculated on 5,500 idealized atherosclerotic vessel models that had the original feature of mimicking the positive arterial remodeling process described by Glagov. Twenty-four nonruptured plaques acquired by intravascular ultrasound on patients were used to test the performance of the associated idealized morphological models. Taking advantage of the extensive simulations, we investigated the effects of anatomical plaque features on Cap(stress). It was found that: 1) at the early stages of positive remodeling, lesions were more prone to rupture, which could explain the progression and growth of clinically silent plaques and 2) in addition to cap thickness, necrotic core thickness, rather than area, was critical in determining plaque stability. This study demonstrates that plaque instability is to be viewed not as a consequence of fibrous cap thickness alone but rather as a combination of cap thickness, necrotic core thickness, and the arterial remodeling index.     

Affinity parameters of amino acid derivative binding to molecularly imprinted nanospheres consisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)

The binding of L-Boc-phenylalanine anilide (BFA) and L-Boc-phenylalanine (phe) to molecularly imprinted and non-imprinted polymer nanoparticles consisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)] has been investigated by adsorption experiments and mathematical modeling. The experimental isotherms have been mathematically adapted following the models of Freundlich, Langmuir, Langmuir-Freundlich, Bi-Langmuir, and extended Langmuir. The extended Langmuir model differentiated between specific and nonspecific binding of the ligand to the receptor nanoparticles and rendered excellent fitting of the experimental data. It delivered a thermodynamic and kinetic parameter set on the experimental association curves of L-BFA by L-BFA-imprinted nanospheres in suspension experiments with the equilibrium constant KD= 4.09 +/- 0.69 micromol L(-1) and the kinetic association rate constant Ka= 5.60 mL micromol(-1) min(-1).     

The physiological effects of deleting the mouse SLC30A8 gene encoding zinc transporter-8 are influenced by gender and genetic background

Objective

The SLC30A8 gene encodes the islet-specific transporter ZnT-8, which is hypothesized to provide zinc for insulin-crystal formation. A polymorphic variant in SLC30A8 is associated with altered susceptibility to type 2 diabetes. Several groups have examined the effect of global Slc30a8 gene deletion but the results have been highly variable, perhaps due to the mixed 129SvEv/C57BL/6J genetic background of the mice studied. We therefore sought to remove the conflicting effect of 129SvEv-specific modifier genes.

Methods

The impact of Slc30a8 deletion was examined in the context of the pure C57BL/6J genetic background.

Results

Male C57BL/6J Slc30a8 knockout (KO) mice had normal fasting insulin levels and no change in glucose-stimulated insulin secretion (GSIS) from isolated islets in marked contrast to the ∼50% and ∼35% decrease, respectively, in both parameters observed in male mixed genetic background Slc30a8 KO mice. This observation suggests that 129SvEv-specific modifier genes modulate the impact of Slc30a8 deletion. In contrast, female C57BL/6J Slc30a8 KO mice had reduced (∼20%) fasting insulin levels, though this was not associated with a change in fasting blood glucose (FBG), or GSIS from isolated islets. This observation indicates that gender also modulates the impact of Slc30a8 deletion, though the physiological explanation as to why impaired insulin secretion is not accompanied by elevated FBG is unclear. Neither male nor female C57BL/6J Slc30a8 KO mice showed impaired glucose tolerance.

Conclusions

Our data suggest that, despite a marked reduction in islet zinc content, the absence of ZnT-8 does not have a substantial impact on mouse physiology.     

The Use of Cellulase in Inhibiting Biofilm Formation from Organisms Commonly Found on Medical Implants

A study was made of the use of cellulase to inhibit biofilm formation by a pathogenic bacterium commonly found in medical implants. A Pseudomonas aeruginosa biofilm was grown on glass slides in a parallel flow chamber for 4 d with glucose as the nutrient source. Biofilm development was assessed by measuring the colony forming units (CFU) and biomass areal density. Biofilm was grown at pH 5 and 7 in the presence of three different cellulase concentrations, 9.4, 37.6 and 75.2 units ml^{m 1}. In addition, a control study using deactivated cellulase was performed. The results show that cellulase is effective in partially inhibiting biomass and CFU formation by P. aeruginosa on glass surfaces. The effect of cellulase depended on concentration and was more effective at pH 5 than pH 7. The experiment was further extended by investigating the effect of cellulase on the apparent molecular weight of purified P. aeruginosa exopolysaccharides (EPS). The observation of EPS using size exclusion chromatography showed a decrease in apparent molecular weight when incubated with enzyme. An increase in the amount of reducing sugar with time when the purified EPS were incubated with enzyme also supports the hypothesis that cellulase degrades the EPS of P. aeruginosa. While cellulase does not provide total inhibition of biofilm formation, it is possible that the enzyme could be used in combination with other treatments or in combinations with other enzymes to increase effectiveness.     

Universal definition of loss to follow-up in HIV treatment programs: a statistical analysis of 111 facilities in Africa, Asia, and Latin America

Background

Although patient attrition is recognized as a threat to the long-term success of antiretroviral therapy programs worldwide, there is no universal definition for classifying patients as lost to follow-up (LTFU). We analyzed data from health facilities across Africa, Asia, and Latin America to empirically determine a standard LTFU definition.

Methods and Findings

At a set “status classification” date, patients were categorized as either “active” or “LTFU” according to different intervals from time of last clinic encounter. For each threshold, we looked forward 365 d to assess the performance and accuracy of this initial classification. The best-performing definition for LTFU had the lowest proportion of patients misclassified as active or LTFU. Observational data from 111 health facilities—representing 180,718 patients from 19 countries—were included in this study. In the primary analysis, for which data from all facilities were pooled, an interval of 180 d (95% confidence interval [CI]: 173–181 d) since last patient encounter resulted in the fewest misclassifications (7.7%, 95% CI: 7.6%–7.8%). A secondary analysis that gave equal weight to cohorts and to regions generated a similar result (175 d); however, an alternate approach that used inverse weighting for cohorts based on variance and equal weighting for regions produced a slightly lower summary measure (150 d). When examined at the facility level, the best-performing definition varied from 58 to 383 d (mean = 150 d), but when a standard definition of 180 d was applied to each facility, only slight increases in misclassification (mean = 1.2%, 95% CI: 1.0%–1.5%) were observed. Using this definition, the proportion of patients classified as LTFU by facility ranged from 3.1% to 45.1% (mean = 19.9%, 95% CI: 19.1%–21.7%).

Conclusions

Based on this evaluation, we recommend the adoption of ≥180 d since the last clinic visit as a standard LTFU definition. Such standardization is an important step to understanding the reasons that underlie patient attrition and establishing more reliable and comparable program evaluation worldwide. Please see later in the article for the Editors'' Summary