Correlating calcium binding, Förster resonance energy transfer, and conformational change in the biosensor TN-XXL

Genetically encoded calcium indicators have become instrumental in imaging signaling in complex tissues and neuronal circuits in vivo. Despite their importance, structure-function relationships of these sensors often remain largely uncharacterized due to their artificial and multimodular composition. Here, we describe a combination of protein engineering and kinetic, spectroscopic, and biophysical analysis of the Förster resonance energy transfer (FRET)-based calcium biosensor TN-XXL. Using fluorescence spectroscopy of engineered tyrosines, we show that two of the four calcium binding EF-hands dominate the FRET output of TN-XXL and that local conformational changes of these hands match the kinetics of FRET change. Using small-angle x-ray scattering and NMR spectroscopy, we show that TN-XXL changes from a flexible elongated to a rigid globular shape upon binding calcium, thus resulting in FRET signal output. Furthermore, we compare calcium titrations using fluorescence lifetime spectroscopy with the ratiometric approach and investigate potential non-FRET effects that may affect the fluorophores. Thus, our data characterize the biophysics of TN-XXL in detail and may form a basis for further rational engineering of FRET-based biosensors.     

Lack of regeneration and climatic vulnerability to fire of Scots pine may induce vegetation shifts at the southern edge of its distribution

Aim Forest ecosystems dominated by fire‐sensitive species could suffer shifts in composition under altered crown fire regimes mediated by climate change. The aims of this study were to: (1) study the spatio‐temporal patterns and the climatic distribution of fires in Scots pine (Pinus sylvestris) forests during the last 31 years in north‐eastern Spain, (2) evaluate the climatic vulnerability to fire of these forests in Spain, (3) analyse the regeneration of Scots pine after fire, and (4) predict the mid‐term maintenance or replacement of Scots pine in burned areas. Location Catalonia (north‐eastern Spain): the southern distribution limit of Scots pine. Methods We characterized the spatio‐temporal and the climatic distribution of fires that occurred in Catalonia between 1979 and 2009. We used a generalized linear model to characterize the climatic vulnerability to fire of Scots pine in the whole of Spain. We assessed the regeneration of the species after crown fires in nine burned areas in Catalonia. The resulting data were integrated into a stochastic matrix model to predict the mid‐term maintenance or replacement of Scots pine in burned areas. Results During the last three decades, Scots pine forests distributed in dry sites were most affected by fire. Our assessment of the vulnerability to fire of Scots pine forests in Spain as a whole, based on climatic and topographical variables, showed that 32% of these forests are vulnerable to fire, and that this proportion could increase to 66% under a conservative climate change scenario. Field data showed almost no regeneration of Scots pine after crown fires, and a limited capacity to recolonize from unburned edges, even in relatively old fires, with 90% of recruits located in the first 25 m from the edge. This process could be delayed by the elapsed time for new recruits to achieve reproductive maturity, which we estimated to be c. 15 years. Finally, our matrix model predicted the replacement of burned Scots pine forests by oak (Quercus sp.) forests, shrublands or mixed resprouter forests. Main conclusions Increased vulnerability to fire of Scots pine forests under future, warmer conditions may result in vegetation shifts at the southern edge of the distribution of the species.     

Rare Large Scale Subdomain Motions in Prion Protein can Initiate Aggregation

Abstract

The prion protein is thought to induce prion diseases by changing its conformation from the cellular form, PrP^c, into the infectious Scrapie-form, PrP^Sc. Little is known about the structural and dynamical features of this conformational change. We here introduce a novel concept that involves rare large scale motions between the subdomains βl-αl-β2 and α2-α3 in the carboxy-terminal, globular part of PrP. The interface between these two subdomains carries most pathogenic mutations known to be associated with prion diseases. Based on computational simulations as well as experimental results we propose that such a large scale motion subsequently destabilizes large parts of the cellular conformer PrP^c, thus, rendering it prone to structural rearrangements, including aggregation of now partially unfolded parts of the PrP sequence. We hypothesize that such large scale motions occur as a rare event even under equilibrium conditions and that the interaction of such partially destabilized PrP^c-conformers, which we named PrP^c*, contributes to the formation of pathogenic oligomeric species of the prion protein.     

Spatial Memory in Spontaneously Hypertensive Rats (SHR)

The spontaneously hypertensive rat (SHR) is an established animal model of ADHD. It has been suggested that ADHD symptoms arise from deficits in executive functions such as working memory, attentional control and decision making. Both ADHD patients and SHRs show deficits in spatial working memory. However, the data on spatial working memory deficits in SHRs are not consistent. It has been suggested that the reported cognitive deficits of SHRs may be related to the SHRs’ locomotor activity. We have used a holeboard (COGITAT) to study both cognition and activity in order to evaluate the influence of the activity on the cognitive performance of SHRs. In comparison to Wistar-Kyoto (WKY) rats, SHRs did not have any impairment in spatial working memory and reference memory. When the rats’ locomotor activity was taken into account, the SHRs’ working memory and reference memory were significantly better than in WKY rats. The locomotor activity appears to be a confounding factor in spatial memory tasks and should therefore be controlled for in future studies. In the SHR model of ADHD, we were unable to demonstrate an impairment of working memory which has been reported in patients with ADHD.     

Intraindividual Variability in Inhibitory Function in Adults with ADHD – An Ex-Gaussian Approach

Objective

Attention deficit disorder (ADHD) is commonly associated with inhibitory dysfunction contributing to typical behavioral symptoms like impulsivity or hyperactivity. However, some studies analyzing intraindividual variability (IIV) of reaction times in children with ADHD (cADHD) question a predominance of inhibitory deficits. IIV is a measure of the stability of information processing and provides evidence that longer reaction times (RT) in inhibitory tasks in cADHD are due to only a few prolonged responses which may indicate deficits in sustained attention rather than inhibitory dysfunction. We wanted to find out, whether a slowing in inhibitory functioning in adults with ADHD (aADHD) is due to isolated slow responses.

Methods

Computing classical RT measures (mean RT, SD), ex-Gaussian parameters of IIV (which allow a better separation of reaction time (mu), variability (sigma) and abnormally slow responses (tau) than classical measures) as well as errors of omission and commission, we examined response inhibition in a well-established GoNogo task in a sample of aADHD subjects without medication and healthy controls matched for age, gender and education.

Results

We did not find higher numbers of commission errors in aADHD, while the number of omissions was significantly increased compared with controls. In contrast to increased mean RT, the distributional parameter mu did not document a significant slowing in aADHD. However, subjects with aADHD were characterized by increased IIV throughout the entire RT distribution as indicated by the parameters sigma and tau as well as the SD of reaction time. Moreover, we found a significant correlation between tau and the number of omission errors.

Conclusions

Our findings question a primacy of inhibitory deficits in aADHD and provide evidence for attentional dysfunction. The present findings may have theoretical implications for etiological models of ADHD as well as more practical implications for neuropsychological testing in aADHD.     

Non-acylated Wnts Can Promote Signaling

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Remifentanil added to sufentanil-sevoflurane anesthesia suppresses hemodynamic and metabolic stress responses to intense surgical stimuli more effectively than high-dose sufentanil-sevoflurane alone

Background

Even extremely high-doses of the potent opioid, sufentanil, cannot reliably suppress stress responses to intense surgical stimuli such as sternotomy. The chemically related opioid remifentanil with its different pharmacokinetics and binding affinities for delta- and kappa-opioid receptors might be more effective in attenuating these responses.

Methods

ASA I-III patients scheduled for a surgical procedure with sternotomy under balanced anesthesia (sevoflurane and sufentanil 3 μg.kg^-1 bolus, 0.017 μg.kg^-1.min^-1 infusion) were randomized into two groups. Patients in the study group were supplemented with remifentanil (2 μg.kg^-1 bolus, 2–7 μg.kg^-1.min^-1 infusion) starting ten minutes before sternotomy. Heart rate, arterial blood pressures, cardiac index, ejection fraction, systemic vascular resistance index (SVRI), total body oxygen uptake (VO₂) and electric dermal response were measured and compared between the groups.

Results

62 patients were studied (study group 32, control group 30). Systolic and mean arterial blood pressures, SVRI, VO₂ and skin conductance increased during sternotomy and sternal spread in the control group but not in the study group. Systolic blood pressure increase: 7.5 ± 19 mmHg vs. -3.4 ± 8.9 (p = 0.005); VO₂ increase: 31 ± 46% vs. -0.4 ± 32%; incidence of systolic blood pressure increase greater than 15 percent: 20% vs. 3% (p = 0.035) (control vs. study group).

Conclusion

High-dose remifentanil added to sevoflurane-sufentanil anesthesia suppresses the sympathoadrenergic response to sternotomy and sternal spread better than high-dose sufentanil alone.

Trial registration

Clinical Trial number: DRKS00004327, August 31, 2012

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2253-15-3) contains supplementary material, which is available to authorized users.     

Trends in Longevity in the Americas: Disparities in Life Expectancy in Women and Men, 1965-2010

Objective

We describe trends in life expectancy at birth (LE) and between-country LE disparities since 1965, in Latin America and the Caribbean.

Methods & Findings

LE trends since 1965 are described for three geographical sub-regions: the Caribbean, Central America, and South America. LE disparities are explored using a suite of absolute and relative disparity metrics, with measurement consensus providing confidence to observed differences. LE has increased throughout Latin America and the Caribbean. Compared to the Caribbean, LE has increased by an additional 6.6 years in Central America and 4.1 years in South America. Since 1965, average reductions in between-country LE disparities were 14% (absolute disparity) and 23% (relative disparity) in the Caribbean, 55% and 51% in Central America, 55% and 52% in South America.

Conclusions

LE in Latin America and the Caribbean is exceeding ‘minimum standard’ international targets, and is improving relative to the world region with the highest human longevity. The Caribbean, which had the highest LE and the lowest between-country LE disparities in Latin America and the Caribbean in 1965-70, had the lowest LE and the highest LE disparities by 2005-10. Caribbean Governments have championed a collaborative solution to the growing burden of non-communicable disease, with 15 territories signing on to the Declaration of Port of Spain, signalling regional commitment to a coordinated public-health response. The persistent LE inequity between Caribbean countries suggests that public health interventions should be tailored to individual countries to be most effective. Between- and within-country disparity monitoring for a range of health metrics should be a priority, first to guide country-level policy initiatives, then to contribute to the assessment of policy success.     

Sequentially Rejective Test Procedures for Detecting Outlying Cells in One- and Two-Sample Multinomial Experiments

For multiple testing of multinomial models in the case of one or two samples we propose using test procedures based on the principle described by MARCUS, PERITZ and GABRIEL (1976). These methods are based in each step of the sequentially rejective strategy on tests which exhaust the full α level (i.e. which are not conservative). The tests can be performed in a finite or asymptotic version.