The "Transport Specificity Ratio": a structure-function tool to search the protein fold for loci that control transition state stability in membrane transport catalysis

BACKGROUND: In establishing structure-function relationships for membrane transport proteins, the interpretation of phenotypic changes can be problematic, owing to uncertainties in protein expression levels, sub-cellular localization, and protein-folding fidelity. A dual-label competitive transport assay called "Transport Specificity Ratio" (TSR) analysis has been developed that is simple to perform, and circumvents the "expression problem," providing a reliable TSR phenotype (a constant) for comparison to other transporters. RESULTS: Using the Escherichia coli GABA (4-aminobutyrate) permease (GabP) as a model carrier, it is demonstrated that the TSR phenotype is largely independent of assay conditions, exhibiting: (i) indifference to the particular substrate concentrations used, (ii) indifference to extreme changes (40-fold) in transporter expression level, and within broad limits (iii) indifference to assay duration. The theoretical underpinnings of TSR analysis predict all of the above observations, supporting that TSR has (i) applicability in the analysis of membrane transport, and (ii) particular utility in the face of incomplete information on protein expression levels and initial reaction rate intervals (e.g., in high-throughput screening situations). The TSR was used to identify gab permease (GabP) variants that exhibit relative changes in catalytic specificity (k(cat)/K(m)) for [(14)C]GABA (4-aminobutyrate) versus [(3)H]NA (nipecotic acid). CONCLUSIONS: The TSR phenotype is an easily measured constant that reflects innate molecular properties of the transition state, and provides a reliable index of the difference in catalytic specificity that a carrier exhibits toward a particular pair of substrates. A change in the TSR phenotype, called a Δ(TSR), represents a specificity shift attributable to underlying changes in the intrinsic substrate binding energy (ΔG(b)) that translocation catalysts rely upon to decrease activation energy ([Image: see text]). TSR analysis is therefore a structure-function tool that enables parsimonious scanning for positions in the protein fold that couple to the transition state, creating stability and thereby serving as functional determinants of catalytic power (efficiency, or specificity)

Interaction of C-60 molecules on Si(100)

The interactions between pairs of C60 molecules adsorbed upon the Si(1 0 0) surface have been studied via a series of DFT calculations. Configurations which have the fullerene cage located within the dimer trench bonded to four dimers (t4) have been investigated, as these have previously been found to be among the most stable for the C60 molecule. These t4 configurations are explored with all possible pairs of fullerene configuration combinations considered. We have looked at two distinct groups of separation distances between the two C60 molecules. These have the fullerene bonding sites as either adjacent to one another or separated by one Si surface dimer. Comparisons between the two groups confirm the trend of the combinations becoming more favourable at a greater fullerene separation. In the systems with adjacent bonding sites the combined pair of fullerenes were in general less favourable than the two isolated cases. At the longer fullerene separation distance this trend was reversed. The longer fullerene separation distance reflects the experimental separation observed by Moriarty et al. [P. Moriarty, Y.R. Ma, M.D. Upward, P.H. Beton, Surf. Sci. 407 (1998) 27]

Brain-computer interface controlled functional electrical stimulation device for foot drop due to stroke.

Gait impairment due to foot drop is a common outcome of stroke, and current physiotherapy provides only limited restoration of gait function. Gait function can also be aided by orthoses, but these devices may be cumbersome and their benefits disappear upon removal. Hence, new neuro-rehabilitative therapies are being sought to generate permanent improvements in motor function beyond those of conventional physiotherapies through positive neural plasticity processes. Here, the authors describe an electroencephalogram (EEG) based brain-computer interface (BCI) controlled functional electrical stimulation (FES) system that enabled a stroke subject with foot drop to re-establish foot dorsiflexion. To this end, a prediction model was generated from EEG data collected as the subject alternated between periods of idling and attempted foot dorsiflexion. This prediction model was then used to classify online EEG data into either "idling" or "dorsiflexion" states, and this information was subsequently used to control an FES device to elicit effective foot dorsiflexion. The performance of the system was assessed in online sessions, where the subject was prompted by a computer to alternate between periods of idling and dorsiflexion. The subject demonstrated purposeful operation of the BCI-FES system, with an average cross-correlation between instructional cues and BCI-FES response of 0.60 over 3 sessions. In addition, analysis of the prediction model indicated that non-classical brain areas were activated in the process, suggesting post-stroke cortical re-organization. In the future, these systems may be explored as a potential therapeutic tool that can help promote positive plasticity and neural repair in chronic stroke patients

Soil Microbial Networks Shift Across a High-Elevation Successional Gradient.

While it is well established that microbial composition and diversity shift along environmental gradients, how interactions among microbes change is poorly understood. Here, we tested how community structure and species interactions among diverse groups of soil microbes (bacteria, fungi, non-fungal eukaryotes) change across a fundamental ecological gradient, succession. Our study system is a high-elevation alpine ecosystem that exhibits variability in successional stage due to topography and harsh environmental conditions. We used hierarchical Bayesian joint distribution modeling to remove the influence of environmental covariates on species distributions and generated interaction networks using the residual species-to-species variance-covariance matrix. We hypothesized that as ecological succession proceeds, diversity will increase, species composition will change, and soil microbial networks will become more complex. As expected, we found that diversity of most taxonomic groups increased over succession, and species composition changed considerably. Interestingly, and contrary to our hypothesis, interaction networks became less complex over succession (fewer interactions per taxon). Interactions between photosynthetic microbes and any other organism became less frequent over the gradient, whereas interactions between plants or soil microfauna and any other organism were more abundant in late succession. Results demonstrate that patterns in diversity and composition do not necessarily relate to patterns in network complexity and suggest that network analyses provide new insight into the ecology of highly diverse, microscopic communities

Changes in CVD risk factors in the activity counseling trial

Primary care facilities may be a natural setting for delivering interventions that focus on behaviors that improve cardiovascular disease (CVD) risk factors. The purpose of this study was to examine the 24-month effects of the Activity Counseling Trial (ACT) on CVD risk factors, to examine whether changes in CVD risk factors differed according to baseline risk factor status, and to examine whether changes in fitness were associated with changes in CVD risk factors. ACT was a 24-month multicenter randomized controlled trial to increase physical activity. Participants were 874 inactive men and women aged 35–74 years. Participants were randomly assigned to one of three arms that varied by level of counseling, intensity, and resource requirements. Because there were no significant differences in change over time between arms on any of the CVD risk factors examined, all arms were combined, and the effects of time, independent of arm, were examined separately for men and women. Time × Baseline risk factor status interactions examined whether changes in CVD risk factors differed according to baseline risk factor status. Significant improvements in total cholesterol, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol, the ratio of total cholesterol to HDL-C, and triglycerides were seen in both men and women who had high (or low for HDL-C) baseline levels of risk factors, whereas significant improvements in diastolic blood pressure were seen only in those men with high baseline levels. There were no improvements in any risk factors among participants with normal baseline levels. Changes in fitness were associated with changes in a number of CVD risk factors. However, most relationships disappeared after controlling for changes in body weight. Improvements in lipids from the ACT interventions could reduce the risk of coronary heart disease in people with already high levels of lipids by 16%–26% in men and 11%–16% in women. Interventions that can be implemented in health care settings nationwide and result in meaningful population-wide changes in CVD risk factors are needed. The ACT physical activity interventions produced substantial improvements among men and women with elevated CVD risk factors

Boreal forest CO2 exchange and evapotranspiration predicted by nine ecosystem process models: Intermodel comparisons and relationships to field measurements

Nine ecosystem process models were used to predict CO2 and water vapor exchanges by a 150-year-old black spruce forest in central Canada during 1994–1996 to evaluate and improve the models. Three models had hourly time steps, five had daily time steps, and one had monthly time steps. Model input included site ecosystem characteristics and meteorology. Model predictions were compared to eddy covariance (EC) measurements of whole-ecosystem CO2exchange and evapotranspiration, to chamber measurements of nighttime moss-surface CO2release, and to ground-based estimates of annual gross primary production, net primary production, net ecosystem production (NEP), plant respiration, and decomposition. Model-model differences were apparent for all variables. Model-measurement agreement was good in some cases but poor in others. Modeled annual NEP ranged from −11 g C m−2 (weak CO2source) to 85 g C m−2 (moderate CO2 sink). The models generally predicted greater annual CO2sink activity than measured by EC, a discrepancy consistent with the fact that model parameterizations represented the more productive fraction of the EC tower “footprint.” At hourly to monthly timescales, predictions bracketed EC measurements so median predictions were similar to measurements, but there were quantitatively important model-measurement discrepancies found for all models at subannual timescales. For these models and input data, hourly time steps (and greater complexity) compared to daily time steps tended to improve model-measurement agreement for daily scale CO2 exchange and evapotranspiration (as judged by root-mean-squared error). Model time step and complexity played only small roles in monthly to annual predictions

Intrathecal enzyme replacement for Hurler syndrome: biomarker association with neurocognitive outcomes.

PurposeAbnormalities in cerebrospinal fluid (CSF) have been reported in Hurler syndrome, a fatal neurodegenerative lysosomal disorder. While no biomarker has predicted neurocognitive response to treatment, one of these abnormalities, glycosaminoglycan nonreducing ends (NREs), holds promise to monitor therapeutic efficacy. A trial of intrathecal enzyme replacement therapy (ERT) added to standard treatment enabled tracking of CSF abnormalities, including NREs. We evaluated safety, biomarker response, and neurocognitive correlates of change.MethodsIn addition to intravenous ERT and hematopoietic cell transplantation, patients (N = 24) received intrathecal ERT at four peritransplant time points; CSF was evaluated at each point. Neurocognitive functioning was quantified at baseline, 1 year, and 2 years posttransplant. Changes in CSF biomarkers and neurocognitive function were evaluated for an association.ResultsOver treatment, there were significant decreases in CSF opening pressure, biomarkers of disease activity, and markers of inflammation. Percent decrease in NRE from pretreatment to final intrathecal dose posttransplant was positively associated with percent change in neurocognitive score from pretreatment to 2 years posttransplant.ConclusionIntrathecal ERT was safe and, in combination with standard treatment, was associated with reductions in CSF abnormalities. Critically, we report evidence of a link between a biomarker treatment response and neurocognitive outcome in Hurler syndrome