Reduced Tissue Levels of Noradrenaline Are Associated with Behavioral Phenotypes of the TgCRND8 Mouse Model of Alzheimer's Disease

Noradrenergic cell loss is well documented in Alzheimer''s disease (AD). We have measured the tissue levels of catecholamines in an amyloid precursor protein-transgenic ‘TgCRND8'' mouse model of AD and found reductions in noradrenaline (NA) within hippocampus, temporoparietal and frontal cortices, and cerebellum. An age-related increase in cortical NA levels was observed in non-Tg controls, but not in TgCRND8 mice. In contrast, NA levels declined with aging in the TgCRND8 hippocampus. Dopamine levels were unaffected. Reductions in the tissue content of NA were found to coincide with altered expression of brain-derived neurotrophic factor (BDNF) mRNA and to precede the onset of object memory impairment and behavioral despair. To test whether these phenotypes might be associated with diminished NA, we treated mice with dexefaroxan, an antagonist of presynaptic inhibitory α₂-adrenoceptors on noradrenergic and cholinergic terminals. Mice 12 weeks of age were infused systemically for 28 days with dexefaroxan or rivastigmine, a cholinesterase inhibitor. Both dexefaroxan and rivastigmine improved TgCRND8 behavioral phenotypes and increased BDNF mRNA expression without affecting amyloid-β peptide levels. Our results highlight the importance of noradrenergic depletion in AD-like phenotypes of TgCRND8 mice.     

Statistical analysis of ENDOR spectra

Electron–nuclear double resonance (ENDOR) measures the hyperfine interaction of magnetic nuclei with paramagnetic centers and is hence a powerful tool for spectroscopic investigations extending from biophysics to material science. Progress in microwave technology and the recent availability of commercial electron paramagnetic resonance (EPR) spectrometers up to an electron Larmor frequency of 263 GHz now open the opportunity for a more quantitative spectral analysis. Using representative spectra of a prototype amino acid radical in a biologically relevant enzyme, the Y122• in Escherichia coli ribonucleotide reductase, we developed a statistical model for ENDOR data and conducted statistical inference on the spectra including uncertainty estimation and hypothesis testing. Our approach in conjunction with ¹H/²H isotopic labeling of Y122• in the protein unambiguously established new unexpected spectral contributions. Density functional theory (DFT) calculations and ENDOR spectral simulations indicated that these features result from the beta-methylene hyperfine coupling and are caused by a distribution of molecular conformations, likely important for the biological function of this essential radical. The results demonstrate that model-based statistical analysis in combination with state-of-the-art spectroscopy accesses information hitherto beyond standard approaches.

Structural information at atomic resolution is essential for many areas of physics, chemistry, and biology. Electron spin resonance (ESR) or electron paramagnetic resonance (EPR) (1) belongs to a pool of methods, including nuclear magnetic resonance, optical spectroscopy, X-ray diffraction, and cryo-electron microscopy, which can deliver this information. For biophysical applications, the EPR-based electron–nuclear double resonance (ENDOR) technique is particularly suited for mechanistic studies involving endogenous paramagnetic centers, such as redox-active amino acid radical intermediates or metal ions and clusters, as it measures the hyperfine (hf) interaction between these centers and their surrounding magnetic nuclei (2), providing information on the protein architecture and establishing structural to functional relationships. The method has identified and characterized a wealth of paramagnetic intermediates in enzymes; for recent examples see refs. 3–7. Moreover, ENDOR spectroscopy has recently been proposed as a method of choice to measure molecular distances in the angstrom to nanometer range by ¹⁹F spin labeling (8), bridging a critical gap in pulse EPR-based distance measurements in biomolecules and complementing ¹⁹F-based NMR spectroscopy (9, 10).ENDOR spectra of magnetic nuclei are usually recorded in the solid state (a frozen solution of a biological sample) at very low temperatures (T < 80 K) and thus display the full anisotropy of magnetic interactions, resulting in very broad lines. The signals are recorded as a change of the EPR electron-spin echo (11), which is sensitive to drifts in experimental conditions, particularly at low temperatures. Therefore, a model-free analysis of the observed signals constitutes one of the main challenges of ENDOR spectroscopy. In the last decade, progress in EPR instrumentation enabled the implementation of pulsed EPR spectrometers operating in the quasi-optical regime (12). We have recently reported a ¹H ENDOR study of the essential Y122• in the wild-type (WT) β2 subunit of Escherichia coli ribonucleotide reductase (RNR) (Fig. 1A), using a commercial instrument (13) operating at 9.4 T/263 GHz. The strong orientation selection (hole burning) under high field/frequency conditions leads to sharpening of the spectra, strongly facilitating their analysis. We demonstrated the effect with the ¹H spectrum of Y122•, illustrated in Fig. 1B, where the sharp peaks were attributed to internal ¹Hs of the radical. Additional broad resonances became visible, whose attribution presents additional challenges. From visual inspection alone, the significance of these signals could not be inferred given their shallow line shapes and the difficulty to separate them from unknown baseline distortions.Open in a separate windowFig. 1.(A) Structure of Y122 and surrounding amino acids in the E. coli RNR WT-β2 subunit from PDB 1mxr (22) in the reduced state (phenolic proton omitted). Highlighted are interactions with two protons at distances <3 Å from the tyrosine oxygen. Protons were added with Pymol 2.2.2 in positions determined by the overall orientation of the amino acids. The O-O distance (3.8 Å) to the water molecule coordinating the proximal Fe ion is highlighted as well. (B) The 263-GHz ¹H Davies ENDOR spectrum of Y122• adapted from ref. 13. Marked are resonances of the ring protons. Unknown broad features (asterisks) became observable only at 263 GHz. (Inset) Structure of a Y• with labeling of the internal protons.The structure and function of amino acid radicals, particularly Y• in prototype biological machineries such as RNRs or photosystem II, have been the focus of several studies since the 1990s, due to their representative role in biological redox reactions and proton-coupled electron transfer (14, 15). In class Ia RNRs, the Y•/diiron cofactor is essential for enzyme activity and thus for cell survival (16). In contrast to nonprotein Y• model systems (17, 18), the conformation of Y•s in proteins appeared much more constrained (19, 20). This finding was rationalized as due to the interaction with the protein environment that confers redox properties and thus governs biological function. Nevertheless, the mechanism of action of Y• at the diiron cluster in class Ia RNRs remains puzzling. The current model for initiating nucleotide reduction requires that Y122• (E. coli numbering) is reduced in combination with protonation by a water molecule bound to the diferric cluster (16, 21) when the RNR subunits β2, α2 are mixed with substrate and allosteric effector. In all structures of β2 alone, however, the distance between the oxygen of the water and the oxygen of Y122• is too long for this step [3.8 Å in Protein Data Bank (PDB) 1mxr (22); Fig. 1A]. Thus, the model requires a conformational change to shorten this distance, for which to date no experimental evidence exists.Statistical methods are valuable to support the interpretation of biophysical experiments (23, 24). They have already been introduced in EPR spectroscopy for the analysis of distance measurements and distance distributions (25, 26). While adding uncertainty quantification to general spectroscopic practice, these works still hinge on assumptions about functions of interest, whether explicitly made through a Bayesian prior distribution or implicitly included in a choice of a penalization functional or thresholding procedure, some of which are hard to verify in practice. Additionally, ENDOR targets a different object of interest as it uses a different physical method. Therefore, a statistical analysis of ENDOR signals requires different methodology and has been less explored, possibly also due to a high incidence of systematic errors that are hard to model. In this contribution we aim at a statistical model that does not hinge on any specific assumptions regarding the functions of interest, in particular not on any that are hard to verify. Hence, our statistical model can be generalized to other biophysical experiments. In particular, our statistical approach includes careful statistical checking of distributional assumptions and thus lays a strong foundation for our statistical hypothesis tests. Recent advances in microwave (mw) technology, which allow for long-term signal stability and thus accumulation of large datasets, now provide the opportunity to gain additional information from quantitative ENDOR analysis. In this context, development of statistical methods that disentangle signals from noise and systematic error without having to rely on any assumptions regarding the spectrum and that report uncertainty estimates becomes mandatory.The topic of this paper is a statistical analysis of ENDOR spectra and its representative application to assess the significance of broad spectral features observed in the 263-GHz ¹H spectra of the E. coli RNR Y122•. We present a statistical model for the experimental data, which is used to extract the “most likely signal.” This model takes advantage of the information hidden in each individual scan (or batch) of the spectrum that is usually lost in the process of signal averaging. The treatment presents estimation of the uncertainty in the ENDOR spectra and permits subsequent statistical tests. The mathematical approach combined with spectroscopy of various isotopically labeled mutants of Y122• ultimately uncovered a distribution of conformations of Y122•, yielding insight into the mechanism of action of this essential radical.     

Photo-ribonucleotide reductase β2 by selective cysteine labeling with a radical phototrigger

Photochemical radical initiation is a powerful tool for studying radical initiation and transport in biology. Ribonucleotide reductases (RNRs), which catalyze the conversion of nucleotides to deoxynucleotides in all organisms, are an exemplar of radical mediated transformations in biology. Class Ia RNRs are composed of two subunits: α2 and β2. As a method to initiate radical formation photochemically within β2, a single surface-exposed cysteine of the β2 subunit of Escherichia coli Class Ia RNR has been labeled (98%) with a photooxidant ([Re ] = tricarbonyl(1,10-phenanthroline)(methylpyridyl)rhenium(I)). The labeling was achieved by incubation of S355C-β2 with the 4-(bromomethyl)pyridyl derivative of [Re] to yield the labeled species, [Re]-S355C-β2. Steady-state and time-resolved emission experiments reveal that the metal-to-ligand charge transfer (MLCT) excited-state ³[Re ]^∗ is not significantly perturbed after bioconjugation and is available as a phototrigger of tyrosine radical at position 356 in the β2 subunit; transient absorption spectroscopy reveals that the radical lives for microseconds. The work described herein provides a platform for photochemical radical initiation and study of proton-coupled electron transfer (PCET) in the β2 subunit of RNR, from which radical initiation and transport for this enzyme originates.     

Combination therapy in dyslipidemia: Where are we now?

Lowering low-density lipoprotein cholesterol (LDL-C) reduces the risk of cardiovascular disease: each 1.0 mmol/L (38.7 mg/dL) reduction in LDL-C reduces the incidence of major coronary events, coronary revascularizations, and ischemic stroke by approximately 20%. Statins are a well-established treatment option for dyslipidemia, with LDL-C reduction in the range of 27–55%.     

Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis

Although the protective cellular immune response to Mycobacterium tuberculosis requires recruitment of macrophages and T lymphocytes to the site of infection, the signals that regulate this trafficking have not been defined. We investigated the role of C-C chemokine receptor 2 (CCR2)-dependent cell recruitment in the protective response to M. tuberculosis. CCR2(-/-) mice died early after infection and had 100-fold more bacteria in their lungs than did CCR2(+/+) mice. CCR2(-/-) mice exhibited an early defect in macrophage recruitment to the lung and a later defect in recruitment of dendritic cells and T cells to the lung. CCR2(-/-) mice also had fewer macrophages and dendritic cells recruited to the mediastinal lymph node (MLN) after infection. T cell migration through the MLN was similar in CCR2(-/-) and CCR2(+/+) mice. However, T cell priming was delayed in the MLNs of the CCR2(-/-) mice, and fewer CD4(+) and CD8(+) T cells primed to produce IFN-gamma accumulated in the lungs of the CCR2(-/-) mice. These data demonstrate that cellular responses mediated by activation of CCR2 are essential in the initial immune response and control of infection with M. tuberculosis.     

Toe walking: muscular demands at the ankle and knee

OBJECTIVE: To compare the relationship between electromyographic activity and internal moment in heel-toe and toe walking. DESIGN: Simultaneous recording of stride characteristics and kinematic, kinetic, and intramuscular electromyographic data; paired t tests identified significant between-condition differences. SETTING: Gait laboratory. PARTICIPANTS: Ten able-bodied subjects. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Kinematic, moment, power, and electromyographic variables (ankle, knee). RESULTS: Compared with heel-toe walking, toe walking showed greater plantarflexion during stance (P<.001), higher plantarflexor moments (peak, mean) during loading response (P<.001) and midstance (P<.001), lower mean plantarflexor moments during terminal stance (P=.002), premature soleus (P=.001) and gastrocnemius (P<.001) activity, and higher levels of mean soleus and gastrocnemius activity during stance. During toe walking, the peak internal knee extensor moment was lower in midstance (P=.002), and power absorption was reduced in loading response; however, vastus intermedius electromyographic activity was not reduced. CONCLUSIONS: During toe walking, terminal stance soleus and gastrocnemius activity was greater, despite a lower mean internal plantarflexor moment. The dichotomy between internal moments and muscle effort (ie, electromyographic activity) was consistent with the reduction in force-generation capacity of the calf muscles when the ankle was in a plantarflexed position.     

Care coordination for cognitively impaired older adults and their caregivers

Dementia and delirium, the most common causes of cognitive impairment (CI) among hospitalized older adults, are associated with higher mortality rates, increased morbidity and higher health care costs. A growing body of science suggests that these older adults and their caregivers are particularly vulnerable to systems of care that either do not recognize or meet their needs. The consequences can be devastating for these older adults and add to the burden of hospital staff and caregivers, especially during the transition from hospital to home. Unfortunately, little evidence exists to guide optimal care of this patient group. Available research findings suggest that hospitalized cognitively impaired elders may benefit from interventions aimed at improving care management of both CI and co-morbid conditions but the exact nature and intensity of interventions needed are not known. This article will explore the need for improved transitional care for this vulnerable population and their caregivers.     

Effects of a contemporary,exercise-based rehabilitation and cardiovascular risk-reduction program on coronary patients with abnormal baseline risk factors

Phase II cardiac rehabilitation programs are associated with improvements in exercise tolerance, coronary risk factors, and psychosocial well-being. Nevertheless, previous reports have generally evaluated the global effectiveness of these programs (ie, on all subjects, collectively), which may serve to camouflage or attenuate the impact of these interventions on specific patient subsets. In this study, we investigated the effectiveness of a contemporary, exercise-based cardiac rehabilitation program that included a cardiovascular risk-reduction intervention, using a computerized database on 117 patients (average age, 66.5 years; 68% men; 96% white) who completed pre-phase II and post-phase II evaluations. Exercise training involved three 45- to 60-min sessions per week at minimum of 40 to 50% to a maximum of 75% oxygen uptake for 6 to 8 weeks. The effectiveness of the exercise training program was substantiated by significant (p 相似文献