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1.  Axonal amyloid precursor protein and its fragments undergo somatodendritic endocytosis and processing 
Molecular Biology of the Cell  2015;26(2):205-217.
In mouse and human neurons, axonally secreted amyloid precursor protein (APP) fragments are processed in the cell body before being sorted into the axon in a process that requires endocytosis for the processing, but not axonal delivery, of APP.
Deposition of potentially neurotoxic Aβ fragments derived from amyloid precursor protein (APP) at synapses may be a key contributor to Alzheimer's disease. However, the location(s) of proteolytic processing and subsequent secretion of APP fragments from highly compartmentalized, euploid neurons that express APP and processing enzymes at normal levels is not well understood. To probe the behavior of endogenous APP, particularly in human neurons, we developed a system using neurons differentiated from human embryonic stem cells, cultured in microfluidic devices, to enable direct biochemical measurements from axons. Using human or mouse neurons in these devices, we measured levels of Aβ, sAPPα, and sAPPβ secreted solely from axons. We found that a majority of the fragments secreted from axons were processed in the soma, and many were dependent on somatic endocytosis for axonal secretion. We also observed that APP and the β-site APP cleaving enzyme were, for the most part, not dependent on endocytosis for axonal entry. These data establish that axonal entry and secretion of APP and its proteolytic processing products traverse different pathways in the somatodendritic compartment before axonal entry.
PMCID: PMC4294669  PMID: 25392299
2.  A γ-Secretase Inhibitor, but Not a γ-Secretase Modulator, Induced Defects in BDNF Axonal Trafficking and Signaling: Evidence for a Role for APP 
PLoS ONE  2015;10(2):e0118379.
Clues to Alzheimer disease (AD) pathogenesis come from a variety of different sources including studies of clinical and neuropathological features, biomarkers, genomics and animal and cellular models. An important role for amyloid precursor protein (APP) and its processing has emerged and considerable interest has been directed at the hypothesis that Aβ peptides induce changes central to pathogenesis. Accordingly, molecules that reduce the levels of Aβ peptides have been discovered such as γ-secretase inhibitors (GSIs) and modulators (GSMs). GSIs and GSMs reduce Aβ levels through very different mechanisms. However, GSIs, but not GSMs, markedly increase the levels of APP CTFs that are increasingly viewed as disrupting neuronal function. Here, we evaluated the effects of GSIs and GSMs on a number of neuronal phenotypes possibly relevant to their use in treatment of AD. We report that GSI disrupted retrograde axonal trafficking of brain-derived neurotrophic factor (BDNF), suppressed BDNF-induced downstream signaling pathways and induced changes in the distribution within neuronal processes of mitochondria and synaptic vesicles. In contrast, treatment with a novel class of GSMs had no significant effect on these measures. Since knockdown of APP by specific siRNA prevented GSI-induced changes in BDNF axonal trafficking and signaling, we concluded that GSI effects on APP processing were responsible, at least in part, for BDNF trafficking and signaling deficits. Our findings argue that with respect to anti-amyloid treatments, even an APP-specific GSI may have deleterious effects and GSMs may serve as a better alternative.
PMCID: PMC4339551  PMID: 25710492
3.  Real-Space x-ray tomographic reconstruction of randomly oriented objects with sparse data frames 
Optics Express  2014;22(3):2403-2413.
Schemes for X-ray imaging single protein molecules using new x-ray sources, like x-ray free electron lasers (XFELs), require processing many frames of data that are obtained by taking temporally short snapshots of identical molecules, each with a random and unknown orientation. Due to the small size of the molecules and short exposure times, average signal levels of much less than 1 photon/pixel/frame are expected, much too low to be processed using standard methods. One approach to process the data is to use statistical methods developed in the EMC algorithm (Loh & Elser, Phys. Rev. E, 2009) which processes the data set as a whole. In this paper we apply this method to a real-space tomographic reconstruction using sparse frames of data (below 10−2 photons/pixel/frame) obtained by performing x-ray transmission measurements of a low-contrast, randomly-oriented object. This extends the work by Philipp et al. (Optics Express, 2012) to three dimensions and is one step closer to the single molecule reconstruction problem.
PMCID: PMC3927634  PMID: 24663531
(000.2190) Experimental physics; (040.7480) X-rays, soft x-rays, extreme ultraviolet (EUV); (100.6950) Tomographic image processing; (110.4155) Multiframe image processing; (110.4280) Noise in imaging systems; (110.6955) Tomographic imaging; (110.7440) X-ray imaging; (340.7440) X-ray imaging
4.  Structural and Kinetic Effects on Changes in the CO2 Binding Pocket of Human Carbonic Anhydrase II 
Biochemistry  2012;51(45):9156-9163.
This work examines the effect on catalysis of perturbing the position of bound CO2 in the active site of human carbonic anhydrase II (HCA II). Variants of HCA II replacing Val143 with hydrophobic residues, Ile, Leu, and Ala, were examined. The efficiency of catalysis in the hydration of CO2 for these variants was characterized by 18O exchange mass spectrometry, and their structures determined by X-ray crystallography at 1.7 to 1.5 Å resolution. The most hydrophobic substitutions V143I and V143L showed decreases in catalysis, as much as 20-fold, while the replacement by the smaller V143A showed only a moderate two-fold decrease in activity. Structural data for all three variants show no significant change in overall position of amino-acid side chains in the active site compared with wild type. However, V143A HCA II showed additional ordered water molecules in the active site compared to wild type. To further investigate the decrease in catalytic efficiency of V143I HCA II, an X-ray crystallographic CO2 entrapment experiment was performed to 0.93 Å resolution. This structure revealed an unexpected shift of the CO2 substrate towards the zinc bound solvent, placing it ~0.3 Ǻ closer than previously observed in wild type in conjunction with the observed dual occupancy of the product bicarbonate, presumably formed during the data acquisition. These data suggest that the Ile substitution at position 143 reduced catalytic efficiency is likely due to steric crowding resulting in destabilization of the transition state for conversion of CO2 into bicarbonate and a decreased product dissociation rate.
PMCID: PMC4301431  PMID: 23098192
5.  Determination of crystallographic intensities from sparse data 
IUCrJ  2015;2(Pt 1):29-34.
A demonstration is given of three-dimensional crystal intensity reconstruction from sparse data, of a nature likely to be encountered in serial microcrystallography experiments at synchrotron sources.
X-ray serial microcrystallography involves the collection and merging of frames of diffraction data from randomly oriented protein microcrystals. The number of diffracted X-rays in each frame is limited by radiation damage, and this number decreases with crystal size. The data in the frame are said to be sparse if too few X-rays are collected to determine the orientation of the microcrystal. It is commonly assumed that sparse crystal diffraction frames cannot be merged, thereby setting a lower limit to the size of microcrystals that may be merged with a given source fluence. The EMC algorithm [Loh & Elser (2009 ▶), Phys. Rev. E, 80, 026705] has previously been applied to reconstruct structures from sparse noncrystalline data of objects with unknown orientations [Philipp et al. (2012 ▶), Opt. Express, 20, 13129–13137; Ayyer et al. (2014 ▶), Opt. Express, 22, 2403–2413]. Here, it is shown that sparse data which cannot be oriented on a per-frame basis can be used effectively as crystallographic data. As a proof-of-principle, reconstruction of the three-dimensional diffraction intensity using sparse data frames from a 1.35 kDa molecule crystal is demonstrated. The results suggest that serial microcrystallography is, in principle, not limited by the fluence of the X-ray source, and collection of complete data sets should be feasible at, for instance, storage-ring X-ray sources.
PMCID: PMC4285878  PMID: 25610625
X-ray serial microcrystallography; sparse data; reconstruction of diffraction intensity; EMC algorithm
6.  Graphene as a protein crystal mounting material to reduce background scatter 
Journal of Applied Crystallography  2013;46(Pt 5):1501-1507.
A new method is presented to reduce background scatter by use of graphene-based mounting of protein crystals.
The overall signal-to-noise ratio per unit dose for X-ray diffraction data from protein crystals can be improved by reducing the mass and density of all material surrounding the crystals. This article demonstrates a path towards the practical ultimate in background reduction by use of atomically thin graphene sheets as a crystal mounting platform for protein crystals. The results show the potential for graphene in protein crystallography and other cases where X-ray scatter from the mounting material must be reduced and specimen dehydration prevented, such as in coherent X-ray diffraction imaging of microscopic objects.
PMCID: PMC3778323  PMID: 24068843
protein crystallography; graphene; X-ray diffraction
7.  Room-temperature serial crystallography using a kinetically optimized microfluidic device for protein crystallization and on-chip X-ray diffraction 
IUCrJ  2014;1(Pt 5):349-360.
An emulsion-based serial crystallographic technology has been developed, in which single crystals are grown in nanolitre-sized droplets inside an X-ray semi-transparent microfluidic chip exploiting a negative feedback mechanism. Diffraction data are measured, one crystal at a time, from a series of room-temperature crystals stored in the chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different unoriented crystals to solve the structure of glucose isomerase to 2.1 Å.
An emulsion-based serial crystallographic technology has been developed, in which nanolitre-sized droplets of protein solution are encapsulated in oil and stabilized by surfactant. Once the first crystal in a drop is nucleated, the small volume generates a negative feedback mechanism that lowers the supersaturation. This mechanism is exploited to produce one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room-temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different unoriented crystals. As proof of concept, the structure of glucose isomerase was solved to 2.1 Å, demonstrating the feasibility of high-throughput serial X-ray crystallography using synchrotron radiation.
PMCID: PMC4174877  PMID: 25295176
protein crystallization; X-ray diffraction; serial crystallography; microfluidic devices
8.  High-dynamic-range coherent diffractive imaging: ptychography using the mixed-mode pixel array detector 
Journal of Synchrotron Radiation  2014;21(Pt 5):1167-1174.
The advantages of a novel wide dynamic range hard X-ray detector are demonstrated for (ptychographic) coherent X-ray diffractive imaging.
Coherent (X-ray) diffractive imaging (CDI) is an increasingly popular form of X-ray microscopy, mainly due to its potential to produce high-resolution images and the lack of an objective lens between the sample and its corresponding imaging detector. One challenge, however, is that very high dynamic range diffraction data must be collected to produce both quantitative and high-resolution images. In this work, hard X-ray ptychographic coherent diffractive imaging has been performed at the P10 beamline of the PETRA III synchrotron to demonstrate the potential of a very wide dynamic range imaging X-ray detector (the Mixed-Mode Pixel Array Detector, or MM-PAD). The detector is capable of single photon detection, detecting fluxes exceeding 1 × 108 8-keV photons pixel−1 s−1, and framing at 1 kHz. A ptychographic reconstruction was performed using a peak focal intensity on the order of 1 × 1010 photons µm−2 s−1 within an area of approximately 325 nm × 603 nm. This was done without need of a beam stop and with a very modest attenuation, while ‘still’ images of the empty beam far-field intensity were recorded without any attenuation. The treatment of the detector frames and CDI methodology for reconstruction of non-sensitive detector regions, partially also extending the active detector area, are described.
PMCID: PMC4151683  PMID: 25178008
pixel array detectors; coherent X-ray diffractive imaging; ptychography
9.  A prototype direct-detection CCD for protein crystallography 
Journal of Applied Crystallography  2013;46(Pt 4):1038-1048.
The fabrication and testing of a prototype deep-depletion X-ray CCD are described. Calibration measurements and the results of initial protein crystallography experiments are described.
The fabrication and testing of a prototype deep-depletion direct-conversion X-ray CCD detector are described. The device is fabricated on 600 µm-thick high-resistivity silicon, with 24 × 24 µm pixels in a 4k × 4k pixel format. Calibration measurements and the results of initial protein crystallography experiments at the Cornell High Energy Synchrotron Source (CHESS) F1 beamline are described, as well as suggested improvements for future versions of the detector.
PMCID: PMC3769057  PMID: 24046505
instrumentation; CCD area detectors; protein crystallography
10.  Structure of a pseudokinase domain switch that controls oncogenic activation of Jak kinases 
Nature structural & molecular biology  2013;20(10):10.1038/nsmb.2673.
The V617F mutation in the Jak2 pseudokinase domain causes myeloproliferative neoplasms, and the equivalent mutation in Jak1 (V658F) is found in T-cell leukemias. Crystal structures of wild type and V658F mutant human Jak1 pseudokinase reveal a conformational switch that remodels a linker segment encoded by exon 12, which is also a site of mutations in Jak2. This switch is required for V617F-mediated Jak2 activation, and possibly for physiologic Jak activation.
PMCID: PMC3863620  PMID: 24013208
11.  A high-pressure cryocooling method for protein crystals and biological samples with reduced background X-ray scatter 
Journal of Applied Crystallography  2012;46(Pt 1):234-241.
A new crystal-hydration method has been developed for high-pressure cryocooling of protein crystals.
High-pressure cryocooling has been developed as an alternative method for cryopreservation of macromolecular crystals and successfully applied for various technical and scientific studies. The method requires the preservation of crystal hydration as the crystal is pressurized with dry helium gas. Previously, crystal hydration was maintained either by coating crystals with a mineral oil or by enclosing crystals in a capillary which was filled with crystallization mother liquor. These methods are not well suited to weakly diffracting crystals because of the relatively high background scattering from the hydrating materials. Here, an alternative method of crystal hydration, called capillary shielding, is described. The specimen is kept hydrated via vapor diffusion in a shielding capillary while it is being pressure cryocooled. After cryocooling, the shielding capillary is removed to reduce background X-ray scattering. It is shown that, compared to previous crystal-hydration methods, the new hydration method produces superior crystal diffraction with little sign of crystal damage. Using the new method, a weakly diffracting protein crystal may be properly pressure cryo­cooled with little or no addition of external cryoprotectants, and significantly reduced background scattering can be observed from the resulting sample. Beyond the applications for macromolecular crystallography, it is shown that the method has great potential for the preparation of noncrystalline hydrated biological samples for coherent diffraction imaging with future X-ray sources.
PMCID: PMC3547228  PMID: 23396891
high-pressure cryocooling; crystal hydration; protein crystallography; coherent X-ray diffraction imaging
12.  High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography 
Science (New York, N.Y.)  2012;337(6092):362-364.
Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.
PMCID: PMC3788707  PMID: 22653729
13.  Single-crystal CVD diamonds as small-angle X-ray scattering windows for high-pressure research 
Journal of Applied Crystallography  2012;45(Pt 3):453-457.
Single-crystal chemical vapor deposition (CVD) diamonds have been investigated using small-angle X-ray scattering, X-ray topography and photoluminescence techniques. An undoped single-crystal CVD diamond proved to be an excellent window material in the characterization of protein unfolding at high pressures.
Small-angle X-ray scattering (SAXS) was performed on single-crystal chemical vapor deposition (CVD) diamonds with low nitrogen concentrations, which were fabricated by microwave plasma-assisted chemical vapor deposition at high growth rates. High optical quality undoped 500 µm-thick single-crystal CVD diamonds grown without intentional nitrogen addition proved to be excellent as windows on SAXS cells, yielding parasitic scattering no more intense than a 7.5 µm-thick Kapton film. A single-crystal CVD diamond window was successfully used in a high-pressure SAXS cell.
PMCID: PMC3359725  PMID: 22675230
small-angle X-ray scattering; chemical vapor deposition; single-crystal diamonds; microwave plasma; high pressure
14.  Solving structure with sparse, randomly-oriented x-ray data 
Optics Express  2012;20(12):13129-13137.
Single-particle imaging experiments of biomolecules at x-ray free-electron lasers (XFELs) require processing hundreds of thousands of images that contain very few x-rays. Each low-fluence image of the diffraction pattern is produced by a single, randomly oriented particle, such as a protein. We demonstrate the feasibility of recovering structural information at these extremes using low-fluence images of a randomly oriented 2D x-ray mask. Successful reconstruction is obtained with images averaging only 2.5 photons per frame, where it seems doubtful there could be information about the state of rotation, let alone the image contrast. This is accomplished with an expectation maximization algorithm that processes the low-fluence data in aggregate, and without any prior knowledge of the object or its orientation. The versatility of the method promises, more generally, to redefine what measurement scenarios can provide useful signal.
PMCID: PMC3635695  PMID: 22714341
(040.7480) X-rays, soft x-rays, extreme ultraviolet (EUV); (110.7440) X-ray imaging; (000.2190) Experimental physics; (110.3055) Information theoretical analysis; (110.4155) Multiframe image processing; (040.0040) Detectors
15.  Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells 
Nature  2012;482(7384):216-220.
Our understanding of Alzheimer’s disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer’s disease, both caused by a duplication of the amyloid-β precursor protein gene1 (APP; termed APPDp), two with sporadic Alzheimer’s disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APPDp patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1–40), phospho-tau(Thr 231) and active glycogen synthase kinase-3β (aGSK-3β). Neurons from APPDp and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr 231) and aGSK-3β levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-β, in GSK-3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer’s disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer’s disease, even though it can take decades for overt disease to manifest in patients.
PMCID: PMC3338985  PMID: 22278060
16.  Systemic inflammation and circadian rhythm of cardiac autonomic modulation 
Systemic inflammation (SI) is associated with impairment of cardiac autonomic modulation (CAM), which is associated with cardiac disease. However, there is limited data about SI on CAM circadian pattern, which this study is aimed to investigate in a middle-aged sample. C-reactive protein (CRP) was used as a SI marker. We performed HRV analysis on each 5-minute segment RRs from of a 24-hour 12-lead ECG to obtain time and frequency domain HRV indices as measures of CAM. The circadian pattern of CAM was analyzed by a two-stage modeling. Stage one, for each individual we fit a cosine periodic model based on the 288 segments of 5-minute HRV data to produce three individual-level cosine parameters that quantity the circadian pattern: mean (M), amplitude (Â), and acrophase time (θ), measure the overall average, the amplitude of the oscillation, and the timing of the highest oscillation, respectively. Stage two, we used random-effects-meta-analysis to summarize the effects of CRP on the three circadian parameters obtained in stage one. CRP was adversely associated with lower M of log-HF, log-LF, SDNN, and RMSSD [β (SE): −0.22 (0.07) ms2, −0.20 (0.06) ms2, −3.62 (0.99) ms, and −2.32 (0.73) ms, respectively, with all p-values<0.01]. More importantly, CRP was also adversely associated with lower  of SDNN and RMSSD [β (SE): −0.84 (0.44) ms and −0.86 (0.38) ms, respectively, both p-values<0.05]. SI is adversely associated with circadian pattern of CAM, suggesting that the cardiac risk associated with SI may be partially mediated via inflammation-related changes in CAM.
PMCID: PMC3111893  PMID: 21444250
C-reactive Protein; Inflammatory Marker; Heart Rate Variability; Periodic Regression; Random-effects Model; Meta-analysis; Community Population
17.  Highly Aminated Mesoporous Silica Nanoparticles with Cubic Pore Structure 
Mesoporous silica with cubic symmetry has attracted interest from researchers for some times. Here we present the room temperature synthesis of mesoporous silica nanoparticles possessing cubic Pm3̄n symmetry with very high molar ratios (>50%) of 3-aminopropyl triethoxysilane. The synthesis is robust allowing, e.g. for co-condensation of organic dyes without loss of structure. By means of pore expander molecules, the pore size can be enlarged from 2.7 to 5 nm, while particle size decreases. Adding pore expander and co-condensing fluorescent dyes in the same synthesis reduces average particle size further down to 100 nm. After PEGylation, such fluorescent aminated mesoporous silica nanoparticles are spontaneously uptaken by cells as demonstrated by fluorescence microscopy.
PMCID: PMC3084371  PMID: 21158438
18.  Small-angle solution scattering using the mixed-mode pixel array detector 
Journal of Synchrotron Radiation  2010;18(Pt 2):148-156.
A fast-readout pixel array detector is evaluated for small-angle X-ray scattering experiments.
Solution small-angle X-ray scattering (SAXS) measurements were obtained using a 128 × 128 pixel X-ray mixed-mode pixel array detector (MMPAD) with an 860 µs readout time. The MMPAD offers advantages for SAXS experiments: a pixel full-well of >2 × 107 10 keV X-rays, a maximum flux rate of 108 X-rays pixel−1 s−1, and a sub-pixel point-spread function. Data from the MMPAD were quantitatively compared with data from a charge-coupled device (CCD) fiber-optically coupled to a phosphor screen. MMPAD solution SAXS data from lysozyme solutions were of equal or better quality than data captured by the CCD. The read-noise (normalized by pixel area) of the MMPAD was less than that of the CCD by an average factor of 3.0. Short sample-to-detector distances were required owing to the small MMPAD area (19.2 mm × 19.2 mm), and were revealed to be advantageous with respect to detector read-noise. As predicted by the Shannon sampling theory and confirmed by the acquisition of lysozyme solution SAXS curves, the MMPAD at short distances is capable of sufficiently sampling a solution SAXS curve for protein shape analysis. The readout speed of the MMPAD was demonstrated by continuously monitoring lysozyme sample evolution as radiation damage accumulated. These experiments prove that a small suitably configured MMPAD is appropriate for time-resolved solution scattering measurements.
PMCID: PMC3042325  PMID: 21335900
small-angle X-ray scattering; detector development
19.  X-ray analog pixel array detector for single synchrotron bunch time-resolved imaging 
Journal of Synchrotron Radiation  2010;18(Pt 2):157-164.
An analog integrating pixel array detector sufficiently fast to isolate synchrotron bunches is presented and tested. The device is appropriate for time-resolved single-crystal Bragg reflection experiments or high-speed beam characterization.
Dynamic X-ray studies can reach temporal resolutions limited by only the X-ray pulse duration if the detector is fast enough to segregate synchrotron pulses. An analog integrating pixel array detector with in-pixel storage and temporal resolution of around 150 ns, sufficient to isolate pulses, is presented. Analog integration minimizes count-rate limitations and in-pixel storage captures successive pulses. Fundamental tests of noise and linearity as well as high-speed laser measurements are shown. The detector resolved individual bunch trains at the Cornell High Energy Synchrotron Source at levels of up to 3.7 × 103 X-rays per pixel per train. When applied to turn-by-turn X-ray beam characterization, single-shot intensity measurements were made with a repeatability of 0.4% and horizontal oscillations of the positron cloud were detected.
PMCID: PMC3042326  PMID: 21335901
time-resolved measurements; area detector; pump–probe; hybrid pixel detector
20.  Sleep Disordered Breathing and Cardiac Autonomic Modulation in Children 
Sleep medicine  2010;11(5):484-488.
To investigate the adverse cardiac autonomic effects of sleep-disordered breathing (SDB) in a large population-based sample and a clinical sample of children.
Subjects were based a population-based sample of 700 and a clinically diagnosed sample of 43 SDB children. SDB was defined based on the Apnea Hyponea Index (AHI) hour over one night of polysomnography. Cardiac autonomic modulation was measured by heart rate variability (HRV) analysis of the beat-to-beat RR interval data collected during the polysomnography.
The mean (SD) age was 112 (21) months, with 49% male and 25% non-white. 73.0% had AHI < 1 (No SDB), 25.8% had 1–5 AHI (Mild SDB), and 1.2% had ≥ 5 AHI (Moderate SDB). Among individuals with moderate SDB in the population-based sample and the clinically diagnosed SDB patients, the mean (SE) of HRV-high frequency power (HF) were significantly lower compared to children without SDB [6.00 (0.32) and 6.24 (0.14), respectively, vs. 6.68 (0.04) ms2, p < 0.05 and p < 0.01, respectively], whereas the low frequency power to high frequency power ratio (LF/HF) were significantly higher [1.62 (0.20) and 1.74 (0.09), respectively, vs. 0.99 (0.02), both p < 0.01)].
SDB in healthy young children and in clinical patients is significantly associated with impaired cardiac autonomic modulation, i.e., sympathetic overflow and weaker parasympathetic modulation, which may contribute to increased risk of acute cardiac events in persons with SDB, even before reaching the “high risk age.”
PMCID: PMC2857753  PMID: 20362503
Sleep Disordered Breathing; Sleep Apnea; Cardiac Autonomic Modulation; Heart Rate Variability; Population-based Study
21.  Fast X-ray microdiffraction techniques for studying irreversible transformations in materials 
Journal of Synchrotron Radiation  2011;18(Pt 3):464-474.
Techniques are described for X-ray diffraction combining micrometer-scale spatial resolution with microsecond-scale temporal resolution for studying rapid localized irreversible transformations in materials.
A pair of techniques have been developed for performing time-resolved X-ray microdiffraction on irreversible phase transformations. In one technique capillary optics are used to focus a high-flux broad-spectrum X-ray beam to a 60 µm spot size and a fast pixel array detector is used to achieve temporal resolution of 55 µs. In the second technique the X-rays are focused with Kirkpatrick–Baez mirrors to achieve a spatial resolution better than 10 µm and a fast shutter is used to provide temporal resolution better than 20 µs while recording the diffraction pattern on a (relatively slow) X-ray CCD camera. Example data from experiments are presented where these techniques are used to study self-propagating high-temperature synthesis reactions in metal laminate foils.
PMCID: PMC3083916  PMID: 21525656
X-ray diffraction; phase transformations
22.  A Short, Strong Hydrogen Bond in the Active Site of Human Carbonic Anhydrase II 
Biochemistry  2010;49(2):249-251.
The crystal structure of human carbonic anhydrase II (HCA II) obtained at 0.9 Å resolution reveals that a water molecule, termed deep water, Dw, and bound in a hydrophobic pocket of the active site forms a short, strong hydrogen bond with the zinc-bound solvent molecule, a conclusion based on the observed oxygen-oxygen distance of 2.45 Å. This water structure has similarities with hydrated hydroxide found in crystals of certain inorganic complexes. The energy required to displace Dw contributes in significant part to the weak binding of CO2 in the enzyme-substrate complex, a weak binding that enhances kcat for the conversion of CO2 into bicarbonate. In addition, this short, strong hydrogen bond is expected to contribute to the low pKa of the zinc-bound water and to promote proton transfer in catalysis.
PMCID: PMC2810610  PMID: 20000378
23.  Insulin resistance and circadian rhythm of cardiac autonomic modulation 
Insulin resistance (IR) has been associated with cardiovascular diseases (CVD). Heart rate variability (HRV), an index of cardiac autonomic modulation (CAM), is also associated with CVD mortality and CVD morbidity. Currently, there are limited data about the impairment of IR on the circadian pattern of CAM. Therefore, we conducted this investigation to exam the association between IR and the circadian oscillations of CAM in a community-dwelling middle-aged sample.
Homeostasis models of IR (HOMA-IR), insulin, and glucose were used to assess IR. CAM was measured by HRV analysis from a 24-hour electrocardiogram. Two stage modeling was used in the analysis. In stage one, for each individual we fit a cosine periodic model based on the 48 segments of HRV data. We obtained three individual-level cosine parameters that quantity the circadian pattern: mean (M), measures the overall average of a HRV index; amplitude (Â), measures the amplitude of the oscillation of a HRV index; and acrophase time (θ), measures the timing of the highest oscillation. At the second stage, we used a random-effects-meta-analysis to summarize the effects of IR variables on the three circadian parameters of HRV indices obtained in stage one of the analysis.
In persons without type diabetes, the multivariate adjusted β (SE) of log HOMA-IR and M variable for HRV were -0.251 (0.093), -0.245 (0.078), -0.19 (0.06), -4.89 (1.76), -3.35 (1.31), and 2.14 (0.995), for log HF, log LF, log VLF, SDNN, RMSSD and HR, respectively (all P < 0.05). None of the IR variables were significantly associated with  or θ of the HRV indices. However, in eight type 2 diabetics, the magnitude of effect due to higher HOMA-IR on M, Â, and θ are much larger.
Elevated IR, among non-diabetics significantly impairs the overall mean levels of CAM. However, the  or θ of CAM were not significantly affected by IR, suggesting that the circadian mechanisms of CAM are not impaired. However, among persons with type 2 diabetes, a group clinically has more severe form of IR, the adverse effects of increased IR on all three HRV circadian parameters are much larger.
PMCID: PMC3017516  PMID: 21134267
24.  Ordered three- and five-ply nanocomposites from ABC block terpolymer microphase separation with niobia and aluminosilicate sols 
We report the first use of a non-frustrated block terpolymer for the synthesis of highly ordered oxide nanocomposites containing multiple plies. The morphological behavior of 15 ISO-oxide nanocomposites was investigated spanning a large range of compositions along the ƒI=ƒS isopleth using aluminosilicate and niobia sols. Morphologies were determined by TEM and SAXS measurements. Four morphologies were identified, including core-shell hexagonal, core-shell double gyroid, three-domain lamellae, and core-shell inverse-hexagonal, in order of increasing O+oxide vol fraction. All of the resulting nanocomposites had three- or five-ply morphologies containing domains that were continuous in one, two, or three dimensions. The five-ply core-shell double gyroid phase was only found to be stable when the O+oxide domain was a minority. Removal of the polymer enabled simple and direct synthesis of mesoporous oxide materials while retaining the ordered network structure. We believe that advances in the synthesis of multi-ply nanocomposites will lead to advanced materials and devices containing multiple plies of functional materials.
PMCID: PMC2832291  PMID: 20209023
25.  Metal Nanoparticle/Block Copolymer Composite Assembly and Disassembly 
Ligand-stabilized platinum nanoparticles (Pt NPs) were self-assembled with poly(isoprene-block-dimethylaminoethyl methacrylate) (PI-b-PDMAEMA) block copolymers to generate organic-inorganic hybrid materials. High loadings of NPs in hybrids were achieved through usage of N,N-di-(2-(allyloxy)ethyl)-N-3-mercaptopropyl-N-3-methylammonium chloride as the ligand, which provided high solubility of NPs in various solvents as well as high affinity to PDMAEMA. From NP synthesis, existence of sub-1 nm Pt NPs was confirmed by high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) images. Estimations of the Pt NP ligand head group density based on HAADF-STEM images and thermogravimetric analysis (TGA) data yielded results comparable to what has been found for alkanethiol self-assembled monolayers (SAMs) on flat Pt {111} surfaces. Changing the volume fraction of Pt NPs in block copolymer-NP composites yielded hybrids with spherical micellar, wormlike micellar, lamellar and inverse hexagonal morphologies. Disassembly of hybrids with spherical, wormlike micellar, and lamellar morphologies generated isolated metal-NP based nano-spheres, cylinders and sheets, respectively. Results suggest the existence of powerful design criteria for the formation of metal-based nanostructures from designer blocked macromolecules.
PMCID: PMC2988680  PMID: 21103025
block copolymer phases; self-assembly; metal nanoparticles; nano-objects; ligand density

Results 1-25 (52)