Cyclic AMP-induced conformational changes in mycobacterial protein acetyltransferases

The activities of a number of proteins are regulated by the binding of cAMP and cGMP to cyclic nucleotide binding (CNB) domains that are found associated with one or more effector domains with diverse functions. Although the conserved architecture of CNB domains has been extensively studied by x-ray crystallography, the key to unraveling the mechanisms of cAMP action has been protein dynamics analyses. Recently, we have identified a novel cAMP-binding protein from mycobacteria, where cAMP regulates the activity of an associated protein acetyltransferase domain. In the current study, we have monitored the conformational changes that occur upon cAMP binding to the CNB domain in these proteins, using a combination of bioluminescence resonance energy transfer and amide hydrogen/deuterium exchange mass spectrometry. Coupled with mutational analyses, our studies reveal the critical role of the linker region (positioned between the CNB domain and the acetyltransferase domain) in allosteric coupling of cAMP binding to activation of acetyltransferase catalysis. Importantly, major differences in conformational change upon cAMP binding were accompanied by stabilization of the CNB and linker domain alone. This is in contrast to other cAMP-binding proteins, where cyclic nucleotide binding has been shown to involve intricate and parallel allosteric relays. Finally, this powerful convergence of results from bioluminescence resonance energy transfer and hydrogen/deuterium exchange mass spectrometry reaffirms the power of solution biophysical tools in unraveling mechanistic bases of regulation of proteins in the absence of high resolution structural information.     

Crystal Structure and Functional Characterization of the Complement Regulator Mannose-binding Lectin (MBL)/Ficolin-associated Protein-1 (MAP-1)

The human lectin complement pathway activation molecules comprise mannose-binding lectin (MBL) and ficolin-1, -2, and -3 in complex with associated serine proteases MASP-1, -2, and -3 and the non-enzymatic small MBL associated protein or sMAP. Recently, a novel plasma protein named MBL/ficolin-associated protein-1 (MAP-1) was identified in humans. This protein is the result of a differential splicing of the MASP1 gene and includes the major part of the heavy chain but lacks the serine protease domain. We investigated the direct interactions of MAP-1 and MASP-3 with ficolin-3 and MBL using surface plasmon resonance and found affinities around 5 nm and 2.5 nm, respectively. We studied structural aspects of MAP-1 and could show by multi-angle laser light scattering that MAP-1 forms a calcium-dependent homodimer in solution. We were able to determine the crystal structure of MAP-1, which also contains a head-to-tail dimer ∼146 Å long. This structure of MAP-1 also enables modeling and assembly of the MASP-1 molecule in its entirety. Finally we found that MAP-1 competes with all three MASPs for ligand binding and is able to mediate a strong dose-dependent inhibitory effect on the lectin pathway activation, as measured by levels of C3 and C9.     

Multimodal imaging of stem cell implantation in the central nervous system of mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s). Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting. BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin). MRI on the other hand is a non-invasive technique which is clinically applicable and can be used to precisely locate cellular grafts with very high resolution, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies. In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).     

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants'' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop¹ and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional ''word'' across the affective faces or the facial ''expressions'' of the face stimuli^1,2. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong ''stimulus-response (SR)'' associations; hence inhibiting these strong SR''s is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task^3,4,5,6, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials⁷ which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.     

血管性认知功能障碍的研究进展

血管性认知功能障碍(VCI)是目前严重危害老年人健康的疾病之一。随着人类寿命的延长、人口老龄化的增加,将会有越来越多的老年人患者患上VCI。对于VCI的早期、有效的干预有重要的临床意义,也受到人们的广泛关注。如能预防甚至治疗VCI的产生,可从很大程度上提高老年人的生活质量,减轻家庭和社会的负担。现就近年来对血管性认知功能障碍的相关研究予以综述。     

利用共振频率分析牙种植体骨愈合期稳定性变化 与早期边缘骨吸收的关系

目的:利用共振频率测量仪(Osstell)连续监测骨愈合期种植体稳定性变化与早期边缘骨吸收的关系。方法:本研究于2010-2011年期间根据纳入及排除标准连续纳入32名成年男性患者作为实验对象共植入45枚Strauman种植体,每名患者选择一颗(4.8mm×10mm)种植体,共计32颗种植体,种植区位于下颌后牙(骨质均为Ⅱ或Ⅲ类骨)。利用共振频率分析仪(Osstell)测量种植体的稳定性,测量时间点为植入时以及术后第1,2,3,4,6,8,12周。另外,影像学分析测量32颗种植体12周时的边缘骨吸收;结果:本实验中所有种植体在12周均实现骨结合,并成功完成种植修复。通过重复性方差分析,见表1,在种植体植入时,初期稳定系数(ISQ)均值为(79.03±6.756)。术后一周,种植体稳定系数(ISQ)均值均呈下降趋势,至术后第2周时达到最低点,与植入时稳定性有统计学差异(P<0.05)。从术后第3周开始种植体稳定系数(ISQ)均值逐渐上升。其中,稳定系数(ISQ)均值在第6周时与第12周无统计学差异,已达到延期稳定期。32颗种植体在第12周的边缘骨吸收均值为(0.86±0.068mm),而在第12周的种植体的稳定系数均值与种植体植入时的稳定系数均值无统计学差异。结论:本实验通过共振频率测量仪(OsstellTM)连续监测,目前的结果认为种植体愈合期边缘骨吸收对种植体愈合期稳定性变化没有影响。     

Delineating biased ligand efficacy at 7TM receptors from an experimental perspective

During the last 10 years, the concept of “biased agonism” also called “functional selectivity” swamped the pharmacology of 7 transmembrane receptors and paved the way for developing signaling pathway-selective drugs with increased efficacy and less adverse effects. Initially thought to select the activation of only a subset of the signaling pathways by the reference agonist, bias ligands revealed higher complexity as they have been shown to stabilize variable receptor conformations that associate with distinct signaling events from the reference. Today, one major challenge relies on the in vitro determination of the bias and classification of these ligands, as a prerequisite for future in vivo and clinical translation. In this review, current experimental considerations for the bias evaluation related to choice of the cellular model, of the signaling pathway as well as of the assays are presented and discussed.     

Probing inter-ligand excited state interaction in homo and heteroleptic ruthenium(II) polypyridyl complexes using selective deuteriation

The effect of deuteriation on the photophysical properties of two series of regioselectively deuteriated Ru(II) complexes ([Ru(bipy)_x(ph₂phen)_3−x]²⁺, where x = 0-3 and ph₂phen is 4,7-diphenyl-1,10-phenanthroline and [Ru(bipy)₂(dcbipy²⁻)], where H₂dcbipy is 4,4′-dicarboxy-2,2′-bipyridyl) is reported. Although overall, deuteriation results in an increase in emission lifetime for all complexes, the effect of substitution of hydrogen for deuterium shows strong regioselectivity both in terms of the ligand and the position on individual ligands that are exchanged.